hAGT inhibitors as chemotherapy enhancers Maria Tintor? Gazulla Aquesta tesi doctoral est? subjecta a la llic?ncia Reconeixement 3.0. Espanya de Creative Commons. Esta tesis doctoral est? sujeta a la licencia Reconocimiento 3.0. Espa?a de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution 3.0. Spain License. UNIVERSITAT DE BARCELONA FACULTAT DE FARM?CIA ?hAGT inhibitors as chemotherapy enhancers? Maria Tintor? Gazulla 2015 UNIVERSITAT DE BARCELONA FACULTAT DE FARM?CIA Programa de Doctorat en Biotecnologia hAGT inhibitors as che motherapy enhancers Mem?ria pres entada per Maria Tintor? Gazulla per a optar al t?tol de doctor per la Universitat de Barcelona Dr. Carme F?brega Claveria Dr. Ram?n Eritja Casadell? Dr. Gl?ria Rosell Pellis? Directora Director Tutora Maria Tintor? Gazulla Barcelona, 2015 The work des cribed in this thesis has been performed in the Inst itute for Advanced Chemistry of Catalonia (CSIC) and in the Institute for Research in Biomedicine of Barcelona, IRB Barcelona. Chapter 5 is the result of a short stay of 4 months in the University of Milan. I am grateful for a FIS contract (grant PI06/1250 , Fondo de Investigaciones Sanitarias) and a predoctoral fellowship from MINECO (Spanish Ministry of Economy and Competitiv ene ss) FPI (BES- 2011-043815) associated to the project CTQ2010-20541-C03-01 and for a short stay grant from MINECO (EEBB-C- 13-00369) . Durant aquest temp s al laboratori he tingut la sort de con?ixer molta gent maca. Voldria agrair esp ecialment als meus companys del laboratori de Qu?mica dels ?cids Nucleics tot el que he apr?s, tant cient?fica com personalment. Gr?cies al Ramon per donar- me ??oport?nitat ??entrar en a??est ma?n??ic ?r?p? A ?a arme? ??e em ?a ensenyar ?aireb? a utilitzar una pipeta i de qui he apr?s que el m?s important en la recerca s?n la paci?ncia i e? ri?or? A ??Anna? ??e m?ha c?i?at com ?na mare? A? anti? per ?a se?a paci?ncia amb e?s meus m?ltiples dubtes sobre s?ntesi qu?mica; a la S?nia, sempre disposada a donar un cop de m? amb els HPLC, els MALDIs o el que faci falta; i a ??A?e?e, per la seva disponibilitat i ajuda amb els cultius cel?lulars. Al Rub?n per la seva ajuda amb els exp eriments de masses i per tots els bons moments i consells. Al Nacho, ??e m?a??anta amb a?e?ria ?es in?asio s de papers a la seva taula i els robatoris de calculadores i bolis . I a la resta de persones que han passat pel lab durant aquest temp s , de qui he pogut aprendre moltes coses. Vull agrair molt a la Stefi la seva carinyosa acollida durant la meva estada al seu laboratori a Mil?. I a la Laura, el Leo, la Gigliola, la Sabrina i ??An?e?a. Tamb? vull mencionar els meus companys estudiants de doctorat, en esp ecial a ??A?bert, per tots els b erenars en qu? he m convertit un moment d e des ?nim per exp eriments que no sortien en unes bones rialles . A? ichae? i ??An?rey pe?s ?inars i pin?-po gs compartits. I als meus companys i amics del m?ster, en esp ecial a la Laura per la seva ajuda ??rant ??escript?ra ?e ?a tesi i al Jes?s p er donar-me un cop de m? amb ??estad?stica. Moltes gr?cies als meus amics de farm?cia pel seu suport, amb ells vaig comen?ar ??a?ent?ra ?e ?a ci?ncia? Agraeixo molt al Germ?n la seva ajuda amb el disseny de la portada de la tesi. I gr?cies tamb? al Carlos Roca, la Laia i ?a resta ??amics per la seva curiositat i inter?s, em fa molta il?lusi? que amics que treballen en camps tan allunyats tinguin tantes ganes de saber el qu? faig. Per ?ltim, vull agrair molt?ssim als meus pares i als meus germans la seva implicaci? i il?lusi? i sobre tot, al Xavi, ??e m?ha a???at? m?ha ?onat ?nims? m?ha pre??ntat i s?ha esco?tat les presentacions? m?ha preparat e? t?pper mo?t?ssimes ?e?a?es i sobre tot, m?ha reco??at en tot moment. INDEX Introduction..... . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. . . .. . .. .. .. .. .. . .. . . .. .. .. .. . .. .. .. .. .. . .. .. ... . .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .. . 1 Objectives. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .29 1. Potential inhibitors of hAGT?s DNA repair activity: study of the hAGT-compound complex formation by ESI-MS and toxicity in cell culture?????????????.....................................33 1.1. Study of the hAGT-compound complex formation by ESI-MS and toxicity in cell c??t?re??????????????????????????????????????????????35 1.2. Appendix 1. Receptor- based virtual screening and biological characterization of new inhibitors of Human Apurinic/Apyrimidinic Endonuclease Enzyme ?Ape?????????????????????????????????????????????? 2. Development of a novel fluorescence assay based on the use of the Thrombin Binding Aptamer for the detection of O6-alkylguanine?DNA-alkyltransferase activity???????????????????????????????????????????????75 2.1. Development of a novel fluoresc ence assay based on the u se of the Th rombin Binding Aptamer for the d etection of O 6 - alk ylguanine ?DNA Alkyltransferase acti?ity???????????????????????????????????????. ?79 2.2. ?pp?ementary in?ormation?????????????????????????????. .. .. .. . .. .. .. .. 89 2.3. Appendix 2: Thrombin Binding A ptamer, more than a simple aptamer: chemically m odified derivatives and biomedical applications... .. .. . .. .. .. .. ... . .. .. .. .. . .. .. . . .. .. . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. . . .. 9 3 3. DNA Origami as DNA repair nanosensor at the single-molecule level???.????.............109 3.1. DNA Origami as DNA repair nanosensor at the single-molecule l e?e???.. ????????113 3.2. ?pp?ementary in?ormation??????????????????????????.. .. . .. .. .. .. .. . .. . 117 3.3. Appen?i? ? ? A nanoarchitect?res? steps to?ar?s bio?o?ica? app?ications?????????1 4. A fluorescence biosensor for hAGT activity????????????????????....................163 4.1. A ???orescence biosensor ?or hAGT acti?ity?????????????????????. . .. .. .. .. .. . .. .167 4.2. ?pp?ementary in?ormation????????????????????????????????179 4. 3. Appendix 4: In vitro assay to evaluate potential inhibitors of hAGT by a new fluoresc ence method: preliminary results ????????????????????????????????203 5. Molecular biosensing using gold-coated superparamagnetic nanoparticles functionalized with DNA aptamers???????????????????????????????????????.209 Genera? ?isc?ssion?????????????????????????????????????????????221 onc??sions????????????????????????????????????????????????237 ?mmary???????????????????????????????????????????. ??241 Resum ???????????????????????????????????????????????????245 ABBREVIATIONS 3 H -MNU: Tritiated 1-methyl-1-nitrosourea ??-Dabsyl CPG: 1- ?????-Dimethoxytrityloxy-3- [O - (N - 4' -sulfonyl-4-(dimethylamino)-azobenzene)- 3-aminopropyl]-propyl-2-O-succinoyl-long chain alkylamino-CPG 3' -Dabcyl CPG: 1-(4, 4' -Dimethoxytrityloxy)-3- [O- (N - 4' -carboxy-4-(dimethylamino)- azobenzene)- 3-aminopropyl)] -propyl-2-O-succinoyl-long chain alkylamino-CPG ??-FAM: 6-???????- dipivaloylfluoresc einyl)- carboxamido]-hexyl-1-O-[(2-cyanoethyl)-(N,N - diisopropyl)] -phosphoramidite Ac: acetyl Ac 2O: acetic anhydride ACN: acetonitrile AcOEt: ethyl acetate AFM: atomic force microscopy anh: anhydrous Ape1: human apurinic/apyrimidinic endonuclease Ar: aromatic Au(OOCCH 3 ): gold acetate AuNPs: gold nanoparticles AuSPIONs: gold-coated superparamagnetic iron oxide nanoparticles BCNU: 1,3-B is (2 -chloroethyl)-1-nitrosourea or carmustine BME: 2-mercaptoethanol Bz: benzyl Bzl: benzoyl Cpd: compound CPG: controlled pore glass Dabcyl: 4-(4 - (dimethylamino)phenyl-azobenzoic acid Dabsyl: N - ??- sulfonyl-4-(dimethylamino)- azobencene)-3-aminopropyl DCM: dichloromethane DEAD: diethyl azodicarboxylate (IUPAC diethyl diazenedicarboxylate) DIEA: N,N -Diisopropylethylamine DLS: dynamic light scattering dmf : dimethylformamidino group DMAP: N,N - dimethylaminopyridine DMF: N,N - dimethylformamide DMSO: dimethylsulfoxide DMT: ????- dimethoxytrityl DMT-Cl: ????- dimethoxytrityl chloride DTT: dithiothreitol EDTA: ethylenediaminetetraacetic acid ESI: electrospray ionization EtTFA: ethyl trifluoroacetate FAM/F: fluoresc eine FG: O 6 - fluoresc ein-benzylguanosine FITC: fluoresc ein isothiocyanate FL: full length FRET: fluoresc ence resonance energy transfer GST: gluthatione-S-transferase hAGT: human O 6 -alkylguanine-DNA alkyltransferase HBA: hydrogen bond acceptors HBD: hydrogen bond donors HPLC: high performance liquid chromatography IC50 : inhibitory concentration 50% IPTG: isopropyl- ?- D- 1- thiogalactopyranoside LCAA: long chain amino alkyl LD50 : lethal dose 50% MALDI: matrix-assisted laser diserption/ionization MeOH: methanol MB: molecular beacon MNU: 1-methyl-1-nitrosourea MTT: (3 -(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) NMR: nuclear magnetic resonance. NPs: nanoparticles OD: optical density O6 -MeG: O 6 - methylguanine PAGE: polyacrylamide gel electrophoresis PBS: phosphate-buffered saline buffer PPh 3 : triphenylphosphine PrePro: prescission protease Pyr: pyridine Q T: (N- 4' -carboxy-4-(dimethylamino)- azobenzene)-aminohexyl-3-acrylamido]- 2' - deoxyuridine RP -HPLC: reverse phase high pressure liquid chromatography rt: room temperature SPIONs : superparamagnetic iron oxide nanoparticles TBA: thrombin-binding aptamer cTBA: complementary TBA TCA: trichloroacetic acid TEA: triethylamine TCEP?HCl: tris(2-carboxyethyl)phosphine hydrochloride TEAAc: triethylammonium acetate TEM: transmission electron microscopy TFA: trifluoroacetic acid THAP: ????????-trihydroxyacetophenone monohydrat e THF: tetrahydrofurane Thr? ?-thrombin TLC: thin-layer chromatography Tm: melting temperature TMAOH: tetramethylammonium hydroxide TOF: time of flight Tris: tris(hydroxymethyl)aminomethane UV: ultraviolet VS: virtual screening Introduction 1 2 Deoxyribonucleic acid (DNA) The DNA double helix was first described by Watson and Crick in 1953.[1] Since then, the scientific advances based in DNA have been prodigious and far beyond expectations in many fields, reaching areas as diverse as medicine, basic biology, genetics, forensics, archeology among others, and culminating in the sequencing of the human genome.[2] Deoxyribonucleic acid is a self-assembling biopolymer that encodes the genetic information used in the development and functioning of all known living organisms and many viruses. DNA, together with proteins and carbohydrates, compose the three major macromolecules essential for all forms of life. DNA forms double helices directed by canonical Watson-Crick base pairing and is stabilized by hydrogen bonds, ?-? stacking and hydrophobic interactions.[1] The two DNA strands are composed of simpler units called nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase?either guanine (G), adenine (A), thymine (T), or cytosine (C)?as well as a monosaccharide sugar called deoxyribose and a phosphate group. The four nucleobases are classified in pyrimidines and purines, depending on their structure: pyrimidines (T and C) consist of a single heterocycle of six atoms and purines (A and G) are composed by a pyrimidine ring fused to an imidazole ring (figure 1). Nucleotides are joined to one another by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in a chain formed by an alternating sugar-phosphate backbone. According to base pairing rules (A with T and C with G), hydrogen bonds bind the nitrogenous bases of the two separate polynucleotide strands to make double-stranded DNA. This double helix is further stabilized by ?-? stacking and hydrophobic interactions. Figure 1. 1A. Representation of the four nucleotides, covalently linked by phosphate bonds. 1B. Watson-Crick canonical base pairing, C-G and A-T. Non-covalent hydrogen bonds between the pairs are shown as dashed lines. Adapted from ref [3].[3] 3 DNA structures DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms (figure 2), although only B-DNA and Z-DNA have been directly observed in functional organisms.[4] The conformation adopted by DNA depends on its hydration level, its sequence, the amount and direction of supercoiling, the chemical modifications of its bases, the type and concentration of metal ions, as well as the presence of polyamines in solution.[5] The more compact A form of DNA has 11 base pairs per turn and exhibits a large tilt of the base pairs with respect to the helix axis. In addition, the A form has a central whole (figure 2, left). This helical form is adopted by RNA?DNA and RNA?RNA helices.[6] The B-form of DNA, usually found in cells, consists of well-defined structures that are repeated along the strands: the helical turn measures ? 3.4 nm, the helical diameter ? 2.0 nm and the twist angle between base-pairs in solution ? 34.3? (figure 2, centre).[1] In contrast, the Z-DNA helix is left-handed and has a structure that is repeated every 2 base pairs (figure 2, right). The major and minor grooves, unlike A- and B-DNA, show little difference in width.[7] Formation of this structure is generally unfavourable, as it is a form of higher energy than B-DNA, although certain conditions can promote it, such as alternating purine-pyrimidine sequence, negative DNA supercoiling or high salt and some cations, all requiring physiological conditions. Z-DNA can form a junction with B- DNA (called a "B-to-Z junction box") in a structure which involves the extrusion of a base pair.[8] The Z-DNA conformation has been difficult to study because it does not exist as a stable feature of the double helix. Instead, it is a transient structure that is occasionally induced by biological activity and then quickly disappears.[9] Figure 2. Different types of DNA conformation. A-DNA is the more compact form of DNA, with 11 base-pairs per turn, and it is typical of RNA-DNA and RNA-RNA lattices. B-DNA, the most common conformation of DNA in 4 nature, is right-handed and possesses a well-defined structure. Z-DNA is conferred as a zig-zag and is a form of higher energy than B-DNA. The remarkable flexibility of nucleic acids allows them to form a great variety of structures, in addition to the double helix described above, as for example triplexes, i- motifs and G-quadruplexes. This flexibility is caused mainly by the high degree of freedom of the deoxyribose ring, and secondly, by the rotation of the glycosidic bond and the phosphate backbone. Triplex forming oligonucleotides (TFOs)[10] bind in the major groove of duplex DNA with high specificity and affinity. A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double helix forming Hoogsteen pairs with purines of the Watson-Crick base-pairs. Intermolecular triplexes are formed between triplex forming oligonucleotides (TFO) and target sequences on duplex DNA[11] (figure 3).[12] TFO should have the identical sequence of the complementary strand of the associating strand in the DNA duplex, while the direction of the TFO relies on the type of DNA triplex. Depending on which bases (pyrimidine or purine) of the TFO interact with the purine base of the duplex, there are two kinds of DNA triplexes: parallel and anti-parallel, respectively.[12] Because of these characteristics, TFOs have been proposed as homing devices for genetic manipulation in vivo to alter gene expression and mediate genome modification.[13] Figure 3. Parallel triplex which consists of T ? A?T and C+ ? G?C triads (light green). Triplex forming oligonucleotide (TFO), bound at the major groove of the DNA duplex, is coloured in pink. Adapted from ref. [12]. In contrast, the i-motif is an intercalated structure formed by association in a head to tail orientation of two parallel duplexes whose strands are held together by hemiprotonated C:C(+) pairs [14] (figure 4). Due to the hemiprotonated nature of the C:C(+) pairs, the formation of i-motifs often requires acidic conditions. However, depending on 5 particular C-rich sequences, i-motifs can fold close to neutral pH.[15] The i-motif may be formed by a single strand containing four cytidine repeats, by association of two strands containing two cytidine repeats or by four strands containing a single cytidine stretch. In vivo, intramolecular i-motifs can be found in C-rich sequences of centromeric and telomeric sequences in the chromosome. Figure 4. Representation of an i-motif quadruplex structure, with its hemiprotonated C:C+ base pair in cyan. Adapted from ref. [12]. G-quadruplexes and TBA G-quadruplexes are a family of four-stranded DNA structures stabilized by the stacking of guanine tetrads, in which four planar guanines form a cyclic array of Hoogsteen hydrogen bonds stabilized by the presence of monovalent cations[16] (figure 5A). However, most divalent cations have the capacity to induce the dissociation of G-quadruplex structures.[17] These G-rich regions are connected by lateral, central or diagonal loops of diverse sizes and composition that form base-pairing alignments, which in turn stack with the terminal G-tetrads, thus further stabilizing G-quadruplex structures.[18] G- quadruplexes can be folded from a single G-rich sequence intramolecularly or by the intermolecular association of two (dimeric) or four (tetrameric) separate strands (figure 5B). In addition, quadruplexes can adopt different topologies with varying loop configurations, depending on how the guanine bases are arranged.[16d] In the case of intermolecular quadruplexes, if the 5'-3? direction of all the strands is the same, the quadruplex is termed parallel (figure 5C). In contrast, intermolecular G-quadruplexes are antiparallel if the strands are arranged in different directions. For intramolecular quadruplexes, if one or more of the runs of guanine bases has a 5?-3? direction opposite to the other runs of guanine bases, the quadruplex is said to have adopted an antiparallel topology (figure 5C). 6 Figure 5. A. Structure of a G-quadruplex structure with the square and planar arrangement of four guanines forming a G-quartet. Adapted from ref. [12] B. Schematic representation of intermolecular quadruplexes, tetra and bimolecular. C. Schematic representation of intramolecular quadruplexes, antiparallel and parallel respectively. Adapted from ref. [19][19] Modifications in the base composition of the tetrads are poorly tolerated by these structures. As an example, inosine [20] and O6-methylguanosine [21], (figure 6) both nucleosides containing non-natural bases, can form a smaller number of hydrogen bonds, provoking the loss of the quadruplex conformation.[22] Figure 6. Chemical structure of methylguanosine and inosine. There is evidence that G-quadruplexes can be formed in vivo[23] and present great biological relevance. They can be found in the telomere, the final region of the chromosome composed by repetitions of non-coding G-rich sequences, which protects the end of the chromosome from deterioration or from fusion with neighbouring chromosomes.[24] In addition, these sequences can be found in gene promoters regions, suggesting that their function may be related with gene regulation at the transcription level.[25] Furthermore, a great variety of proteins can recognize and interact with 7 oligonucleotide aptamers that possess a G-quadruplex structure, as the G-quadruplex aptamers that inhibit the HIV integrase.[26] In particular, the ?-thrombin binding aptamers (TBA) are well characterized chair- like, antiparallel quadruplex structures that bind specifically to ?-thrombin at nanomolar concentrations and therefore have interesting anticoagulant properties.[27] ?-thrombin is able to interact with two different TBAs in two binding sites of the protein.[28] These two sequences are known to bind specifically and cooperatively to two specific and almost opposite epitopes of ?-thrombin when folded into their quadruplex structure.[28b] TBA1 is a 15mer nucleotide composed of two G-tetrads that are connected by three edge-wise loops, forming a well-characterized intramolecular chair-like, antiparallel quadruplex which binds ?-thrombin in its primarily fibrinogen exosite.[28a] In contrast, TBA2, a 29mer nucleotide that forms a combined quadruplex/duplex structure, interacts with ?-thrombin in its heparin-binding exosite, placed in the opposite side of the protein[28b] (figure 7). Figure 7. ?-thrombin interaction with the ?-thrombin binding aptamers. The fibrinogen exosite (I) of ?- thrombin interacts with the 15mer aptamer while the heparine exosite (II) is recognized by the longer aptamer (29mer). DNA stability, damage and repair DNA may suffer structural damage that can alter or eliminate the cell's ability to transcribe the encoded genes.[29] DNA damage occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day, which constitutes only 0.000165% of the human genome,[30] and plays a major role in mutagenesis, carcinogenesis and ageing. These reactions are triggered by exposure to environmental factors and exogenous chemicals, or they can result from metabolic processes inside the cell.[31] Environmental and exogenous DNA damage can be caused by the UV radiation of the sun,[32] other radiation frequencies, as X- 8 rays and gamma radiation,[33] and human-made mutagenic chemicals,[34] specially aromatic compounds that act as intercalating agents.[35] Some viruses can also cause mutations in DNA during their replication cycle inside the host cell.[36] In contrast, endogenous damage is caused by spontaneous mutations and replication errors,[37] or by reactive oxygen species produced from normal metabolic byproducts.[38] There are several types of damage to DNA due to exogenous factors and endogenous cellular processes:[39] oxidation of bases, as for example 8-oxo-7,8- dihydroguanine (8-oxoG);[40] alkylation of bases[41] (usually methylation), such as formation of 7-N-methylguanine,[42] 3-N-methyladenine[43] and 6-O-methylguanine;[44] or hydrolysis of bases, such as deamination,[45] depurination,[46] and depyrimidination.[47] Other damage includes bulky adduct formation, mismatch of bases due to errors in DNA replication and crosslinks of different types.[48] Cells must repair DNA damage to prevent mutations from propagating and accumulating, and to maintain genome integrity and stability. The persistence of unrepaired DNA damage results in accumulation of mutations, cell cycle arrest and apoptosis.[49] DNA damage alters the spatial configuration of the helix and such alterations can be detected by the cell, inducing several mechanisms for DNA repair.[50] The ATM and ATR genes often initiate the DNA damage response, activating signal transduction pathways that arrest the cell cycle and increase the expression of DNA repair genes.[51] Enzymes involved in different pathways as the base-excision, the nucleotide excision, the mismatch repair, the double-strand break, and other DNA repair processes (figure 8) all respond in a pre- and post-transcriptionally regulated fashion to DNA damage. Nevertheless, cells unable to sense and repair DNA damage may continue to grow and divide, eventually causing cellular dysfunction and death, a hallmark of diseases such as neurological defects and infertility. In addition, deregulation of DNA damage sensing can also yield an increased mutation rate, potentially causing uncontrolled cell growth and cancer. 9 Figure 8. Scheme of the different types of damage that DNA can suffer with their according DNA repair pathways. Adapted from ref. [52] The base excision repair (BER) pathway is a major mechanism for dealing with a variety of lesions in DNA produced by alkylation.[53] This pathway is initiated by specific DNA glycosylases, which recognize and excise the damaged base to generate an apurinic/apyrimidinic (AP) site. AP endonuclease 1 (Ape1)[54] cleaves the phosphodiester backbone adjacent to the 5? site of the AP site, generating a 3? hydroxyl and 5?-deoxyribose phosphate termini.[54d] Polymerase ? removes the 5?-deoxy ribose phosphate, fills in the one-nucleotide gap, and the consequent nick is ligated by DNA ligase I or by DNA ligase III/XRCC1[47, 55] (figure 9). Ape1 is a fundamental protein in this essential repair pathway and it is thought to be responsible for 95% of total AP endonuclease activity in human cell lines.[56] 10 Figure 9. Schematic representation of the enzymes involved in the base excision repair pathway. Other DNA repair mechanisms are based on a single reaction modulated by a single enzyme. For example, the DNA repair O6-alkylguanine DNA alkyltransferase (AGT or MGMT) is in charge of repairing alkylating damage, removing alkyl adducts from the O6 position of guanines. The human AGT (hAGT), the most thoroughly characterized AGT protein, repairs alkylated DNA by flipping the damaged base out of the helix, and the alkyl group is transferred from the point of lesion to the active site Cys145 residue to be repaired.[57] Once alkylated, this protein is degraded by the ubiquitin pathway.[58] hAGT is normally present in all cells, but its overexpression can be triggered in tumoral cells that are being attacked by alkylating chemotherapeutic agents. DNA alkylating agents as chemotherapy for cancer Alkylating agents are chemotherapeutic anticancer drugs that produce their cytotoxic effect by generating alkylation and adducts at multiple sites in DNA.[59] DNA repair pathways induced by chemotherapeutic drugs as alkylating agents have a key role in the prognosis and evolution of multiple cancers. [60] The simultaneous treatment with alkylating agents and inhibitors of DNA repair proteins sensitizes tumoral cells to chemotherapy and results in a better outcome and prognosis.[61] An example of it is the identification and characterization of small molecules that inhibit the repair endonuclease activity of APE-1,[62] including methoxyamine (MX), lucanthone and 7-nitroindole-2- carboxilic acid (NCA). All three compounds were able to enhance the effects of 11 methylmethane sulfonate (MMS) or temozolamide (TMZ) in ovarian,[63] breast,[63b, 64] colon[65] and HT1080 fibrosarcoma cancer cells.[66] Another subset of alkylating agents, which includes nitrosoureas and temozolamide, have a preference for alkylating guanine at the O6 position, which is the most important in terms of mutagenesis and carcinogenesis.[42, 44, 67] In particular, 1,3-bis- (2-chloroethyl)-1-nitrosourea (BCNU or carmustine) attacks initially at the O6 guanine position, causing its rearrangement in a cyclic intermediate due to the attack at the N1 position of guanine, giving rise to N1,O6-ethanoguanine.[68] Finally, a cross-link with the opposite cytosine is formed (figure 10) and, as a consequence, DNA replication is blocked, producing G2/M arrest.[69] N H N N N H 2 N H O N H N N N H 2 N ON O N H ClCl N O BC N U Cl N H N N N H 2 N O D NA DNA DNA N N O H 2 N D NA N H N N N H 2 N O D NA N N O DNA HN Figure 10. Mechanism of action of 1,3-bis-(2-chloroethyl)-1-nitrosourea, which alkylates the O6 position of guanines, causing a crosslink with the opposite cytosine. The DNA-repair O6-alkylguanine DNA alkyltransferase (hAGT) is in charge of removing alkyl adducts from the O6 position of guanines, blocking their cytotoxic effects and playing an important role as a chemotherapy resistance mechanism (figure 11).[70] Figure 11. Mechanism of action of the repair reaction of hAGT. A. Carmustine or BCNU (bis- chloroethylnitrosourea) introduces alkyl groups in the O6 position of guanines. B. The active site cystine 145 removes the alkylation through a nucleophilic attack SN2. C. The guanine is restored while the active site of hAGT remains covalently linked to the alkyl group and the protein undergoes a process of ubiquitination and degradation. 12 It is well established that tumor cells frequently express higher levels of this protein, which appears to be predictive of poor response to chemotherapeutic drugs. This effect has been observed in a large number of cancers, ranging from colon cancer, lung tumors, breast cancer, pancreatic tumors, non-Hodgkin?s lymphoma, myeloma and glioblastoma multiforme, among others.[71] Additionally, methylation of hAGT promoter and consequently hAGT complete depletion has been associated with longer survival in patients with gliomas under radiation-chemotherapy combining treatment.[72] Therefore, pharmacological inhibition of hAGT has the potential to enhance cytotoxicity of a diverse range of anticancer agents.[73] Adducts formed at the O6 position of the guanine are of major importance in both the initiation of mutations and in the cytotoxic effects of these agents. For all these reasons, hAGT is considered relevant as a prognosis marker of cancer and represents a potential therapeutic target.[70c] Several research groups have focussed their research on the identification of small molecules capable of inhibiting hAGT activity and enhancing the cytotoxic effect of the alkylating agents in tumour cells.[74] Over the last years, a number of drugs have been shown to inactivate hAGT in cells, human tumour models and cancer patients, and O6-benzylguanine and O6-[4- bromothenyl]guanine have been used in clinical trials. These agents also inactivate MGMT in normal tissues and hence exacerbate the toxic side effects of the alkylating drugs, requiring dose reduction.[75] In addition, they possess poor pharmacokinetic conditions[76] and also produce myelosupression.[77] 13 DNA Nanotechnology At the end of the XX century, Ned Seeman set the bases for the use of DNA as a scaffold for nanoscale building material.[78] The remarkable specificity of the molecular recognition between complementary nucleotides in the DNA base pairing has made it an attractive molecule for scientists and engineers interested in micro- and nano-fabrication. Its predictability, rigidity, and precise structural control, as well as the creation of algorithms for de novo design of new self-assembled structures,[79] make it a useful building material to develop different kinds of nanotechnological platforms. Compared to other self- assembling molecules, DNA nanostructures offer programmable interactions and surface features for the precise positioning of other nanoparticles and biomolecules.[80] Seeman?s original goal was the creation of regular 3D lattices of DNA which could be used as scaffolding for the rapid, orderly binding of biological macromolecules to speed the formation of suitable crystals for 3D protein structure elucidation in x-ray diffraction studies.[78] This concept gave rise to the tile-based assembly method, used to synthesize two-dimensional periodic lattices[81] and three-dimensional architectures as for example a cube in solution[82] and different polyhedra in solid phase.[83] Some examples of 3D DNA constructs are represented in figure 12.[84] Figure 12. A. Schematic representation of a tridimensional cube, containing twelve equal-length double- helical edges arranged around eight vertices. Adapted from ref. [82] B. Double-helical representation of an ideal truncated octahedron. Adapted from ref. [83b] C. Schematic representation of a DNA tetrahedron. Adapted from ref. [83c] D. Reconstituted 3D image of an octahedron. Secondary structures of the octahedron consisted of different DNA motifs for 3D formation. Adapted from ref. [83a] E. Scheme of the polyhedral structures: a tetrahedron, a dodecahedron and a bucky ball were assembled from three-point star building blocks. Adapted from reference [83d]. DNA Origami Another important breakthrough in the structural DNA nanotechnology field has been the development of DNA origami by Paul Rothemund,[85] where a long scaffold strand of 7 14 kilobase, the M13 phage genome, is folded with the help of hundreds of short ?staples? to create a rational desired two-dimensional shape (figure 13). Since then, various DNA motifs have been designed in 2D and 3D, and extensive studies are currently ongoing to apply these nanostructures to a large amount of biomedical, computational and molecular motor purposes. Figure 13. Scheme of the formation of a DNA origami by the annealing of a viral circular scaffold and the complementary staple strands. Adapted from ref [87]. DNA origami is a versatile tool for the self-assembly of other molecular species[86] and constitutes an excellent platform to create a variety of new nanoscale devices[87] with great biological potential and applications.[88] As examples, Yao and co-workers have used the DNA origami as an addressable support for label-free detection of RNA hybridization[89] and later, Seeman and co-workers have developed a nanosensor to detect single nucleotide polymorphism (SNP).[90] Both strategies represent an innovative way to use the DNA origami methodology to create a nanosensor for biomedical applications at the single molecule level using atomic force microscopy (AFM) (figure 14A). In addition, DNA origami has been applied for the study of DNA repair proteins, as represented in figure 14B, where the methyl transfer reaction of EcoRI is visualized in a frame-like DNA origami. In this particular case, Sugiyama and collaborators studied by AFM the regulation of DNA methylation using different tensions of double strands. They introduced two different double helical tensions (tense and relaxed) into an origami frame, to control the methyl transfer reaction of EcoRI and examine the structural effect of this methylation.[91] Endo et al. further evolved this idea to create a versatile nanochip for direct analysis of DNA base- excision repair. In this case, the studied enzymes were 8-oxoguanine glycosylase (hOgg1) and t4 pyrimidine dimer glycosylase (PDG).[92] The exact positioning and displacement of the enzymes in the reaction can be monitored and analyzed. Another relevant application of the DNA origami is its potential use as drug delivery systems with high biocompatibility: for example, a logic-gated DNA nano-pill was designed for the selective delivery of molecular payloads to the cell and is represented in figure 14C.[93] These nanopills are capable of inducing a great variety of responses in cell behavior. Another cutting-edge design of DNA origami related with cellular biology was 15 the design of DNA-based channel that can punch pores into the lipidic cell membrane which are able to discriminate single DNA molecules (figure 14D).[94] This could be of great interest for the delivery of oligonucleotide-based drugs for gene therapy, which do not enter the cell easily due to their chemical features. Figure 14. A. Detection of single molecule polymorphisms via atomic force microscopy over an origami surface. Adapted from ref. [90]. B. Methyl transfer reaction of EcoRI and analysis of the structural effect of this methylation using a same frame-like design of DNA origami. Adapted from ref. [91] C. DNA-origami based channel that can punch pores into the lipidic cell membrane, with ability to discriminate single DNA molecules. Adapted from ref. [93]. D. Logic-gated DNA nano-pill for the selective delivery of molecular payloads to the cell. Adapted from ref. [94]. Further development of DNA nanotechnology holds great potential for biological applications, hopefully for biomedical approaches with highly programmable and controllable abilities in the very near future. 16 DNA Nanoparticles In recent years, a great variety of chemical methods has been developed to synthesize functionalized nanoparticles for biomedical applications such as drug delivery, cancer therapy, diagnostics, tissue engineering and molecular biology, and the structure- function relationship of these functionalized nanoparticles has been extensively examined.[95] Nanoparticles are particles between 1 and 100 nanometres in size. In nanotechnology, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are classified according to their composition and diameter. Their properties allow the functionalization with diverse biomolecules, including DNA, by different chemical means. In particular, the controlled assembly and disassembly of gold nanoparticles (AuNPs) has been a subject of great interest over the past decade due to the potential applications of these particles in nanobiotechnology.[96] Their unique physical properties,[97] particularly their localized surface plasmon resonance (LSPR)[98] and their efficient interaction with molecules with a free thiol group, make AuNPs attractive building blocks for nanoscale electronic and photonic devices.[99] Due to the strong interaction between gold and thiols (-SH), a single monolayer of DNA around the gold particle can be obtained. The negative charge repulsion of the phosphate backbone of DNA orients the DNA strands out into solution with a unique footprint that is dependent on gold nanoparticle size and packing density. Since the first DNA sensor was designed by Mirkin and co-workers,[100] the development of AuNP-based colorimetric biosensors has been increasingly applied for the detection of a large variety of targets, including nucleic acids, proteins, saccharides, small molecules, metal ions, and even cells. This technique takes advantage of the colour change that arises from the interparticle plasmon coupling during AuNP aggregation (red-to-purple or blue) or redispersion of an AuNP aggregate (purple-to-red) (figure 15).[96a, 96b, 96g] It is quickly becoming an important alternative to conventional detection techniques, as fluorescence-based assays, and holds great potential in clinical diagnostics, drug discovery, and environmental contaminant analysis, among others. 17 Figure 15. Schematic representation of the colorimetric change suffered by gold colloid upon aggregation and its UV spectra displacement. Adapted from ref [103].[101] In contrast, super paramagnetic iron oxide nanoparticles possess different interesting features for nanomedicine. SPION are one of the most promising agents in diagnostics, due to their advantages as MRI contrast agents.[102] Under an applied magnetic field, SPION shorten the spin-spin relaxation time (T2) of the proton, which results in darkening of MR images. A schematic representation of this process is shown in figure 16.[103] Figure 16. Super paramagnetic iron oxide nanoparticles as negative contrast agents. Under an applied magnetic field, SPION shorten the spin-spin relaxation time (T2) of the proton, which results in darkening of MR images. Adapted from ref. [103] In comparison with the traditional gadolinium-based contrast agents, SPION produce lower toxicity, stronger enhancement of proton relaxation and have a lower detection limit.[104] Furthermore, SPION have several other applications in biomedicine, especially for delivery purposes and biosensing, due to their reduced size and ability to be transported in biological systems.[105] Their functionalization with DNA is not straightforward, as iron oxide interacts poorly with biomolecules. For this reason, organic compounds are used for their coating, focusing in two major objectives: preserve the magnetic properties of magnetic iron oxides and enhance their biocompatibility.[106] 18 Gold and iron-based magnetic nanoparticles seem to be one of the most promising nanoparticles for biomedical applications for their unique properties. The combination of them through a gold coating over the magnetic core provides the benefits from both nanoparticles, adding the magnetic properties to the robust chemistry provided by the thiol functionalization of the gold coating (figure 17).[107] As a result, an increasing number of laboratories is working on the synthesis and applications of this type of gold-coated nanoparticles.[105c, 105d, 108] Figure 17. Steps in the synthesis and functionalization of gold-coated magnetic nanoparticles. Adapted from ref [107]. As mentioned before, DNA functionalized nanoparticles are suitable for several applications, as drug delivery or the development of sensors for the detection of biomolecules. Nanoparticles provide the benefits from controlled drug delivery and cell- specific targeting, compared to the traditional forms of drug administration. A drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower.[109] However, for nanoformulations used in drug delivery the focus in most researches is mainly on the reduction of toxicity of the incorporated drug, whereas the possible toxicity of the carrier used is not considered. Their toxicity due to bioaccumulation still needs to be studied and minimized, as the kind of hazards they may cause are beyond that produced by the chemicals or biomolecules with which they are functionalized. The detection potency of nanoparticles is multidisciplinary, as the combination of their features permits the use of different techniques. SPIONs? and AuSPIONs? magnetic properties allow the detection of biomolecules by means of Magnetic Resonance Imagining, because they are contrast agents for image enhancement and the change in contrast (T2) caused by biological reactions resulting in nanoparticles aggregation can be easily detected.[105e] AuSPIONs and AuNPs have a surface plasmon maximum at 520 nm that shifts to higher wavelengths when precipitation occurs, allowing the use of UV- 19 spectroscopy as a detection method. And finally, aggregation of the three types of nanoparticles can be detected by Dynamic Light Scattering, measuring the differences in diameter upon several changes caused by biological reactions. Due to all their features, nanoparticles can provide promising and multidisciplinar detection systems for biomedical applications. 20 References: [1] J. D. Watson, F. H. Crick, Nature 1953, 171, 737-738. [2] a) F. Sanger, G. M. Air, B. G. Barrell, N. L. Brown, A. R. Coulson, C. A. Fiddes, C. A. Hutchison, P. 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Irudayaraj, Anal Chem 2006, 78, 3234-3241; d) R. Hao, R. Xing, Z. Xu, Y. Hou, S. Gao, S. Sun, Adv Mater 2010, 22, 2729-2742; e) M. V. Yigit, D. Mazumdar, Y. Lu, Bioconjug Chem 2008, 19, 412-417. [106] W. Wu, Q. He, C. Jiang, Nanoscale Res Lett 2008, 3, 397-415. [107] L. Y. Zhou, J.; Wei, J., J. Mater. Chem. 2011, 21, 2823-2840. [108] a) I. Robinson, D. Tung le, S. Maenosono, C. Walti, N. T. Thanh, Nanoscale 2010, 2, 2624-2630; b) L. Wang, J. Luo, Q. Fan, M. Suzuki, I. S. Suzuki, M. H. Engelhard, Y. Lin, N. Kim, J. Q. Wang, C. J. Zhong, J Phys Chem B 2005, 109, 21593-21601. [109] W. H. De Jong, P. J. Borm, Int J Nanomedicine 2008, 3, 133-149. 27 28 Objectives 29 30 OBJECTIVES: The long term objective of this thesis is the search for inhibitors of hAGT repair activity to be used as adjuvants in chemotherapy treatment. In order to achieve this goal, in this thesis we have evaluated potential inhibitors of hAGT and we have developed several analytical methods to find a robust technique for the in vitro evaluation of hAGT activity that will help us to characterize hAGT inhibitors. The specific objectives of this thesis are: A. Primary screening of the compounds with potential capacity to inhibit hAGT. 1. Evaluation of the complex formed by hAGT and its potential inhibitors by ESI-MS. 2. Evaluation of the toxicity and efficacy of the candidate compounds in cell culture. B. Development of methods for the detection of hAGT repair activity in vitro: 1. Methods based on the conformational change of a G-quadruplex: 1.1. Fluorescence assay based on the conformational change of a G-quadruplex oligonucleotide. 1.2. Design of a supramolecular platform that allows the detection of hAGT activity through the direct monitoring of the conformational change of a pair of G-quadruplexes using Atomic Force Microscopy. 2. New method to detect hAGT activity by transfer of fluorescence from the substrate oligonucleotide to the active site of the protein. C. Comparative study of the properties of different type of nanoparticles to detect a single methylation that implies a conformational change in a G-quadruplex, to set the bases for a new detection platform for hAGT activity over a nanoparticle system. 31 32 Chapter 1 Potential inhibitors of hAGT?s DNA repair activity: study of the hAGT-compound complex formation by ESI-MS and toxicity in cell culture. 33 In this first chapter, we evaluate the potency of small molecules which can be inhibitors of the DNA repair activity of the protein hAGT. A virtual screening was realized in collaboration with the Bioinformatics Unit of the CMBSO . A library of 4 million compounds was screened for shape complementarity to the active site of the pro tein, assuming that the best candidates could be good hits for hAGT inhibition due to their ability to enter the active site and occupy the space where the alkylated guanine of the DNA needs to enter in order to be repaired , as well as for their interactio n with the amino acids implied in the repair. A set of 15 compounds with potential inhibitory activity against hAGT w as selec ted. From these 15 compounds, only 10 were commercial ly available. These 10 compounds were studied by ESI - MS to pre - establish their ability to form a complex with hAGT in vitro , and subsequently, their toxicity was assess ed in cell culture by MTT and colony formation assays. In this chapter, we include as an annex a work we realized related to the search of inhibitors for another DNA repair protein, Ape1, involved in the base excision repair pathway. This paper reports the identification of new compounds as potential Ape1 inhibitors selected by a docking-based virtual screening and characterized following similar techniques as the ones described in chapter 1 for hAGT. 34 Potential inhibitors of hAGT?s DNA repair activity: study of the hAGT- compound complex formation by ESI-MS and its toxicity in cell culture. Table of contents: 1. Introduction: inhibition of hAGT 2. hAGT: structure, overexpression and purification 3. Mass Spectrometry study of the complex formation between hAGT and its potential inhibitors 4. Toxicity in cell culture 4.1. MTT assays 4.2. Colony formation assays 5. Conclusions 6. Experimental details 35 1. Introduction: inhibition of hAGT. The human O6-alkylguanine-DNA-alkyltransferase (hAGT)[1] is a DNA repair protein that removes alkyl mutations from the O6 position of guanines, restoring the DNA which has been damaged by alkylating agents. This group of chemotherapeutic anticancer drugs, alkylating agents, produce their cytotoxic effects by introducing alkyl groups which generate adducts and crosslinks at multiple sites in DNA.[2] For this reason, hAGT represents a relevant pharmacological target in the fight against chemotherapy resistance and patients? survival.[3] Chemotherapy based on alkylating agents, together with the inhibition of hAGT, may result in a higher efficiency of the treatment, due to the blocking in the repair of the alkylated-DNA by hAGT that leads to apoptosis of cancer cells.[3] hAGT is a suicidal enzyme which repairs alkyl damage from the O6 position of guanines, and in minor rate, from the O4 position of thymidines. It has 207 amino acids, a molecular weight of 21645.9 Da and a theoretical isoelectric point of 8.28. Its crystallographic structure was solved by Daniels and coworkers in 2000.[1b] hAGT is a monomer and has 2 domains, the N- and the C-terminal, where the active site cysteine is found (Figure 1). Figure 1. hAGT structure, described by Daniels et. al. The two domains are clearly seen. The violet sphere corresponds to a zinc atom present in the N-terminal domain. PBD code 1EH8. Later on, the same group solved the structure of hAGT together with DNA, enlightening the mechanism of action and the amino acids involved in the repair reaction.[1c] Arginine 128 plays an important role in the dealkylation process, entering the DNA double strand and pulling out the damaged guanine. In this way, the damaged guanine can accommodate in the active site close to cysteine 145 to be repaired (figure 2). 36 Figure 2. hAGT (green) bound to DNA (red) , with Arginine 128 (yellow, left ) entering the double helix and pushing outside the alkylated guanine (blue) , which interacts with the active site, Cysteine 145 (yellow, right). PDB code 1T39. The first obje ctive of this thesis is to evaluate the potency of small molecules whic h can be inhibitors of the repair activity of hAGT. Previously, a virtual screening was realized in collaboration with the Bioinformatics Unit of the CMB SO. A library of 4 million compounds was screened for shape complementarity to the active site of the protein, assuming that the best candidates could be good hits for hAGT inhibition due to their ability to enter the active site and occupy the space where the alkylated guanine of the DNA need s to enter in order to be repaired , as well as for their interaction with the amino acids implied in the repair. A set of 15 compounds with the best free energy of binding with hAGT w as selected . We did an exhaustive s earch for companies that che mically synthesize and sell these compounds and finally, from these 15 compounds, only 10 were commercially available. Table 1 shows the chemical structure and properties of thes e 10 compounds, which were numbered from 1 to 10. 37 Compound Chemical structure xlogP HBD HBA Charge Molecular Weight 1 1.78 3 13 0 453.492 2 3.68 3 11 0 471.572 3 2.93 3 13 0 467.519 4 1.91 4 14 0 483.518 5 2.93 3 12 - 1 463. 459 38 Compound Chemical structure xlogP HBD * HBA ** Charge Molecular Weight 6 2.05 3 13 0 467.519 7 1.15 3 14 - 1 478. 430 8 1.79 3 15 - 1 516.454 9 1.96 3 12 - 1 383. 348 10 1.90 1 2 0 148.205 Table 1. Chemical structure and properties of the compounds tested in this the sis as potential hit s of hAGT activity, found by a virtual screening. *HBD: hydrogen bond donors. **HBA: hydrogen bond acceptors. In order to confirm the results obtained by the virtual screening about the possible interaction of this set of compounds with hAGT, we undertook a first evaluation of their ability to form a complex with the protein by mass sp ectrometry, and their toxicity and effectiveness as potential inhibitors was test ed in cell culture assays. 39 2. hAGT: structure, overexpression and purification Re combinant hAGT full length (FL) (Scheme 1A) was used for the MS study of the complex formation with the potential inhibitors, but here we also describe in full detail the overexpression and purification of a mutant version of hAGT which will be used in the following chapters of this thesis. This inactive mutant of hAGT (hAGT C145S) had a shorter sequence, wh ere 30 amino acids were removed from its C-terminal end without affecting its folding, as represented in scheme 1B. I ts active site cysteine was replaced by a s erine, ca using the loss of its repair activity but maintaining its capacity to recognize and bind alkylguanine-DNA. [4] This hAGT mutant was designed in order to use it as a negative control for the different in vitro assays. Scheme 1. Amino acid se quence of the hAGT proteins . 1A. hAGT FL with the active site , cysteine 145, in red . 1B. hAGT ???? C145S, the shorter version of hAGT FL with a mutation at the active site (serine 145) in red. The two types of hAGT were overexpressed and purified following the protocol described by Ruiz et al . [5] This protocol implied the introduction of a hystidine tag with affinity for nickel at the N terminal end of the sequence of the protein, to enable its purification by a nickel HiTrap chelating column figure 3A. After that, the resulting sample was loaded into a Size exclusion column to further purify the protein and to ex change the buffer used in the chelating column to a more convenient buffer for storing the protein (figure 3B). hAGT FL was obtained in high yield and purity, as shown by polyacrylamide gel electrophoresis (PAGE) which was performed to follow all the purification process (figure 3C). 40 Figure 3. Purification of hAGT full length with Hys-tag. 3A. Chromatogram of the nickel HiTrap-column purification. 3B. Chromatogram of the size exclusion column, where a single pure peak can be observed. 3C. PAGE of the purification process. In the case of the hAGT C145S, a dyalisis at 4?C with prescission protease (PrePro) was carried out after the nickel HiTrap column. PrePro is a protease which cuts a specific sequence introduced between the hystidine tag and the N-terminal of hAGT, removing the Hys tag. Finally, a Size exclusion column was used to further purify the protein and to slowly exchange the buffer to the storing buffer. Figure 4. Purification of hAGT C145S with Hys-tag. 4A. Nickel HiTrap purification chromatogram. 4B. Size exclusion purification chromatogram. In this case, two peaks are observed: the first and smaller one corresponds to a portion of hAGT which is aggregated and the second one corresponds to the well folded hAGT C145S. 4C. PAGE of the purification process. The top gel shows the sample preparation prior to the nickel column, the nickel column and the GST column. The bottom gel corresponds to the analysis of the fractions collected from the size exclusion column, showing the pure hAGT C145S. Both proteins were concentrated in storing buffer and stored in 40% glycerol at -20?C until further use. All buffers and conditions are detailed in the experimental section of this chapter. 41 3. Mass Spectrometry analysis of the non-covalent complex between hAGT and the lead compounds. In order to study the ability of the preselected compounds to form a complex with hAGT and pre-establish that they are molecules with potential capacity to inhibit hAGT, we performed a Mass Spectrometry analysis of their interaction. Mass sp ectrometry exp eriments were done using electrospray ionisation, a travelling wave ion mobility cell and a time- of -flight mass analyzer. This technique allows the detection of an increase in the mass of hAGT when the compound shows a strong interaction with the protein, which may indicate that the active site of hAGT is occupied with the compound . It does not permit to calculate a constant of dissociation nor an inhibitory activity, but is useful to predetermine the disposition of these molecules to be better or worse candidates for hAGT inhibition, based on their chemical structures. The modification of the mass sp ectra indicates a complexation between hAGT and the studied compounds, but not the nature of this interaction nor the degree of interaction. The presence of several new peaks with a delay corresponding to the addition of the m/z of the compound may indicate that the complex is not specific and more than one molecule can enter the active site of hAGT or interact with its surface instead of entering the active site. The hAGT buffer was exchanged to 100 mM ammonium acetate pH =7 using BioSpin columns P-6, to prevent buffer interferences in the mass sp ectrometry exp eriments. All the compounds are only soluble in DMSO at high concentrations. For this reason, we first realized the mass analysis of hAGT in the presence or absence of DMSO, in order to see how DMSO affects the Mass Spectrometry analysis (figure 5). We observed that a volume of DMSO higher than 1.5% v/ v caused the loss of hAGT spectrum and thus determined the maximum concentration of inhibitor which can be used in th ese exp eriments, being it 150 ?M. This concentration is optimal for our exp eriments, because it is within a ratio of 1:5 of protein/inhibitor, given that the ideal concentration of hAGT to perform mass exp eriments was found to be around 30 ?M. For inhibitors which require higher concentrations to be active, this technique cannot be used if they are not soluble in aqueous buffer. 42 Figure 5. Mass spectra of hAGT 30 ?M in absence (green) or presence of DMSO 1.5% v/v (violet). The mass analysis of hAGT also allowed us also to select the m/z area were the complex formation was observed with clarity, which is from 1800 to 3000. Then, hAGT was mixed with each inhibitor in DMSO on a 1:5 ratio. Figure 6 shows all the results of the mass analysis. Five main peaks of m/z can be observed at 1749.69, 1893.29, 2065.33, 2271.60 and 2530.79. Changes in the chromatogram of hAGT, as the appearance or displacement of the peaks a number of units corresponding to the m/z of the inhibitors, represent an interaction. Our results show that in the case of compounds 1, 2 and 3, no changes in the chromatogram were observed compared with native hAGT, meaning that these compounds are not able to form a complex with hAGT in these conditions. Compound 4 does not seem to interact with the active site of hAGT, neither, however in this case a variation in the peaks? form is seen in comparison with native hAGT. This replacement of the peaks by a softened curve indicates denaturation of the protein, and we can presume that compound 4 is interacting in some way with hAGT in vitro, but it is impossible to determine by these means if the interaction is due to a binding with the active site or with some other amino acids producing its denaturation or unfolding. The chromatogram of hAGT in the presence of compound 10 was not obtained, probably due to a high rate of denaturation of hAGT, which indicates some kind of interaction between compound 10 and hAGT. Smaller concentrations of inhibitor 10 were tested without achieving a stable signal. In contrast, the rest of the compounds (5, 6, 7, 8 and 9) were able to form a complex with hAGT, some of them in a specific way and some others in a non-specific. Compound 9 forms a clearly non-specific complex with hAGT, as various shifts for each peak of the hAGT chromatogram appear. In the case of compounds 5, 6 and 7, it is very difficult to see if the formed complex is specific or non-specific, as two shifts appear for the fourth peak but only one for the rest of the peaks. This probably means that we would be able to see a 43 non-specific complex formation upon increase of the inhibitor concentration, which is currently impossible to perform due to the constraints in DMSO concentration tolerated . Finally, compound 8 forms a clearly sp ecific complex with hAGT at 150 ?M concentration. Figure 6 . Mass spectra of hAGT and its interaction with the different compounds. hAGT was mixe d with each inhibitor in DMSO on a 1:5 ration, being hAGT concentration 30 ?M. 44 4. Cell culture assays : effect of cytotoxicity and enhancement of BCNU on human cancer cells. All cell culture assays were performed in colon cancer cells ( HT - 29), des cribed as a cellular line which overexpress hAGT. Carmustine (or BCNU) is a chemotherapeutic drug which alkylates and cross-links DNA during all phases of the cell cycle, resulting in disruption of DNA function, cell cycle arrest, and apoptosis. [6] This agent also carbamoylates proteins, including DNA repair enzymes , resulting in an enhanced cytotoxic effect. [7] Carmustine is highly lipophilic and crosses the blood -brain barrier readily. [8 ] Inhibition of hAGT in cell culture has be en shown to increase sensitivity of human cells to BCNU, and to enhance its efficiency. [9] Th ese exp eriments were conducted to establish an IC 50 value (inhibitory concentration 50%) for all the compounds, which allows to compare its efficiency in enhancing BCNU. According to the FDA, IC 50 represents the concentration of a drug that is required for 50% inhibition in vitro. [10] This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is need ed to inhibit a given biological process . In the same line, we have calculated the LD 50 (lethal dose 50%) for all the compounds, which is the required dose to produce a 50% of cell death. [11] This value was estimated to establish the toxicity of the compounds by thems elv es . 4.1. MTT as s ays . The MTT cytotoxicity assays were performed to evaluate the toxicity of the compounds per se or in combination with carmustine. MTT ( 3 - ( 4 ,5 - Dimethylthiazol- 2- yl) - 2,5 - diphenylt etrazolium bromide) is a bright yellow compound which is reduced t o purple formazan in living cells ( figure 7 ) . This reduction and change of color allows the detection by UV absorption of the remaining living cells after a certain treatment and is widely used for the toxicity evaluation of new drugs. [12] Figure 7. Reduction of MTT (yellow) to formazan ( purple blue ) by the mitochondr ial reductase in li ving cells. 45 We first tested the toxicity of carmustine per se , observing that BCNU produced around 30% of cell death at 160 ?M in 2 hours . This c oncentration was found to be optimal for our MTT assays, as smaller concentrations of carmustine did not kill the cells and higher concentrations of carmustine did not show a significant increase in cell death . Figure 8 shows the c ell survival at different concentrations o f BCNU and the optimal concentration of BCNU required for this assay. Figure 8. Increasing concentrations of BCNU treatment on cells see de d at 6000 cells/ well (2 hours of exposure) . Cells were incubated with the 10 compounds de scribed before, during and after BCNU treatment, to ensure that inhibitors were present during the entire period of time need ed for DNA damage to be formed and repair ed . As negative controls we evaluated the effect of DMSO and the solvent used as vehicle for BCNU (EtOH:PBS 1:1), which were found to be negligible. Scheme 2 explains the protocol followed for these exp eriments. Cells incubated with compound alone were used to evaluate its toxicity per se. Scheme 2. Schematic representation of the MTT assay for the study of the toxicity of each compound per se ( compound + vehicle) or in combination with BCNU (compound + BCNU). Light and dark colours refer to the colour coding of figures 8 and 9. 46 From the 10 potential inhibitors, compounds 5 and 8 were non-toxic by thems elves , as the survival of the cells was not effected in the range of 0 to 100 ?M concentrations . The rest of them were toxic, but at different ranges of c oncentrations (f igure 9 and table 2 ) . Compound s 1, 3 and 10 were found to be slightly toxic from 0,1 - 1 ?M concentrations, producing over 20, 30 and 10% of cell death resp ectively and their LD 50 was found to be > 100 ?M . However, the y maintained the lev el of toxicity constant upon concentration. Compound 2 was slightly toxic at 0.1 - 1 ?M, causing less than 20% of the cell death, but at more than 50 ?M it was found to be very toxic, pr oducing a 60% of cell death per se . Its LD50 was estimated to be around 5 ?M. Instead, compound 4 was progressively toxic while increasing its concentrations, reaching a lev el of 80% of cell death (LD 50 ~ 2.5 ?M) , and compounds 6 and 7 were toxic from 1 ?M but their LD 50 differed , as compound 6 requires higher concentrations than 100 ?M and in contrast the LD 50 found for compound 7 was around 10 ?M. C ompound 9 was toxic from 0.1 ?M (40% of cell death) but its toxicity was not significantly affected by incre asing concentrations . These results suggest that compounds 2, 4 and 7 are clearly toxic by thems elves and compounds 1, 3, 6, 9 and 10 were slightly toxic, too. Only compounds 5 and 8 were found to be non - toxic per se . Figure 9. MTT toxicity assays of the diff erent compounds (1- 10). Light colours represent the toxicity of the compounds by the m selve s, while dark colours indicate the toxicity of the dif ferent compounds in the presence of 160 ?M of Carmustine. The dashe d lines indicate the 50% of the cell survival. The p values were calculated 47 ?sin? the t??ent?s t test comparing cells treated with inhibitor in the absence and presence of BCNU. * p ? ???, ** p ? 0.05 and *** p ? ?????. Regarding their enhancement of BCNU toxicity, compounds 5 and 8, which were non-toxic per se , were found to enhance significantly the effect of carmustine , with IC 50s of around 10 and 50, resp ectively. Compounds 2, 4, 6 and 7 enhanced the effect of carmustine with IC 50s around 1 ?M , even if they were toxic per se . The behaviour of compound 2 at low micromolar concentrations is not toxic while the others presented toxicity by thems elves . Compound 1 exhibited a relative capacity to stimulate cell death caused by carmustine, without reaching an IC 50 at this range of concentrations. Compound 9 showed an increased effect in carmustine toxicity, but this effect is due to its increasing toxicity upon concentrations. In contrast, compounds 3 and 10 were unable to change the effect of carmustine significantly, remaining stable upon increasing concentrations. IC50 values were estimated from these results and are shown in table 2. t??ent?s t test was performed for all the compounds. The p values demonstrate that the enhancement of carmustine toxicity produced by the compounds 5 and 8 were significant. Compound LD50 (toxicity per se ) IC50 ( enhancement of BCNU) 1 >100 ?M >100 ?M 2 5 ?M 1.5 ?M 3 >100 ?M >100 ?M 4 2.5 ?M 1 ?M 5 NT ~ 10 ?M 6 >100 ?M <1 ?M 7 ~10 ?M <1 ?M 8 NT ~ 50 ?M 9 >100 ?M >1 ?M 10 >100 ?M >1 00 ?M Table 2. Estimated LD50 and IC50 of the 10 compounds studied by MTT assay. NT = non-toxic. From these results, we can conclude that compounds 5 and 8 are good candidates to enhance the effect of BCNU in cell culture, as both were non-toxic at 100 ?M and they were able to improve the rate of cell death significantly when incubated with carmustine . 48 4.2. Colony formation ass ays . The colony formation assay was used in order to study in more detail the capability of compounds 5, 6, 7 and 8 to enhance BCNU cytotoxicity in colorectal cells. Th es e compounds were selected due to the ir previous good performance in the MS analysis and MTT toxicity assays . The mass sp ectrometry analysis points to compounds 5, 6, 7, 8 and 9 as capable of forming a non-covalent complex with hAGT. From this five, compounds 5, 6, 7 and 8 were found to enhance ?s to?icity in the MTT assays , and 5 and 8 were non- toxic for the cells by thems elves . Compounds 6 and 7 were found to be very toxic for the cells , and will allow us to compare the results obtained. Cells were incubated with the 4 compounds mentioned above, before, during and after BCNU treatment, to ensure that inhibitors were present during the entire period of time need ed for DNA damage to be formed and repaired by hAGT. Th en, cells were replated and left to grow for a week with periodic replacem ent of media. We analysed the colony formation efficiency of HT - 29 colorectal cells exposed to inhibitor alone, and in combination with BCNU . In this case, we added 240 ?M of carmustine, enough to produce a 50% of cell death (see figure 8) . Figure 10 shows the remaining cells stained with crystal violet after treatment with the compounds in absence or presence of carmustine . Figure 10. Pictures of a colony formation assay, with the remaining cells after treatment and a we ek of growth stained with crystal violet. 49 Figure 11. Colony formation assay of compounds 5, 6, 7 and 8. Light colours represent the cell viability of the cells incubated with the compounds alone, while dark colours indicate the survible of the cells in presence of the diff erent compounds and 240 ?M of carmustine. The dashed lines indicate the 50% of the cell survival. The p values were calculated us in? the t??ent?s t test comparing cells treated with inhibitor in the absence and presence of BCNU. * p ? ???? ?? p ? ???? an? ??? p ? ?????? As shown in Figure 11, all compounds except compound 6 enhanced BCNU cytotoxicity. However, compound 5 reduced cell viability by itself in this case , showing a LD50 of around 0.5 ?M and enhancing carmustine . The other 3 compounds were found to be non-toxic by these means. Compound 6 seemed not toxic but had little effect in enhancing carmustine. Even though, this results cannot be considered final as the effect of carmustine in this assay was very limited. In contrast, compound 7 is not toxic per se but enhances carmustine, showing an IC 50 around 0.1 ?M even if higher concentrations do not seem to potentiate this effect . Compound 8 is slightly more toxic than compound 7 per se but strongly enhances BCNU toxicity. IC 50 values were estimated from these graphical results and are shown in table 3. Compound LD50 (toxicity per se ) IC50 ( enhancement of BCNU) 5 >0.5 ?M < 0.05 ?M 6 NT > 100 ?M 7 NT ~0.1 ?M 8 NT ~7.5 ?M Table 3. Estimated LD50 and IC50 of the 4 compounds studied by colony formation assays. NT = non-toxic. 50 5. Conclusions This set of 10 compounds were selected as candidates for the inhibition of hAGT in a virtual screening. We have tested their complex formation with hAGT by mass sp ectrometry, and their toxicity to tumoral cells, as well as their capacity to enhance the effect of an alkylating agent, carmustine. Table 1 summarizes the results obtained for the three exp eriments: ESI- MS MTT assays : Colony formation assays : Toxicity per se (LD 50 ) Enhancement of BCNU (IC 50 ) Toxicity per se (LD 50 ) Enhancement of BCNU (IC 50 ) Cpd 1 Non observed Slightly t oxic ( > 100 ?M) Low (>100 ?M) - - Cpd 2 Non observed Toxic (5 ? M) Low (1.5 ?M) - - Cpd 3 Non observed Slightly toxic (>100 ?M) Low (>100 ?M) - - Cpd 4 Denaturation of hAGT Toxic (2.5 ?M) Significant (1 ?M) - - Cpd 5 Complex (Non specific) Non Toxic (>>100 ?M) Significant (~10 ?M) Toxic (0.5 ?M) Significant (<0.05 ?M) Cpd 6 Complex (Non specific) Slightly toxic (>100 ?M) Significant (<1 ?M) Non toxic (>>100 ?M) Low (>100 ?M) Cpd 7 Complex (Non specific) Toxic (10 ?M) Significant (<1 ?M) Non toxic (> > 100 ?M) Significant ( ~ 0. 05 ?M) Cpd 8 Complex (Specific) Non toxic (>>100 ?M) Significant (>50 ?M) Non toxic (> > 100 ?M) Significant ( ~ 7 .5 ?M) Cpd 9 Complex (Non specific) Slightly toxic (>100 ?M) None (>100 ?M) - - Cpd 10 Precipitation of the sample Slightly toxic (>100 ?M) None (>100 ?M) - - Table 1. Summary and comparison of the results obtained for the mass spe ctrometry and the cell culture studies. Combining the results obtained by ESI-MS and by cell culture assays, we can conclude that 2 compounds out of our first ten are able to form a specific (8) or non- specific (5) complex with hAGT and are non-toxic per se in MTT toxicity assays . In addition, they seem to enhance the effect of carmustine, both in MTT and in colony formation assays. However, compound 5 showed toxicity per se in the colony formation 51 assays. The differences in toxicity found by MTT and colony formation assays may be due to the fact that the latter studies the cell viability during 10 days, while MTT is applied 24h after the treatment with compounds and BCNU. Even though, the colony formation assays are preliminary and should be reproduced before considering these results conclusive. Compound 8 represents the best candidate for the study of hAGT inhibition for chemotherapy enhancement, as it formed a specific complex with hAGT, was not found to be toxic per se neither by MTT nor by colony formation assays and showed a clear enhancement of carmustine at high concentrations . Following the line of this thesis, thes e potential inhibitors should be tested by the methods des cribed subsequently to reveal their active conce ntrations and their suitability to continue preclinical studies in animal models. 52 6. Experimental details: 1. hAGT overexpression and purification. hAGT- FL protein cloned in the pet -21a(+) (Novagen) vector was expressed in the E. coli strain Rosetta. Once the culture reached an OD 600 value of 0.98, hAGT was induced by adding 1 mM IPTG (Sigma) and left to grow for 4 h at 30 ?C. The pellet from a 1- L culture was disrupted by sonication and centrifuged . The supernatant was filtered, loaded into a HiTrap TM FF column (GE Healthcare) with buffer 350 mM NaCl, 20 mM Tris pH 8, 20 mM imidazole, and 1 mM BME, and then eluted with an imidazole (Fluka) gradient up to 500 mM in the same buffer. Finally, the protein was loaded into a Superdex 75 16/60 column (GE Healthcare) with the following buffer: 200 mM NaCl (Merck), 20 mM Tris pH 8.0 (Merck), 10 mM DTT (Sigma) and 0.1 mM EDTA (Sigma). The protein was concentrated to 2 mg/ ml in this buffer and kept at -20 ?C in the presence of 40 % glycerol. A similar protocol was used for the purification of the inactive mutant hAGT-C145S, cloned in the pet-28a (+) vector (Novagen) and expressed in the E. coli strain BL21. However, in this case the hystidine tag was cut using PrePro during a 4h dyalisis at 4?C. 2. Compounds. 10 mg of the c om pounds 1 to 9 were purchase from IBScreen. ompo?n? ?? ???- aminoquinoline) was obtained from TCI Europe. Their reference numbers are shown in this list. Compound 1: STOCK5S - 10931 Compound 2: STOCK4S - 24457 Compound 3: STOCK4S - 9879 9 Compound 4: STOCK4S - 90 334 Compound 5: STOCK4S - 14286 Compound 6: STOCK4S - 97010 Compound 7: STOCK4S - 94755 Compound 8: STOCK4S - 96314 Compound 9: STOCK4S - 9986 8 Compound 10: A0417 All of the m were dissolved in DMSO (Sigma) at a final stock concentration of 1 mM and kept at -20?C until used . 53 3. Mas s spectra of the complex with hAGT . Mass sp ectrometry exp eriments were performed on a Waters (Manchester, UK) instrument equipped with electrospray ionisation, a travelling wave ion mobility cell and a time- of -flight mass analyser. Synapt G1 was interfaced to a chip -based nanoESI device, (Advion, Triversa Nanomate). This device contains a 384 -w ell-sample plate, 38 4 disposable spraying tips and an ESI chip with 100 nozzles in front of the inlet of the mass sp ectrometer. The instrument was operated in the automatic mode using a contact closure signal. A spraying voltage of +1.76 kV and a sample pressure of 5.80 psi were applied . Each well was loaded with 10?l of sample, from which a total of 4 ?l was infused during 10 min. Operating conditions for the Synap G1 mass sp ectrometer were: cone voltage = 20V; extraction cone = 5.5 V; source temp erature = 20 ?C; trap and transfer voltages = 6 V and 4V, resp ectively. The ion mobility cell was filled with N 2 and an electric field was applied to the cell in the form of waves (wave height = 9.5 V) that passed thought the cell at 300 m/s. The bias voltage for ion introduction into the IMS cell was 15V. The hAGT buffer was exchanged to 100 mM ammonium acetate pH =7 using BioSpin columns P-6, to prevent buffer interferences in the mass sp ectrometry exp eriments. hAGT in the presence of DMSO was used as Mass sp ectrum control. Different concentrations of DMSO were studied to find the optimal conditions. Once the mass sp ectrum for the native form of hAGT was confirmed , the non-covalent inhibitor-protein complexes were analyzed. hAGT (30 ?M) was mixed with (150 ?M of) each inhibitor in ammonium acetate buffer at a final volume of DMSO of 1.5% v/ v (1, 2, 3, 4, 5, 6, 7, 8, , 9 and 10) separately and left to equilibrate at rt for one hour. Each study was performed in triplicates . 4. C ell cu lture as s ays . HT- 29 colon cancer cells were purchased from ATCC (catalog No. HTB- 38 ) and cultured in RPMI 1640 medium (Gibco) supplem ented with 10% fetal bovine serum (Gibco) at 37?C and 5% CO 2 . BCNU (1,3-bis- (2 -chloroethyl) - 1-nitrosourea) was obtained from Sigma and dissolved in 50% phosphate-buffered saline buffer (PBS)-50% ethanol at 4 mM (10 mg/ ml) stock solution. 4.1. MTT toxicity as s ays . The optimal concentration of BCNU was explored using MTT assays. 6000 cells per well (in 100 ?L RPMI medium) were seed ed in a 96 -w ell plate and left to grow for 24 hours. Then, cells were incubated during 2 h with concentrations of carmustine ranging 54 from 40 to 240 ?M and after that, cells were rinsed with PBS and media was replaced . The day after, 20 ?L of MTT (2mg/ ml in PBS) were adde d and left to react for 4 hours. At the end of the reaction blue crystals inside the living cells could be observed at the microscope. The m edium + MTT was removed and th e crysta?s res?spen?e? in ??? ? o? DMSO and agitated until reaching homogeneity. Absorbance was read at 560 nm using a ELx 800 Universal Microplate Reader (Bio-Tek Instruments INC). The cytotoxic effects of compounds 1 to 10 in HT - 29 human colorectal tumoral cells were analyzed by m eans of MTT toxicity assay. 6000 cells per well (in 100 ?L RPMI medium) were seed ed in a 96 -w ell plate and left to grow for 24 hours. The day after, the cells were incubated for 6 hours with different concentrations of compounds 1 to 10 (0, 0.01, 0.1, 1, 10, 50 and 100 ?M). Then, enough quantity of BCNU to reach a concentration of 160 ?M (or the equivalent volume of the ve hicle as negative control) was added and left to react during 2 hours. Afterwards, the medium was replaced by compound-containing RPMI (using the same concentrations as before the treatment) and left overnight. The next morning, the medium was replaced by fresh RPMI and cells were left to grow for 24 hours. Then, 20 ?L of MTT (2mg/ ml in PBS) were added and left to react for 4 hours. The m edium ? TT ?as absorbe? an? the crysta?s res?spen?e? in ??? ? o?  an? s haked until reaching homogeneity. Absorbance was read at 570 nm using a ELx 800 Universal Microplate Reader (Bio-Tek Instruments INC) and toxicity of the compounds per se or in combination with BCNU was determined relative to untreated cells. All these exp eriments were performed in triplicates and repeated three times. IC50 values were estimated from the graphical results. The p values were calculated usin ? the t??ent?s t test (2 tails, equal variance) comparing cells treated with inhibitor in the absence and presence of BCNU . 4.2. Colony formation ass ays . The effe ct of compounds 6 to 9 on the sensitivity of human colorectal ( HT - 29 ) cells to BCNU was determined using colony formation assays as it has been previously des cribed . [5] HT - 29 cells were seed ed at 10 x 10 3 cells per well density in 24-well, flat- bottomed plates and incubated in a humidified , 5% CO 2 incubator at 37?C for 48 h. Compound solutions were diluted in the culture medium RPMI at final concentrations of 100, 50, 10, 5, 1, 0.5, 0.1, and 0.05 ?M, and were immediately used to treat the cells. Cells ?ere inc?bate? ?ith these compo?n?s? so??tions ?or ? h an? then  ?or the e??i?a?ent volume of the vehicle as negative controls) was add ed to a final concentration of 160 ?M. After 2 hours of incubation, the medium was replaced with fresh medium containing the same compound concentration, and cells were left to grow for an additional 16 hours. The 55 cells were then replated at densities of 2000 cells p er well in 24 -well plates and grown for 1 week until discrete colonies were formed . Colonies were washed twice with PBS and stained with a 0.5% crystal violet - 20% ethanol solution. Cells were rinsed with deionized water and air dried. Stained colonies were counted in a ELx 800 Universal Mi croplate Reader (Bio-Tek Instruments INC) and clonogenic survival was determined relative to untreated cells. Samples were assayed in triplicates and exp eriments repeated three times. IC50 values were estimated from the graphical results. The p values were calculated using the t??ent?s t test (2 tails and equal variance), comparing cells treated with inhibitor in the absence and presence of BCNU . 56 References: [1] a) D. S. Daniels, J. A. Tainer, Mutat Res 2000, 460 , 151-163; b) D. S. Daniels, C. D. Mol, A. S. Arvai, S. Kanugula, A. E. Peg g , J. A. Tainer, E MBO J 2000, 19, 1719-1730; c) D. S. Daniels, T. T. Woo, K. X. Luu, D. M. Noll, N. D. Clarke, A. E. Peg g , J. A. Tainer, Nat Struct Mol Biol 2004 , 11 , 714- 720. [2] M. R. Middleton, G. P. Margison, Lancet Oncol 2003, 4 , 37 -44 . [3] T. P. Brent, P. J. Houghton, J. A. Houghton, Proc Natl Acad Sci U S A 1985, 82 , 2985- 2989 . [4] J. J. Rasimas, A. E. Peg g , M. G. Fried, J Biol Chem 2003 , 278 , 79 73- 7980. [5] F. M. Ruiz, R. Gil-Redondo, A. Morreale, A. R. Ortiz, C. Fabrega, J. Bravo, J Chem Inf Model 2008 , 48 , 844-854. [6] http:/ /pubchem.ncbi.nlm.nih.gov/compound/2578#x291. [7] E. L. Schaefer, R. I. Morimoto, N. G. Theodorakis, J . Seidenfeld, C arcinogenesis 1988, 9 , 1733-1738. [8] A. B. Fleming, W. M. Saltzman, C lin Pharmacokinet 2002, 41 , 403- 419. [9] L. Yan, J. R. Donze, L. Liu, Oncogene 2005, 24 , 2175-2183. [10] http:/ /w ww .fda.gov/ohrms/ dockets/ac/00/slides/3621s1d/sld036.htm [11] http:/ /w ww .d erangedp hysiology.com/p hp / Pharmacodynamics/ Pharmacolog y --- Pharmacodynamics --- Definitions- of -median-doses ---ED50-LD50-and-TD50.php . [12] T. Mosmann, J Immunol Methods 1983 , 65 , 55-63 . 57 58 Appendix 1 Receptor-based virtual screening and biological characterization of new inhibitors of Human Apurinic/Apyrimidinic Endonuclease Enzyme (Ape1) 59 60 Receptor-based virtual screening and biological characterization of new inhibitors of Human Apurinic/Apyrimidinic Endonuclease Enzyme (Ape1) Federico M. Ruiz, 1 Sandrea M. Francis, 2 Maria Tintor?, 3 Rub?n Ferreira, 3 Rub?n Gil - Redondo, 4 , 5 Antonio Morreale, 4 ?ngel R. Ortiz, 4 Ramon Eritja 3 and Carme F? brega 3 * ChemMedChem, (2012) Volume 7, Issue 12, pages 2168 ?2178 . DOI: 10.1002/cmd c.201200372 . Impact factor: 3.046 1 Chemical and Physical Biology, CIB (CSIC), 2 Institute of Biomedicine of Valencia, (IBV - CSIC) Valencia 3 Institute for Research in Biomedicine of Barcelona, IQAC-CSIC, CIBER-BBN Networking, Centre on Bioengineering Biomaterials and Nanomedicine. Cluster Building, Baldiri i Reixach 10, E-08028 Barcelona 4 Bioinformatics Unit, CBMSO (CSIC-UAM), Universidad Aut?noma de Madrid, Cantoblanco, 28049 Madrid. 5 SmartLigs Bioinform?tica S.L., Fundaci?n Parque Cient?fico de Madrid, c/ Faraday, 7. Campus de Cantoblanco UAM, E-28049. Madrid, Spain. 61 62 DOI: 10.1002/cmdc.201200372 Receptor-Based Virtual Screening and Biological Characterization of Human Apurinic/Apyrimidinic Endonuclease (Ape1) Inhibitors Federico M. Ruiz,[a] Sandrea M. Francis,[b] Maria Tintor,[c] Rubn Ferreira,[c] Rubn Gil- Redondo,[d, e] Antonio Morreale,*[d] ngel R. Ortiz,[d] Ramon Eritja,[c] and Carmen Fbrega*[c] Introduction Chemo- and radiotherapy are the two main currently available treatments to improve the outcome in cancer patients. The cy- totoxicity of many of these agents is directly related to their propensity to induce genomic DNA damage.[1] The persistence of unrepaired DNA damage results in accumulation of muta- tions, cell cycle arrest, and apoptosis.[2] However, the ability of cancer cells to recognize this damage and initiate DNA repair is an important mechanism that impacts negatively upon ther- apeutic efficacy.[3] The base excision repair (BER) pathway is the major mecha- nism for dealing with a variety of lesions in DNA produced by alkylating agents. This pathway is initiated by specific DNA gly- cosylases, which recognize and excise the damaged base to generate an apurinic/apyrimidinic (AP) site. AP endonuclease 1 (Ape1) cleaves the phosphodiester backbone adjacent to the 5? site of the AP site, generating a 3?-hydroxy and 5?-deoxyri- bose phosphate termini.[4] Polymerase b removes the 5?-deoxy- ribose phosphate, fills in the single-nucleotide gap, and the consequent nick is ligated by DNA ligase I or by DNA ligase III/ XRCC1.[5] Ape1 is a fundamental protein in this essential repair pathway and it is thought to be responsible for 95% of total AP endonuclease activity in human cell lines.[6] Ape1 also pos- sesses 3?-phosphodiesterase, 3?-phosphatase, RNase H, and 3?!5? exonuclease functions.[7] In addition to its DNA repair ac- tivities, Ape1 acts as a redox factor for a variety of important transcription factors such as NF-kB, p53, c-Fos, and c-Jun.[8] The DNA repair and the redox activities of Ape1 are distinct, both structurally and functionally. Ape1 protein levels and intracellular distribution have been related to the pathogenesis of several human tumors[5a,9] and its expression pattern appears to have a prognostic signifi- cance in cancer cells, including breast,[9b] lung,[10] ovarian,[9e] gastro-esophageal[11] and pancreatic?biliary,[11] and bone[12] tumors. For this reason, several preclinical and clinical studies have suggested that Ape1 may be an attractive target for anti- cancer drug development. Using either antisense oligonucleo- tides or RNA interference approaches, different groups have re- ported that depletion of intracellular Ape1 sensitizes mammali- an cells to a variety of DNA-damaging agents.[5a,13] In pancreat- ic cancer cell lines for example, down-regulation of Ape1 po- tentiated the cytotoxicity of gemcitabine[14] and The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio- and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various che- motherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 in- hibitors ; however, those compounds are in the early stages of development. Herein we report the identification of new com- pounds as potential Ape1 inhibitors through a docking-based virtual screening technique. Some of the compounds identified have in vitro activities in the low-to-medium micromolar range. Interaction of these compounds with the Ape1 protein was ob- served by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl metha- nesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors. [a] Dr. F. M. Ruiz+ Chemical and Physical Biology, CIB (CSIC) (Spain) [b] S. M. Francis+ Institute of Biomedicine of Valencia, (IBV-CSIC) Valencia (Spain) [c] M. Tintor, R. Ferreira, Prof. R. Eritja, Dr. C. Fbrega Institute for Research in Biomedicine of Barcelona, IQAC-CSIC CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nano- medicine, Cluster Building, Baldiri i Reixach 10, 08028 Barcelona (Spain) E-mail : carme.fabrega@irbbarcelona.org [d] Dr. R. Gil-Redondo, Dr. A. Morreale, Prof. . R. Ortiz Bioinformatics Unit, CBMSO (CSIC-UAM) Universidad Autnoma de Madrid, Cantoblanco, 28049 Madrid (Spain) E-mail : amorreale@cbm.uam.es [e] Dr. R. Gil-Redondo SmartLigs Bioinformtica S.L. , Fundacin Parque Cientfico de Madrid c/Faraday, 7 Campus de Cantoblanco UAM, 28049 Madrid (Spain) [+] These authors contributed equally to this work. 2168  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2012, 7, 2168 ? 2178 MED 63 troxacitabine.[15] Ape1 down-regulation has also been shown to block ovarian cancer cell-growth.[16] In melanoma and colon cell lines, Ape1 down-regulation alone or in combination with bleomycin led to an increase in apoptosis,[2a,17] whereas its overexpression conferred protection from cisplatin- or H2O2-in- duced apoptosis.[17a] Attempts to create Ape1 knockout mice were embryonically lethal, suggesting that Ape1 is crucial for embryonic development.[18] Heterozygous Ape1 mice were viable but abnormally sensitive to oxidative stress and prone to cancer progression.[19] Recently, there has been a significant effort toward identify- ing inhibitors of DNA repair proteins in keeping with the emerging concept that sensitizing cancer tissue to core che- motherapeutic regimens by simultaneous targeting DNA repair may result in enhanced treatment outcomes.[20] As a result of the promising therapeutic potential of the inhibition of Ape1 DNA repair activity, several reports have described the identifi- cation and characterization of small molecules that inhibit its repair endonuclease activity,[21] including methoxyamine (MX), lucanthone and 7-nitroindole-2-carboxilic acid (NCA). All three compounds were able to enhance the effects of methyl metha- nesulfonate (MMS) or temozolamida (TMZ) in ovarian,[22] breast,[22b,23] colon,[24] and HT1080 fibrosarcoma cancer cells.[25] In addition, Lucanthone was able to potentiate the effects of radiation therapy in patients with brain metastasis.[26] However, the evidence of lucanthone topoisomerase inhibitory activity raised concerns regarding an off-target effect[27] and the syner- gistic cell killing effects observed by NCA with TMZ or MMS were unclear, as recent data showed that its specificity for Ape1 is controversial.[22b,28] By using a fluorescence-based high- throughput assay, Kelley and co-workers described the identifi- cation of 2,4,9-trimethylbenzo[b][1,8]napthyridin-5-amine (AR03), which was found to be active in the low micromolar range in vitro against purified Ape1 and inhibited AP site inci- sion activity and its repair in SF67 glioblastoma cells.[29] Other research groups using high-throughput screening (HTS) approaches reported Ape1 inhibitors including com- pounds containing arylstibonic[30] or bis-carboxylic acid,[31] Re- active blue 2, 6-hydroxy-d,l-DOPA, and myricetin.[32] The bis- carboxylic acid derivatives were not tested in cell-based assays,[31] and no information about its in vivo activity can be inferred. The arylstibonic acid compounds effectively inhibited Ape1 in vitro, but were ineffective in vivo due to poor cellular uptake.[30] Moreover, antimony-containing compounds are gen- erally considered from a probe development standpoint due to their possible promiscuity akin to the effect of heavy metal ions and their occasional high toxicity.[33] Reactive blue 2, 6-hy- droxy-d,l-DOPA, and myricetin were found to have numerous other targets besides Ape1 in cells ; therefore, they lack the se- lectivity and specificity required for promising Ape1 inhibi- tors.[32] We recently developed a flexible, fully automated virtual screening computational platform (VSDMIP)[34] to identify in- hibitors of protein targets from libraries of millions of com- pounds. This computational platform has been successfully ap- plied to the discovery of new inhibitors of the DNA repair pro- tein O6-alkylguanine DNA alkyltransferase[35] and in the devel- opment of small molecules that compete with ubiquitin E2 variant for its interactions with ubiquitin-conjugating enzyme UBC13, inhibiting its enzymatic activity,[36] among others. Herein we report the identification of new Ape1 inhibitors by using a docking-based virtual screening technique. The ac- tivity of these compounds has been experimentally proved both in vitro and in vivo. These molecules are promising lead candidates to establish quantitative structure?activity relation- ship models for further development of clinically relevant Ape1 inhibitors as co-adjuvants in cancer chemotherapy. Results and Discussion Virtual screening (VS) The virtual screening protocol employed here is summarized in Scheme 1 and briefly described in the Experimental Section. An essential part of the procedure is to characterize the shape of the active site. For this purpose we used GAGA algorithm (see Experimental Section) to obtain a sort of a negative image of the binding site (Figure 1). Overall, the shape of the active site does not undergo significant conformational changes even after binding to the DNA, as very low RMSD values were ob- tained after superimposing the different Ape1 structures de- posited in the PDB (IDs: 1BIX,[37] 1DE9,[38] 1DEW,[38] 1DE8,[38] 1E9N,[39] 2ISI, and 1HD7[39]). This fact is reassuring taking into account that the protein flexibility is not explicitly considered during the docking process. The active site is a well-defined deep V-shaped cleft, with an Mg2+ cation located in its ?elbow?. Upon characterizing the binding site, the free database of commercially available compounds ZINC[40] was first screened Scheme 1. Flowchart of the virtual screening procedure applied in this study. ChemMedChem 2012, 7, 2168 ? 2178  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemmedchem.org 2169 MEDHuman Ape1 Inhibitors 64 using DOCK[41] and the negative image of the binding site. We applied a ZScore (see Experimental Section) cutoff value of 4 on the DOCK-ranked list, and a set of 2288 molecules were taken through to the next step. These molecules were then re- docked with CDOCK[42] and scored with its molecular mechan- ics energy function, which specifically includes solvent and H- bonding terms. The 100 highest-scoring compounds were sub- mitted to MD simulation in explicit solvent and their binding energies estimated and pairwise decomposed by MM-GBSA[43] calculation over a large collection of snapshots homogenously sampled along the trajectories. Finally, from the top-scoring compounds, and upon visual examination, a total of 15 candidates were selected, pur- chased, and tested experimentally. For those showing in vitro activity against Ape1, the MM-GBSA[43] method was used to es- timate the binding free-energies from molecular dynamics (MD) trajectories. The physicochemical properties of the mole- cules as stored in the ZINC database[40] are listed in Table 1. Ape1 endonuclease in vitro assays Fifteen top-ranked compounds selected from the VS computa- tions were first dissolved in DMSO. The ability of those 15 can- didates to inhibit the recombinant Ape1 activity in vitro was determined by a fluorescence-based assay described by differ- ent groups.[25,30] In brief, a double-stranded DNA (dsDNA) sub- strate was used containing, in one of the strands, an internal ri- bitol (THF) AP site mimic,[44] and a 5?-6-carboxyfluorescein (FAM) label, whereas the complementary strand was labeled with a 3?-Dabsyl fluorescence-quencher moiety. The close prox- imity of the fluorophore and the quencher results in a damp- ened signal upon light excitation. Following DNA-backbone cleavage by Ape1, a short single-stranded DNA fragment 5?- tagged with the FAM group is released from the duplex DNA substrate, resulting in an increase in the fluorescence. Because assay variability is to be expected, as a comparative control we included a compound that was previously described as Ape1 inhibitor (data not shown).[30] Ape1 (at 590 pm concentration) was incubated with the dsDNA at 37 8C for 30 min in the pres- ence of each of the 15 compounds selected during the VS at concentrations of 100 and 200 mm. The results of this first assay showed that compounds 11 to 15 did not exhibit any in- hibitory action against Ape1 at these concentrations (Table 1). Compounds 7?10 showed an inhibitory effect versus Ape1 at concentration between 150 and 200 mm and therefore they were not evaluated further. On the other hand, compounds 1?6 showed inhibitor activi- ty at concentrations below 100 mm and were further analyzed to determine the concentration dependence for the inhibition of Ape1 endonuclease activity. All of them inhibited Ape1 with IC50 values in the low-to-medium micromolar range. Com- pounds 1 and 2 were identified as the most potent inhibitors of Ape1 with an IC50 of 1.8 and 6.8 mm, respectively. These two lead compounds had compara- tive or slightly higher effects in blocking Ape1 activity than thi- olactomycin, methyl 3, 4-de- phostatin and better than other compounds found by Simeonov and colleagues.[32] Compounds 3 and 4 had an intermediate effect (IC50 : 17?39.5 mm), where- as compounds 5 and 6 were weakly active (IC50>50 mm). The resultant IC50 values obtained for all active compounds are listed in Table 1 and in Figure 2. The chemical structures of these compounds are shown in Figure 3, prioritized by low mi- cromolar IC50 values. Figure 1. Inside view of the Ape1 active site with the ligand negative spheres image computed with GAGA. Table 1. The 15 top-ranked compounds obtained in the virtual screen.[a] ZINC ID logP HBD[b] HBA[c] Charge Mr [Da] IC50 [mm][d,f] EC50 [mm][e,f] 1 ZINC08790444 1.57 3 10 0 497.89 1.80.5 >100 2 ZINC09086704 1.82 0 9 0 497.54 6.80.5 7.3 3 ZINC00708759 6.37 0 9 1 543.58 17.30.6 >50 4 ZINC00870176 6.39 0 9 1 543.58 39.20.8 >100 5 ZINC00730105 4.79 1 8 1 487.56 52.80.9 28.5 6 ZINC02118845 1.97 2 8 1 473.51 55.70.8 >100 7 ZINC04059809 5.19 0 8 1 427.48 >130 ND 8 ZINC08854784 2.61 0 12 0 477.50 >150 ND 9 ZINC08877288 2.12 3 9 1 493.58 >150 ND 10 ZINC04060698 4.63 0 8 1 413.45 >150 ND 11 ZINC03583501 3.69 0 9 0 523.95 >200 ND 12 ZINC08932744 3.48 2 6 0 440.52 >200 ND 13 ZINC09042551 1.91 3 9 1 423.45 >200 ND 14 ZINC04202254 3.97 0 8 1 456.53 >200 ND 15 ZINC00706278 2.61 0 8 1 421.41 >200 ND [a] The computed chemical properties (as found in the ZINC database) and the in vitro and in vivo activities of the active compounds are listed. [b] H-bond donors. [c] H-bond acceptors. [d] in vitro IC50 value: compound concentration required to decrease Ape1 activity by 50%. [e] in vivo EC50 value: compound concentration re- quired to kill 50% of cells in the presence of 300 mm MMS; ND: not determined. [f] Values represent the aver- age standard error of the mean (SEM) of three separate experiments. 2170 www.chemmedchem.org  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2012, 7, 2168 ? 2178 MED A. Morreale, C. Fbrega, et al. 65 DNA intercalation binding To test if these compounds inhibit Ape1 activity through non- specific DNA binding rather than by direct inhibition of the en- zymatic activity, we employed a fluorescent-dye displacement assay.[32,45] Briefly, if a compound acts nonspecifically by associ- ation with DNA, it would displace a DNA-bound fluorophore such as thiazole orange (ThO) or ethidium bromide. As these molecules emitted fluorescence upon intercalation to DNA, its displacement should produce a decrease in the fluorescence of the complex dye-DNA when increasing amounts of the compounds were added. We selected ThO, which binds nonco- valently to DNA with high affinity instead of ethidium bromide, due to the fact that its fluorescence excitation and emission are red-shifted, which ensures a decreased susceptibility to compound autofluorescence.[32] As shown in Figure 4, com- pounds 1?6 did not displace bound ThO at any range of con- centrations starting from 100 pm to 100 mm. This result suggests that these molecules do not inhibit the Ape1 DNA repair activity by a noncompeti- tive effect such as DNA binding. In contrast, mitoxantrone, de- scribed in Simeonov et al.[32] as a DNA binding compound, was able to displace ThO producing a 100% decrease in fluores- cence in the same concentra- tion range. Complex formation detected by MS Electrospray ionization mass spectrometry (ESIMS) has become an established tool for the investigation of macromo- lecular complexes.[46] ESI is a gentle ionization method that enables the analysis of proteins without causing internal frag- mentation of the molecule. Their typical multiply charged ions result in low mass-to- charge values that allow an ac- curate mass determination.[47] The noncovalent complex for- mation of Ape1 with com- pounds 1?6 was analyzed by this means. The mass spectrum of Ape1 alone exhibited a char- acteristic series of multiply charged positive ions (Fig- ure 5a). A narrow range of charge state is a good indica- tion that Ape1 has retained its folded or native conformation. Moreover, the ion mobility measurements further confirmed that the protein was folded during the nano-ESIMS experi- ments. The direct observation of the protein?ligand complex ion can be easily achieved for tight-binding complexes. However, weakly bound complexes, particularly those dependent on hy- drophobic effects, are fragile and may dissociate during desol- vation,[48] as observed with compounds 2?4 (data not shown). The mass spectra of Ape1 in complex with compound 5 (Fig- ure 5a) showed that although the peaks are too complex to be observed in detail, the free-protein peaks shifted to a higher mass-to-charge value and this fact may indicate an in- hibitor?protein interaction. In the mass spectrum of Ape1 in presence of compounds 1 and 6, a clear signal of complex for- mation was observed. The upper part of Figure 5a shows the ESIMS peaks corresponding to free protein (P) and protein in Figure 2. Concentration curves showing the inactivation of human Ape1 by compounds 1?6 [panels a)?f), respec- tively] . Remaining Ape1 activity as a function of compound concentration is plotted relative to untreated control samples. Compounds 1?3 show inhibitory effects on Ape1 activity in the low micromolar range, whereas com- pounds 4?6 show inhibitory effects in the mid-micromolar range. Data are the averageSEM of three separate experiments performed in triplicate. ChemMedChem 2012, 7, 2168 ? 2178  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemmedchem.org 2171 MEDHuman Ape1 Inhibitors 66 complex with inhibitor (PI). The complex peaks are shown in greater detail in Figure 5b. The estimated stoichiometry of the complex between Ape1 and the three studied compounds (1, 5 and 6) respectively was equimolar (1:1). Docking binding modes For those compounds showing in vitro activity against Ape1, the binding mode was refined by MD simulations. The binding mode of compound 2 with Ape1 highlights the interaction with the metallic cation, which energetically is the most impor- tant. The H-bonding pattern is represented in Figure 6. These interactions were established between: 1) the nitro group of compound 2 and the backbone NH group of residue Asn174; and 2) an oxygen atom from the sulfonyl moiety and the amino group from the side chain of residue Asn212 on the one hand, and the NHe group of residue His309, on the other hand. This binding mode also presented a network of hydro- phobic contacts surrounding the quinoxaline core of com- pound 2 with Phe266, Trp280, and Leu282, and an additional stacking interaction with Tyr269, which stabilizes the benzylpyri- dinium moiety. The other five active com- pounds all have a carboxylic moiety in common, which also presented their main interaction with Ape1 through the Mg2+ cation. However, compared with compound 2, this interaction is somehow skewed. This fact allows a gradual contribution to the stability of the complex from a salt bridge that is formed between the carboxylic moiety and the side chain of residue Lys98, which in turns depends on the position of the Mg2+ cation. Nevertheless, rather similar interaction energies were obtained for the contacts of the ligands with the hydrophobic residues Phe266 and Trp280 and polar Asn174 and Asn212. Fi- nally, an important interaction was established between the central core of the ligands and the side chain of residue Lys98, which is mainly hydrophobic except for compound 1 in which a hydrogen bond is formed with one of the oxygen atoms in the succinimide-like central moiety. Effects of cytotoxicity and enhancement of MMS on human cells The presented data suggested that the six compounds can po- tently and selectively inhibit Ape1 in vitro. However, to have a general utility as BER inhibitors, it is important to confirm that they can block Ape1 function in living cells. A colony- forming assay was used to study the capability of compounds 1?6 to enhance MMS cytotoxicity in HOS cells.[12,22b,23, 25,49] MMS creates base damage by methylation, which is either lost spon- taneously or excised by the alkylpurine DNA glycosylase, re- sulting in a high number of cytotoxic abasic sites.[50] Inhibition of Ape1 in cell culture or the expression of a dominant nega- tive form of the protein has been shown to increase sensitivity of human cells to MMS.[12,22b,23,25,49] Cells were incubated with the six compounds before, during, and after MMS treatment to ensure that inhibitors were present during the entire period of time required for DNA damage to occur. We analyzed the colony formation effi- ciency of HOS cells exposed to MMS alone, each inhibitor alone, or the combination of MMS plus inhibitor, relative to an unexposed control. MMS alone decreased the number of colo- Figure 3. Structures of the six compounds that show Ape1 inhibition in the micromolar range. Figure 4. ThO inhibitor displacement assays were used to evaluate the DNA binding ability of all compounds (1?6) that were active in the Ape1 in vitro assay. No decrease in ThO fluorescence was observed upon addition of the compounds, indicating that they do not bind DNA. Mitoxantrone, a clear DNA binding molecule, was used as positive control (&). The fluorescence signal was normalized against DNA. 2172 www.chemmedchem.org  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2012, 7, 2168 ? 2178 MED A. Morreale, C. Fbrega, et al. 67 nies by 15%, and, as shown in Figure 7, all six compounds en- hance MMS cytotoxicity. However, no significant effect of the compounds alone was observed, indicating that none of them is toxic by itself in the concentrations used. Compounds 1 and 3 are less effective in sensitizing the tumor cell to MMS than the rest, requiring a concentration above 100 mm to obtain a 50% increase in cell death. The lower-sensitizing effect of compound 3 is consistent with its slightly smaller in vitro affini- ty for Ape1. However, the inefficiency of compound 1 was un- expected on the basis of its high ability to inhibit Ape1 activity in vitro. This observation suggested that it may not be able to cross cell membranes or it could be degraded before reaching its final target. Compound 4 had almost no effect on the cyto- toxicity produced by MMS; only a small decrease in cell surviv- al was observed at 50 mm, which was not incremented when cells were incubated with higher concentrations of 4. Com- pound 5 exhibited an increasing capability to enhance the kill- ing effect of MMS with an EC50 value ~50 mm, which is close to the IC50 value obtained in the in vitro experiments. Compound 6 showed a progressive lethal effect (until 50 mm), even if at Figure 5. Complex formation identified by mass spectrometry: Ape1 (28 mm) was mixed with each inhibitor (140 mm) in ammonium acetate buffer (100 mm) and allowed to equilibrate at 4 8C for 1 h as described in the Exper- imental Section. a) ESIMS spectra of Ape1 alone (bottom) or with test com- pounds as indicated; b) zoom-in of the complex regions for Ape1 with com- pounds 6 and 1. Figure 6. Average minimized structure of the Ape1?compound 2 complex after MD simulations. The protein is represented by the ribbon and loop structure in cyan; side chains of main interacting residues are colored by atom type: C cyan, N blue, and O red. For compound 2, C atoms are in orange, N in blue, O in red, and S in yellow. Hydrogen atoms have been omitted for clarity. The grey sphere represents the Mg2+ cation. Dashed lines correspond to H-bond interactions. higher concentrations it was unable to further increase the tox- icity of MMS in the cells. The combination of MMS and compound 2 presented the best result in vivo. Its lethality was found to be in the lower micromolar range (7.25 mm), being consistent with its affinity for Ape1 in vitro. The results observed for this lead compound opens the door for its further optimization as Ape1 inhibitor. Conclusions Several classes of inhibitors of Ape1 have been described pre- viously. However, the specificity of some of these compounds for Ape1 remain unclear[27] or slightly controversial.[22b,28] Here, we have applied a docking-based virtual screening technique ChemMedChem 2012, 7, 2168 ? 2178  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemmedchem.org 2173 MEDHuman Ape1 Inhibitors 68 to select a concrete number of molecules (15 compounds) as hit compounds from a 4 million library, to become new and more potent Ape1 inhibitors. Six of these molecules selected from the ZINC database were found to be active. In particular, two of them are single-digit micromolar inhibitors against puri- fied Ape1 (similar or even more potent than prior reported in- hibitors), with the capacity to potentiate the cytotoxicity of a relevant DNA-damaging agent like MMS. The predicted bind- ing modes highlighted the interaction with the metallic cation, which is energetically the most important, the hydrogen bond- ing with residues Asn174 and Asn212, and a network of hydro- phobic contacts of the ligands with the Phe266 and Trp280. In the case of compound 2, the most important interactions with Ape1 are mainly established through the quinoxaline core. In summary, new compounds have been identified as potent leads of Ape1 DNA repair inhibition. The binding prop- erties, cellular uptake, and solubility of these compounds can be further optimized to produce a subfamily of candidates with improved pharmacological properties. This new genera- tion of compounds could lead to innovative drugs that may act as co-adjuvants in cancer chemotherapy. Experimental Section Materials : The Ape1 gene (Homo sapiens) inserted into the pOTB7 vector was obtained from the Genomics Unit (Clone ID: IMAGE 2823545) at the Centro Nacional de Investigaciones Oncologicas (CNIO). The pet28a plasmid and the competent E. coli strains (DH5a, BL21(DE3), Rosetta) were purchased from Novagen. The en- Figure 7. Effect of compounds 1?6 [panels a)?f), respectively] on HOS cell survival relative to untreated cells. Dark-grey bars show samples in the presence of various compound concentrations as indicated with no MMS added; light-grey bars show the same experiment in the presence of MMS (300 mm). MMS alone decreased the cell number to an average of 15%. Data represent the averageSEM of three separate experiments performed in duplicate. Significance was determined by Student?s t test, comparing cells treated with inhibitor in the absence and presence of MMS: *p0.1, **p0.05, ***p0.01, ****p0.001. 2174 www.chemmedchem.org  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2012, 7, 2168 ? 2178 MED A. Morreale, C. Fbrega, et al. 69 zymes and their corresponding buffers for cloning were purchased from Fermentas or New England Biolabs. The chemical reagents used for the expression and purification steps were purchased from Sigma, Merck, Bio-Rad or Fluka, and the SDS-PAGE standards, gels and buffers, from Invitrogen. HiTrap FF and size-exclusion (HiLoad Superdex 75 16/60) columns were purchased from GE Healthcare. The oligonucleotides 5?-FAM-GAGAA[X]ATAGTCGCG-3? and 3?-Q- CTCTTGTATCAGCGC-5? (in which FAM is fluorescein, Q is Dabsyl, and X is ribitol,[51] an abasic (AP) site analogue) were custom-made by Sigma. Candidate compounds were purchased from various sources; in particular, compounds 1, 5, and 6 were obtained from IBScreen, compound 2 was obtained from Enamine, and com- pounds 3 and 4 were obtained from Specs. Stock solutions were prepared by dissolving them in DMSO at a final concentration of 1 mm and kept at 20 8C until further use. The fluorescence assay was carried out on a spectrofluorimeter multi-detection microplate reader BioTek FLx800 and a Jasco FP6200, in optical 96-well reac- tion plates. Human osteosarcoma cells, (ATCC Number HOS) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Invitrogen). Methyl methanesulfonate (MMS) was obtained from Sigma. Virtual screening (VS): All VS calculations were performed within the automated platform VSDMIP[34] (virtual screening data manage- ment on an integrated platform), see Figure 1. For clarity, we brief- ly describe below the main steps of the protocol (Scheme 1). Receptor preparation : The crystal structure of PDB ID 1HD7[39] was selected as a receptor because no substantial differences were found in the active site amongst the available Ape1 structures and it presented the highest resolution structure (1.95 ). All atoms other than the receptor were deleted except for the divalent metal (Pb2+) in the active site, which, to resemble in vivo conditions, was replaced by magnesium, the preferred metal cofactor of the human Ape1 enzyme.[52] The AMBER8 ff99[53] force field was then used to assign atom types and charges for each atom in the recep- tor. Hydrogen atoms were added assuming standard protonation states of titratable groups, except for the key interacting residues, in which the hydrogen atoms were assigned based on the informa- tion given from the H+ + web server.[54] For this purpose, the receptor was submitted to the server and a Generalized Born (GB) model was used for the pKa calculation at pH 6.5 with 0.15m salt concentration and internal and external die- lectric constants of 4 and 80, respectively. The histidine residue His309, present in the binding site, was found to be protonated, which is consistent with an NMR study done by Lowry et al.[55] Binding site definition and characterization : To delimit the bind- ing site PDB codes 1HD7[39] (Ape1 apo form) and 1DEW[38] (Ape1 bound to DNA, subunit A) were superimposed, (RMSD among Ca atoms 0.28 ) selecting a DNA fragment as the core around which to build the docking box by adding a 5.0  cushion to its maximum dimensions. This DNA fragment consists of an abasic sugar connected to a cytosine nucleotide residue to its 5? end and including the following 5? phosphate. An equally spaced grid of 0.375  was then built, and CGRID[42] was used to calculate recep- tor interaction fields, a 12?6 Lennard?Jones term. The electrostatic term was modeled with a sigmoidal dielectric screening function using typical atom probes (C, H, N, O, S, P, F, Cl, Br, and I) at each grid point. Next benzene, water, and methanol probes were docked with CDOCK[42] to generate intermolecular interaction energy maps aimed to capture the most favorable hydrophobic, hydrophilic, and H-bond interaction areas, respectively. These areas were further compressed into Gaussian functions using GAGA algo- rithm,[56] producing a sort of a negative image of the interaction site. The putative active ligands in the library must conform to this approximate shape. Chemical library preparation : Ligands for VS consisted of a library with more than 4 million (4039777) non-redundant molecules, ob- tained from the publicly available ZINC database[40] in SMILES format.[57] Multiple protonation states and tautomeric forms were considered as implemented by default in ZINC database. The mole- cules were then processed within VSDMIP[34] as follows: a) conver- sion from SMILE to 3D MOL2 using CORINA,[58] b) atomic charges calculations with MOPAC[59] (MNDO ESP method) on every single structure provided by CORINA, and c) atom types assignment ac- cording to the AMBER ff99[53] force field and conformational analy- sis with ALFA.[60] Filter 1: An initial filter was performed with the docking program DOCK[41] to quickly discard those molecules that do not geometri- cally fit within the binding site. The spheres needed by DOCK were generated previously with GAGA. We used DOCK contact as scor- ing function, normalizing the score values (scorei) by converted them into ZScore using mean (average score) and standard devia- tion (s) values (ZScorei= (scoreiaverage score)/s). Only molecules with a ZScore beyond a cutoff value of 4 were selected and 2500 passed onto the next step. Filter 2 : Selected molecules from filter 1 were studied with the more accurate docking algorithm CDOCK.[42] CDOCK exhaustively docks each molecule within the binding site of the receptor using the interaction energy grids previously calculated with CGRID. This was achieved by an exhaustive exploration of the location and ori- entation of each molecule by positioning its centers of mass on grid points, where discrete rotations of 278 arc on each axe are performed. Finally, the energy for each pose was evaluated by the molecular mechanism force-field scoring function implemented in CDOCK, which besides including a 12?6 Lennard?Jones term and an electrostatic term modeled with a sigmoidal dielectric screening function, also accounts for ligand and receptor desolvations as well as for H-bonding interactions.[61] Molecular dynamics simulations : The top ranked 100 molecules (according to the scoring function of CDOCK) were subjected to a more exhaustive binding free-energy estimation by a combination of MD trajectories and MM-GBSA[43] calculation on these trajecto- ries. The 100 complexes were hydrated by using boxes containing explicit water molecules, energy minimized, heated (20 ps), and equilibrated (100 ps). Then, when the equilibration was reached, MD trajectories were continued for 2 ns. Structures were homoge- nously sampled at each ps and stored for post-processing. All the simulations were performed at constant pressure and temperature (1 atm and 300 K) with an integration time step of 2 fs. SHAKE[62] was used to constrain all the bonds involving H atoms at their equilibrium distances. Periodic boundary conditions and the Parti- cle Mesh Ewald methods were applied to treat long-range electro- static effects.[63] AMBER ff99[53] and TIP3P[64] force-fields were used in all cases. Finally, the effective binding free energies were qualita- tively estimated with the MM-GBSA[43] approach, which calculates the free energy of binding as a sum of a molecular mechanics (MM) interaction term, a solvation contribution thorough a General- ized Born (GB) model and a Surface Area (SA) contribution to ac- count for the nonpolar part of desolvation. A 12?6 Lennard?Jones term was used to model the MM contribution. For GB, the solute dielectric constant was set to 4, whereas that of the solvent was set to 80, and the dielectric boundary was calculated using a sol- ChemMedChem 2012, 7, 2168 ? 2178  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemmedchem.org 2175 MEDHuman Ape1 Inhibitors 70 vent probe radius of 1.4 . The SA contribution was approximated as a linear relationship to the change in solvent accessible surface area (SASA): DGnp ? a? bDSASA in which a is 0.092 kcalmol1, b is 0.00542 kcalmol12, DGnp is the SA contribution, and the change in SASA refers to the complex SASA minus the sum of that of the receptor and the ligand alone. In addition, an interaction energy analysis between the ligands and the more relevant residues in the binding site were computed (with MM-GBSA) and are contained in Table 2. All the trajectories and analysis were performed using the AMBER 8 computer pro- gram and associated modules.[65] Selection of candidates : Among the 100 molecules with highest scoring values, a total of 15 were selected upon visual examina- tion. All visualizations were done within the molecular graphics program PyMOL.[66] Averaged structures along the MD trajectories were obtained and minimized in vacuum with the ff99 force field, without periodic boundary conditions and during 1000 steps (the first 500 with the steepest descent method and the rest with the conjugated gradient) solely to alleviate the possible clashes that may be originated by averaging the coordinates. These structures were used for graphical representation and comparison of the binding modes. Finally, those 15 selected compounds were pur- chased and tested experimentally. Inhibition of Ape1 activity Protein production and purification : The in vitro assays were carried out using recombinant Ape1. The cDNA of full-length Ape1 (IMAGE:2823545) was amplified by PCR using primers 5?-CCC GGG CAT ATG CCG AAG CGT GGG AAA AAG-3? and 5?-GCC CTC GAG TCA CAG TGC TAG GTA TAG GGT-3?, which incorporated the NdeI and XhoI restriction enzymes sites, respectively. The PCR product was cloned into the pet-28a(+) (Novagen) vector. The resulting construct, Ape1-pet28a, was verified by nucleic acid sequencing. The protein was expressed in the E. coli strain BL21 and once the culture reached an OD600 value of 0.6 it was induced by adding 1 mm IPTG during 3 h at 30 8C. The pellet from a 2 L culture was disrupted by sonication and centrifuged. The supernatant was fil- tered, loaded into a HiTrap FF column (GE Healthcare), and eluted with an imidazole gradient. Finally, the protein was loaded into a Superdex 75 16/60 column (GE Healthcare), with buffer NaCl (300 mm), Tris pH 8.0 (20 mm), DTT (1 mm), and glycerol (5% v/v). Ape1 activity assay : The in vitro AP site cleavage assay was carried out by using a modified version of the protocol that had been de- scribed previously.[25,30] Briefly, the Ape1 enzyme (590 pm) was incu- bated with or without compounds (at 100 and 200 mm) in a buffer containing Tris?HCl (50 mm, pH 8.0), MgCl2 (1 mm), NaCl (5 mm) and dithiothreitol (DTT; 2 mm) at room temperature for 30 min. The sequence 5?-FAM-GAGAA[X]ATAGTCGCG-3? and its complemen- tary oligonucleotide 3?-Q-CTCTTGTATCAGCGC-5? were annealed to form a double-stranded DNA, and the reaction was initiated by ad- dition of the annealed substrate at 50 nm, considering that this concentration was found to be below the KM value of Ape1 in the reaction buffer and conditions (data not shown). Fluorescence readings were taken continuously after 30 min incubation at 20 8C, at excitation and emission wavelengths of 485 and 535 nm, respec- tively. Hits from the initial screen were analyzed further for inhibi- tory potency using decreasing dilutions of inhibitor. Each com- pound concentration was assayed in triplicate and experiments re- peated three times. The percent of inhibition was calculated rela- tive to DMSO-control samples. Bovine serum albumin (BSA) was used as a negative control (data not shown). The IC50 values were determined using OriginPro8 data analysis and graphic software, using the four-parameter equation as follows: y ? A2 ? A1  A2 1? exp xx0dx   Each value is an average of three independent experiments with their corresponding standard errors. DNA intercalation assay : A modified version of the ethidium bro- mide-based DNA binding assay was carried out essentially as de- scribed previously.[32,45] In brief, a mixture of labeled double-strand- ed DNA (500 nm) and ThO (2.5 mm) in Ape1 reaction buffer was prepared to a total volume of 400 mL. Compounds (at concentra- tions in the range of 100 pm to 100 mm) were added, and the fluo- rescence signal (excitation 501 nm and emission 530 nm) was mea- sured after 10 min of incubation at room temperature in Tris?HCl (pH 7.5, 50 mm), NaCl (50 mm), MgCl2 (10 mm) and EDTA (1 mm). Each compound concentration was assayed in triplicates. The per- centage fluorescence was calculated relative to the total fluores- cence acquired with double-stranded DNA and ThO (2.5 mm). Mass analysis of the noncovalent complex between Ape1 and lead compounds : Mass spectrometry experiments were performed on Waters (Manchester, UK) instrument equipped with electrospray ionization, a travelling wave ion mobility cell and a time-of-flight mass analyzer. The Synapt G1 instrument was interfaced with a chip-based nano-ESI device, (Advion, Triversa Nanomate). This device contains a 96-well sample plate, 96 disposable spraying tips and an ESI chip with 100 nozzles in front of the inlet of the mass spectrometer. The instrument was operated in the automatic mode by using a contact closure signal. A spraying voltage of +1.76 kV and a sample pressure of 5.80 psi were applied. Each well was loaded with 10 mL of sample, from which a total of 4 mL was infused during 10 min. Operating conditions for the Synapt G1 mass spectrometer were as follows: cone voltage=20 V; extraction cone=5.5 V; source temperature=20 8C; trap and transfer voltag- es=6 V and 4 V, respectively. The ion mobility cell was filled with N2, and an electric field was applied to the cell in the form of Table 2. Interaction energies for compounds 1?6 as computed from MD simulations by the MM-GBSA approach. Residue Eint [kcalmol 1] 1 2 3 4 5 6 Lys98 1.75 0.3 5.97 1.93 4.85 1.94 Tyr128 0.23 0.13 0.16 0.87 1.99 0.53 Asn174 0.3 2.45 2.56 1.98 3.23 0.34 Arg177 3.91 0.3 2.81 7.47 4.25 5.42 Asn212 0.34 6.27 1.85 0.17 1.49 0.23 Phe266 1.94 3.04 3.55 1.51 2.94 1.09 Thr268 1.2 0.56 1.92 2.51 1.38 0.84 Tyr269 3.38 3.31 0.38 1.44 0.03 1.22 Trp280 2.1 1.31 2.2 1.43 3.65 0.04 Leu282 0.68 1.4 1.02 0.14 0.78 0.09 Asp308 0.04 6.07 2.33 0.54 0.08 3.46 His309 0.31 7.83 0.66 0.42 0.47 0.5 Mg2+ 59.51 37.66 41.01 56.41 60.33 70.59 2176 www.chemmedchem.org  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2012, 7, 2168 ? 2178 MED A. Morreale, C. Fbrega, et al. 71 waves (wave height=9.5 V) that passed through the cell at 300 ms1. The bias voltage for ion introduction into the IMS cell was 15 V. The Ape1 buffer was exchanged to 100 mm ammonium acetate pH 7 to prevent buffer interferences in the mass spectrometry ex- periments. Once we confirmed the mass spectrum for the native form of Ape1, we proceeded to analyze the noncovalent inhibitor? protein complexes. Ape1 (28 mm) was mixed with each inhibitor (1 to 6 ; 140 mm) separately and let to equilibrate at 4 8C for 1 h. Ape1 in the presence of DMSO or an inactive molecule (16) were used as Mass spectrum controls. Cell culture cytotoxic assay : The effect of compounds 1?6 on the sensitivity of human osteosarcoma cells (HOS) to methyl methane- sulfonate (MMS) was determined using colony formation assays as it has been previously described in Ape1 inhibition studies.[30] HOS cells were seeded at 10103 cells per well density in 24-well, flat- bottomed plates and incubated in a humidified, 5% CO2 incubator at 37 8C for 36 h. Compound solutions were diluted in the culture medium at final concentrations of 100, 50, 30, 20, 5, 2.5, and 1 mm, and were immediately used to treat the cells. Cells were incubated with the drug solutions for 6 h and then MMS (or the equivalent volume of the vehicle as negative controls) was added to a final concentration of 300 mm. After 2 h of incubation, the medium was replaced with fresh medium containing the same compound con- centration, and cells were left to grow for an additional 16 h. The cells were then re-plated at densities of 2000 cells per well in 24- well plates and grown for 1 week until discrete colonies were formed. Colonies were washed twice with PBS and stained with a solution of 0.5% crystal violet and 20% ethanol. Cells were rinsed with deionized water and air dried. Stained colonies were counted in a ELx 800 Universal Microplate Reader (BioTek Instru- ments Inc.) and clonogenic survival was determined relative to un- treated cells. Samples were assayed in duplicates and experiments repeated three times. Acknowledgements This work was supported by ?Fondo de Investigaciones Sanita- rias? (grant PI06/1250), by ?Ministerio Ciencia e Innovacin? (grant CTQ-2010-20541-C03-03), and by ?Comunidad de Madrid? (SBIO-0214-2006 [BIPEDD] and S2010-BMD-2457 [BIPEDD2]). C.F. is grateful to Generalitat de Catalunya and Instituto de Salud Carlos III for an SNS Miguel Servet contract. R.G.-R. appreciates the MICINN contract from ?Programa de Personal Tcnico y de Apoyo 2008?. F.M.R. was supported by a Junta de Ampliacin de Estudios-Doc contract. A.M. acknowledges financial support from Fundacin Severo Ochoa through the AMAROUTO program. We thank Dr. Marta Vilaseca for her advice in the MS experiments. Generous allocation of computer time at the Barcelona Super- computer Center is gratefully acknowledged. Finally, this work is dedicated to the memory of ngel Ramrez Ortiz, who has been a great mentor, colleague, and friend. Keywords: antitumor agents ? cancer ? DNA repair ? drug design ? inhibitors [1] a) M. Christmann, M. T. Tomicic, W. P. Roos, B. Kaina, Toxicology 2003, 193, 3; b) O. 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DeLano, DeLano Scientific, Palo Alto, CA (USA) 2002. Received: August 2, 2012 Revised: September 26, 2012 Published online on October 25, 2012 2178 www.chemmedchem.org  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemMedChem 2012, 7, 2168 ? 2178 MED A. Morreale, C. Fbrega, et al. 73 ???? ?????? ?? ?? ????? ?? ?? ???????? ?? ???????? ?? ????????? ?? ???????????? ?? ?????????? ?? ?? ?????? ?? ??????? ?? ???????? ?? ??? ?????????????? ??????? ????????? ??? ?????????? ???????????????? ?? ????? ????????????????????? ???????????? ?????? ?????????? ???????? ??????? ??? ????????? ???? ?????????? ?? ????????? ????? ????????? ???????????? ???????????? ?????????? ?? ??? ??? ?? ????????????? ??????? ??????? ???? ??? ????????? ???? ?? ????? ??? ???????? ???????? ?? ??? ????????????? ?????????? ????? ??? ???? ?????????? ??? ???????????? ?? ?????? ??????????? ?????? ?? ???????????? ?????? ???? ????? ????? ??? ???? ?? ??? ??????????? ?? ? ??? ?????????? ?? ???? ??????????? ???? ??????????????????? ??????????? ????? ??? ? ? ?? ?? ????? ??? ??? ??? ????? ???? ???????? 74 Chapter 2 Development of a Novel Fluorescence Assay Based on the Use of the Thrombin Binding Aptamer for the Detection of O6-alkylguanine?DNA Alkyltransferase Activity 75 76 Development of a Novel Fluorescence Assay Based on the Use of the Thrombin Binding Aptamer for the Detection of O6-alkylguanine?DNA Alkyltransferase Activity Maria Tintor?, 1 Anna Avi??, 1 Federico M. Ruiz, 2 Ram?n Eritja 1 and Carme F?brega 1* Journal of Nucleic Acids (2010), Article ID 632041, 9 pages . DOI: 10.4061/2010/632041. Estimated impact factor: 1.52 1 Institute for Research in Biomedicine (IRB Barcelona) , IQAC - CSIC, CIBER - BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine, Cluster Building, Baldiri i Reixac 10, E- 08028 Barcelona, Spain. 2 Chemical and Physical Biology CIB (CSIC), Ramiro de Maeztu 9, 28040 Madrid , Spain. 77 In order to find inhibitors of the repair activity of hAGT to be used as adjuvants in chemotherapy , we developed a real - time fluorescence hAGT activity assay for the in vitro evaluation of hAGT activity . This m ethod is based on the detection of the conformational changes of the ?- thrombin - binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine of one of its two tetrads is replaced by an O 6 - methyl - guanine. The TBA sequence contains a fluoro phore and a quencher attached to the opposite ends. In th e unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the alkylated guanine, TBA folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close pr oximity, thereby resulting in a decrease in fluoresc ence intensity. In this chapter we developed a new method to quantify the hAGT activity without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the c onformational change of TBA that is induced by the removal of the O 6 - methyl group. In addition, we include as annex of this chapter a review on the chemical modifications that affect the structure of TBA and its biomedical applications, written by our grou p. 78 SAGE-Hindawi Access to Research Journal of Nucleic Acids Volume 2010, Article ID 632041, 9 pages doi:10.4061/2010/632041 Research Article Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity Maria Tintore?,1 Anna Avin?o?,1 Federico M. Ruiz,2 Ramo?n Eritja,1 and Carme Fa`brega1 1 Institute for Research in Biomedicine (IRB Barcelona) IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine, Cluster Building, Baldiri i Reixac 10, 08028 Barcelona, Spain 2 Chemical and Physical Biology CIB (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain Correspondence should be addressed to Carme Fa`brega, carme.fabrega@irbbarcelona.org Received 11 June 2010; Accepted 17 July 2010 Academic Editor: Ashis Basu Copyright ? 2010 Maria Tintore? et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Human O6-alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein) and a quencher (dabsyl) attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group. 1. Introduction Alkylating agents are chemotherapeutic anticancer drugs that produce their cytotoxic effect by generating adducts at multiple sites in DNA [1]. A subset of alkylating agents, which includes nitrosoureas and temozolamide, have a preference for alkylating guanine at the O6 position, which is the most relevant in terms of mutagenesis and carcinogenesis [2?9]. In particular, 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) attacks initially at the O6 guanine position, causing its rearrangement in a cyclic intermediate thus giving rise to N1,O6-ethanoguanine [10]. Finally, a cross-link with the opposite cytosine is formed, and, as a consequence, DNA replication is blocked, producing G2/M arrest [11]. In addition to the well-known side effects and limitations of chemotherapeutic agents, these substances also present problems of acquired tumor resistance. In particular, the DNA-repair human O6-alkylguanine DNA alkyltransferase (hAGT or MGMT) is responsible for removing alkyl adducts from the O6 position of guanines, thereby blocking the cytotoxic effects of the alkylating agents and making a crucial contribution to the resistance mechanism [12?14]. It is well established that tumor cells show greater expression of this protein than healthy cells. Thus, this increased expression appears to be predictive of a poor response to chemother- apeutic drugs. This effect has been observed in a large number of cancers, ranging from colon cancer, lung tumors, breast cancer, pancreatic tumors, non-Hodgkin?s lymphoma, myeloma, and glioblastoma multiforme, among others [15? 17]. In addition methylation of the hAGT promoter and consequently the complete depletion of hAGT, it has been associated with longer survival in patients with gliomas undergoing combined radiation-chemotherapy treatment [18, 19]. Therefore, pharmacological inhibition of hAGT has the potential to enhance the cytotoxicity of a diverse range of anticancer agents [20]. 79 2 Journal of Nucleic Acids hAGT is a 22-kDa (207 AA) DNA-binding protein that contains a highly conserved internal cysteine, which acts as the acceptor site for alkyl groups. The S-alkyl-Cys formed is not regenerated and the protein, which behaves as a suicidal enzyme, inactivates itself in the dealkylation process [21, 22]. This single turnover of hAGT renders it vulnerable to inactivation. On the basis of this observation, intense research effort has been devoted to the identification of small molecules capable of inhibiting hAGT activity and significantly enhancing the cytotoxic effect of BCNU in prostate, breast, colon, and lung tumor cells [20]. Several methods are available to characterize the mech- anism of action of hAGT and its activity. Moreover, they also have the capacity to evaluate the capacity of small molecules to inhibit hAGT. Most of these methods are based on radioactivity assays while others are based on multiple- step enzymatic reactions [23?26]. G-quadruplexes are a family of four-stranded DNA structures stabilized by the stacking of guanine tetrads, in which four planar guanines form a cyclic array of hydrogen bonds [27]. These G-rich regions are connected by lateral, central, or diagonal loops of diverse sizes and composition that form base-pairing alignments, which in turn stack with the terminal G-tetrads, thus further stabilizing G- quadruplex structures [28]. Another key element in G- quadruplex formation is the presence of monovalent cations, which stabilize the negative electrostatic potential created by the guanine O6 oxygen atoms within the quadruplex core [29, 30]. However, most divalent cations have the capacity to induce the dissociation of G-quadruplex structures [31]. Finally, modifications in the base composition of the tetrads are poorly tolerated by these structures. As an example, inosine [32] and O6-methylguanine [33], both nonnatural bases can form a smaller number of hydrogen bonds and thus destabilize the G-quadruplex. Given the potential relevance of hAGT as a prognostic marker of cancer and as a therapeutic target, and that its substrate O6-methylguanine disrupts the formation of G-quadruplex structures [33], here, we developed a new fluorescence activity assay for hAGT. For this purpose, we selected the thrombin-binding aptamer (TBA) as our G- quadruplex model. TBA is a well-known 15 mer that adopts a chair-like structure consisting of two G-tetrads connected by two lateral TT loops and a central TGT loop [34]. The modification of its sequence in positions 5 or 6 by replacing a guanine of the tetrad for an O6-methylguanine disrupts folding, leaving it in an extended conformation. Giving that the two conformations bring the two ends of TBA together take them further apart, our working hypothesis was that the incorporation of fluorescence probes results in a measurable change in intensity. The final aim of this fluorescence assay was to measure the DNA repair activity of hAGT and thus facilitate the search for new and more potent inhibitors which enhance chemotherapeutic drugs. Although several methods have been described to quantify hAGT activity [23? 26], none of them use the conformational change of G- quadruplex for this purpose. Here, we describe the devel- opment of a straightforward, rapid, one-step fluorescence resonance energy transfer (FRET) assay. 2. Materials and Methods 2.1. Chemicals. 5?-Fluorescein CE phosphoramidite (6- FAM), 3?-Dabsyl CPG, O6-methylguanine (O6-MeG) and Gdmf phosphoramidites were purchased from Link tech- nologies (UK) and Glen Research (USA). O6-methylguanine was protected with the isobutyryl group [35]. Standard phosphoramidites and ancillary reagents were purchased from Applied Biosystems (Europe). The matrices for MALDI-TOF experiments were 2?,4?,6?- trihydroxiacetophenone monohydrate (THAP) and ammo- nium citrate dibasic. Solvents for chromatographic analysis were prepared using triethylamine, acetic acid, and acetonitrile as mobile phase. All other chemicals were of analytical reagent grade and were used as supplied. Ultrapure water (Millipore, USA) was used in all experiments. 2.2. Instrumentation. Semipreparative reverse phase (RP) HPLC was performed on a Waters chromatography system using Nucleosil semipreparative 120 C18 (250 ? 8mm) columns. Analytical RP-HPLC was performed using an XBridge OST C18 2.5 ?m column and a Nucleosil Analytical column 120 C18 (250 ? 4mm). Oligonucleotides were detected by UV absorption at 260 nm. Mass spectra were recorded on a MALDI Voyager DETM RP time-of-flight (TOF) spectrometer (Applied Biosystems, USA) with a nitrogen laser at 337 nm using a 3-ns pulse. Fluorometric measurements were performed on a spectrofluorometer multidetection microplate reader Biotek FL ? 800 and a Jasco FP6200. Molecular absorption spectra between 220 and 550 nm were recorded with a Jasco V650 spectrophotometer. The temperature was controlled with a Jasco ETC 272T Peltier device. Hellma quartz cuvettes (0.5 and 1.0 cm path length, 500 or 1000 ?L volume) were used. 2.3. Oligonucleotide Synthesis. Oligonucleotide sequences (shown in Table 1) were synthesized on an ABI 3400 DNA Synthesizer (Applied Biosystems, USA) using a 200- nmol scale synthesis and following the standard proto- cols. We used dimethylformamidino-protected guanine Gdmf phosphoramidite for all the syntheses. 5?-Fluorescein CE phosphoramidite (6-FAM), O6-methylguanine (O6-MeG) and Gdmf phosphoramidites were from commercial sources. The isobutyryl group was used to protect the amino group of O6-MeG [35]. The quencher group was introduced at the 3? end using the controlled pore glass functional- ized with a 3?-Dabsyl derivative CPG. O6-MeG-containing oligonucleotides were deblocked using concentrated aqueous ammonia overnight at room temperature following the man- ufacturer?s instructions. The resulting products were desalted by Sephadex G-25 (NAP-10, GE Healtcare, USA) and puri- fied by reversed-phase HPLC using Nucleosil columns. The yields and purities obtained for the products were around 85% for 5-O6-MeG-TBA and 6-O6-MeG-TBA, and >98% for the rest of the sequences. The length and homogene- ity of the oligonucleotides were checked by MALDI-TOF. 80 Journal of Nucleic Acids 3 Table 1: Sequences of TBA oligonucleotide derivatives used in the development of the hAGT fluorescence assay. MeG represents O6- methylguanine. MB represents the fluorophore group (FAM), labelled in the 5? end, and the quencher group (Dabsyl), labelled in the 3? end of the sequence. Abbreviation Sequence TBA 5?-GGT TGG TGT GGT TGG-3? 5-O6-MeG-TBA 5?-GGT T MeGG TGT GGT TGG-3? 6-O6-MeG-TBA 5?-GGT TG MeGTGT GGT TGG-3? C-TBA 5?-CCA ACC ACA CCA ACC-3? MB-TBA 5?-FAM-GGT TGG TGT GGT TGG-Dabsyl-3? MB-5-O6-MeG-TBA 5?-FAM-GGT T MeGG TGT GGT TGG- Dabsyl-3? MB-6-O6-MeG-TBA 5?-FAM-GGT TG MeG TGT GGT TGG- Dabsyl-3? 3-HP-TBA 5?-A CCT TTT GGT TGG TGT GGT TGG-3? 6-HP-TBA 5?-C CAA CCT TTT GGT TGG TGT GGT TGG-3? 9-HP-TBA 5?-A CAC CAA CCT TTT GGT TGG TGT GGT TGG-3? 5-O6-MeG-TBA [M] = 4731.7 (expected 4737.8), 6-O6- MeG-TBA [M] = 4729.9 (expected 4737.8), MB-5-O6-MeG- TBA [M] = 5828.49 (expected 5826.0), MB-6-O6-MeG-TBA [M] = 5826.80 (expected 5826.0). The DNA-strand concentration was determined by absorbance measurements (260 nm) by calculating extinc- tion coefficients. Oligonucleotide samples were kept at 4?C until further use. Double-stranded O6-MeG-TBA was formed by annealing equimolar concentrations of comple- mentary oligonucleotide strands at 72?C for 5 min and then allowed to slowly cool to room temperature. 2.4. Melting Temperature Studies. Melting curves were mea- sured by monitoring the absorbance hyperchromicity at 295 and 495 nm in a JASCO V650 spectrophotometer equipped with a Peltier temperature-controlling unit. UV/Vis absorp- tion spectra were recorded at 1?C/min intervals, with a 1- min equilibration time at each temperature; the sample was heated over the range 20?80?C. The buffer solutions used were 10mM sodium cacodylate pH 7.0 and 100mM KCl. Sample concentration was around 4 ?M. Each sample was allowed to equilibrate at the initial temperature without any external control of temperature for 5 min before the melting experiment began. The melting temperatures (Tm) are the average value of at least one pair of Tm experiments. 2.5. CD Spectra. Samples were prepared as described for the melting experiments by UV spectroscopy. Measurements were conducted in 10mM sodium cacodylate pH 7.0 and 100mM KCl. Sample concentration was between 1?4 ?M. The CD spectra were recorded on a Jasco J-810 spectropo- larimeter attached to a Julabo F/25HD circulating water bath in 1 cm path-length quartz cylindrical cells. Spectra were recorded at room temperature using a 100 nm/min scan rate, a spectral band width of 1 nm and a time constant of 4 s. All the spectra were corrected with the buffer blank and plotted using Origin software. 2.6. Human Recombinant hAGT Protein. Full-length hAGT was overexpressed and purified as previously described [36]. Briefly, hAGT protein cloned in the pet-21a (+) (Novagen) vector was expressed in the E. coli strain Rosetta. Once the culture reached an OD600 value of 0.98, hAGT was induced by adding 1mM IPTG (Sigma) and left for 4 h at 30?C. The pellet from a 1-L culture was disrupted by sonication and centrifuged. The supernatant was filtered, loaded into a HiTrap FF column (GE Healthcare) with buffer 350mM NaCl, 20mM Tris pH 8, 20mM imidazole, and 1mM BME, and then eluted with an imidazole (Fluka) gradient up to 500mM in the same buffer. Finally, the protein was loaded into a Superdex 75 16/60 column (GE Healthcare) with the following buffer: 200mM NaCl (Merck), 20mM Tris pH 8.0 (Merck), 10mM DTT (Sigma) and 0.1mM EDTA (Sigma). The protein was concentrated to 2mg/mL in this buffer and kept at ?20?C in the presence of 40 % glycerol. The same protocol was used for the purification of the inactive mutant hAGT-C145S, cloned in the pet-28a (+) vector (Novagen), and expressed in the E. coli strain BL21. 2.7. HPLC hAGT Assay. Assays were conducted using double-stranded 5-O6-MeG and 6-O6-MeG TBA paired with TBA complementary sequence or using single-stranded 5- O6-MeG and 6-O6-MeG TBA. In order to measure the dealkylation of O6-MeG, 50 pmol of the O6-MeG-TBA substrates were incubated with a range of concentration of hAGT (16 nM to 1.6 ?M) to a final volume of 120 ?L in a reaction buffer (200mM NaCl, 50mM Tris pH 8.0, 1mM DTT, and 5mM EDTA). Several incubation times were tested (30, 60, 90, 120, 360, and 1440 min) at 37?C and the reaction was stopped by heating the samples at 72?C for 5 min. The reaction products were analyzed by HPLC on a Nucleosil analytical column at 60?C or room temperature, depending on the substrates used in the experiment (double- or single-stranded TBA). The HPLC flow rate was 1mL/min, and a gradient of 10%?40% acetonitrile for 20 min was used. 2.8. Fluorescence Assay for hAGT Activity. The fluorescence assay was performed in a multidetection microplate reader biotek FLx800 in optical 96-well Optical btm reaction plates 81 4 Journal of Nucleic Acids Me hAGT Me-hAGT 5?-FAM 3?-DABSYL 5?-FAM 5?-FAM 3?-DABSYL 3?-DABSYL Figure 1: Scheme of fluorescence-based hAGT assay. The sub- strate is the thrombin-binding aptamer modified by an O6- methylguanine (Me) in extended conformation, with a fluorophore and quencher in the opposite ends of the sequence. The refold of the G-quadruplex structure of TBA is dependent on the removal of the methyl adduct by alkyl-guanine-DNA-O6-alkyltransferase (hAGT). This repair moves the quencher and the fluorophore molecules in close proximity and blocks fluorescence. (Nunc, USA). Full-length hAGT recombinant protein (207 amino acids) was used for the assay and the hAGT-C145S inactive mutant was used as a negative control. The reaction was performed in a total volume of 50 ?L in each well, incubating increasing concentrations of hAGT (5, 10, 20, 40, 60, and 80 nM) enzyme in reaction buffer (200mM NaCl, 50mM Tris pH 8.0, 1mM DTT, 5mM EDTA, and 20mM KCl). The assay of hAGT was then initiated by the addition of 5 ?L of different concentrations (5, 10, 25, and 50 nM) of fluorescently labelled MB-O6-MeG-TBA substrate and this solution was then placed in a microplate reader system. Fluorescence was measured every minute for 20 min or 40 min at excitation and emission wavelengths of 485 and 535 nm, respectively. Averages over three readings were taken for each condition tested. Each experiment was performed in triplicate. 3. Results and Discussion The aim of this study was to develop a real-time hAGT activity assay based on the detection of differences between extended and folded conformations of TBA. Our working hypothesis was that the quadruplex structure of TBA is 220 240 260 280 300 320 ?2 ?1 0 1 2 3 4 5 ? (nm) MB-TBA MB-5-O6-MeG-TBA ? M (d eg cm 2 dm ol ? 1 ) Figure 2: CD spectra of MB-TBA and MB-5-O6-MeG-TBA at 25?C. Buffer conditions: 10mM sodium cacodylate pH 7.0 and 100mM KCl, sample concentration 1 ?M. disrupted when a central guanine is replaced by an O6- methylguanine. The TBA sequence also contains a fluo- rophore and a quencher group attached to 5? or 3? end, respectively. In the presence of O6-methylguanine, methy- lated TBA unfolds and the fluorophore and the quencher become physically separated beyond the Fo?rster distance. When the repair protein hAGT is added to the methylated aptamer, the enzyme removes the methyl group from the mutated guanine, thus allowing TBA to fold back into its chair-like conformation. As a result, the quencher comes closer to the fluorophore and blocks its fluorescence, as illustrated in Figure 1. This loss of fluorescence is quantified as a direct measurement of hAGT activity. 3.1. Synthesis of Modified G-Quadruplex Sequences. The G- quadruplex sequences used in this study are shown in Table 1. Oligonucleotide synthesis was performed by the solid-phase 2?-cyanoethyl-phosphoramidite method [37]. Ammonia treatment was performed at room temperature overnight to minimize decomposition of O6-methylguanine. For the same reason, the dimethylformamidino group was selected for the protection of 2?-deoxyguanosine [38]. The sequences were characterized by HPLC and mass spectrom- etry, which provided the expected molecular weights. The yields of the isolated molecular beacon oligonucleotides after HPLC purification and desalting were in the range of those obtained for unmodified oligonucleotides. 3.2. Thermal Stability of Methylguanine-Modified TBA. In order to induce the unfolding of the quadruplex structure of TBA, we introduced an O6-methyl-guanine at position 5 or 6 of TBA. Melting curves of the modified sequences were performed by UV spectroscopy and compared with the unmodified sequence. These experiments were carried 82 Journal of Nucleic Acids 5 G1 G1 G1 G1 G2 G5 G6 G8 G11 T1 T1T3 T7 T9 T4 (a) G1 G1 G1 G1 G2 G5 G6 G8 G11 T1 T1T3 T7 T9 T4 T16 T17 T18 T19 C20 C21 A22 (b) G1 G1 G1 G1 G1 G2 G5 G6 G8 T1 T1 T3 T4 T7 T9 T16 T17 T18 T19 C20C21A22A23C24C25 (c) G1 G1 G1 G1 G1 G2 G5 G6 G8 T1 T1 T3 T4 T7 T9 T16 T17 T18 T19 C20C21A22A23C24C25 C27A26 A28 (d) Figure 3: Schematic representation of the structure of several derivatives of thrombin-binding aptamers (TBAs) prepared in this study. TBA sequence is shown in black, and T4 loop with different sizes of the complementary sequences are shown in red. (a) Native TBA; (b) TBA-hairpin containing three overhanging complementary nucleotides (3-HP-TBA); (c) TBA-hairpin containing six overhanging complementary nucleotides (6-HP-TBA); (d) TBA-hairpin containing nine overhanging complementary nucleotides (9-HP-TBA). Between 3?6 complementary nucleotides are required to disrupt the intramolecular quadruplex to form an intramolecular duplex. out at pH 7 in buffer containing 10mM sodium cacody- late and 100mM KCl, which is predicted to stabilize G- quadruplexes structures. We did not observe a melting temperature at 295 nm for 5-O6-MeG-TBA or 6-O6-MeG- TBA and the corresponding molecular beacons (see Figures S1?S4 in Supplementary Data Material available online at doi: 10.4061/2010/632041). The absence of this transition is consistent with the disruption of the quadruplex structure. These results contrast with the melting temperature of native TBA (Tm 48?C) and MB-TBA (Tm 46?C). Moreover, circular dicroism of methylated-TBA derivatives did not show the presence of the maximum at 295 nm, which is characteristic of the antiparallel quadruplex of TBA (Figure 2 and Figure S5). These results confirmed that methylation of guanine in either of the two positions of the TBA sequence prevents the formation of the chair structure. This observation confirms our working hypothesis. Given that the DNA repair activity exerted by hAGT is higher in double-stranded DNA than single-stranded DNA [39], we studied the stability and the quadruplex formation of elongated self-complementary TBA derivatives (see Table 1). We designed several TBA oligonucleotides elongated in their 3? end with a subset of self-complementary sequences of diverse sizes. The purpose of these elongations was to check whether the extended sequences have the capacity to form a double strand helix of different lengths and strengths with themselves without disrupting the chair- like structure. For 6-HP-TBA and 9-HP-TBA, we found that the corresponding Tm at 260 nm were 63oC and 71.8?C, respectively. This observation confirms our hypothesis of a double helix structure that increases in strength as the sequence length increases. However, we did not detect a melting temperature of these two sequences at 295 nm. The absence of a transition at 295 nm is consistent with the absence of an antiparallel quadruplex structure. In contrast, 3-HP-TBA, corresponding to an overhang of only 3 nucleotides, gave a melting temperature of 46?C at 295 nm, which is slightly but similar to that obtained for natural TBA. This result indicates that 3-HP-TBA forms an antiparallel quadruplex instead of a duplex because the overhang is too short to break the chair-like structure. Circular dichroism confirms the quadruplex structure of 3-HP-TBA and the absence of quadruplex structure in 6-HP-TBA and 9-HP- TBA (Figure S6). Models of all these structures are shown in Figure 3. 3.3. HPLC Analysis of DNA Repair Activity of hAGT. In order to observe the efficiency of hAGT to remove alkyl groups from single-stranded oligonucleotides, we performed several assays to determine the optimal conditions of the reaction. For this purpose, the full-length hAGT was first over- expressed and purified as described previously [36]. Increas- ing concentrations of the protein were incubated with double-stranded 5-O6-MeG and 6-O6-MeG TBA, annealed with its complementary sequence. Figure 4(a) shows the HPLC profile of the final product of the reaction with double-stranded 5-O6-MeG-TBA. In order to separate the two strands of the TBA substrate (Figure 4(a) top right panel), HPLC analyses were done at 60?C. After incubation with hAGT, we detected a peak with a shorter retention time, which corresponds to the restored TBA sequence caused by 83 6 Journal of Nucleic Acids 0 5 10 15 20 A bs (2 60 n m ) (minutes) cTBA O6-Me-TBA TBA 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 ?0.01 0 5 10 15 20 (minutes) cTBA O6-Me-TBA ?10?2 ?5 0 5 10 15 20 25 30 35 A bs (2 60 n m ) (a) 0 5 10 15 20 A bs (2 60 n m ) (minutes) O6-Me-TBATBA 0 0.01 0.02 0.03 0.04 0.05 0 5 10 15 20 (minutes) O6-Me-TBA ?10?2 A bs (2 60 n m ) 0.5 ?0.5 0 1 1.5 2 2.5 3 3.5 4 (b) Figure 4: HPLC analysis of hAGT activity over double- and single-stranded TBA before and after incubation with hAGT. (a) Repair of double-stranded 5-O6-MeG-TBA by hAGT. The peak labelled as cTBA corresponds to the complementary sequence of 5-O6-MeG-TBA. The top right panel shows HPLC chromatogram in absence of hAGT. The gradient was from 10%?40% acetonitrile for 20 min at 60?C. (b) Repair of single- stranded 5-O6-MeG-TBA by hAGT. The inserted panel shows HPLC chromatogram in the absence of hAGT. Gradient used: 10%?40% acetonitrile, 20 min at room temperature. 0 20 40 60 80 100 Fl uo re sc en ce (% ) 1 2 3 4 (a) 0 20 40 60 80 100 Fl uo re sc en ce (% ) 0 20 40 60 (nM) (b) Figure 5: Fluorescence intensity of the real-time hAGT assay, measured at excitation and emission wavelengths of 485 nm and 535, respectively. (a) Illustration of the different controls with 5 nM MB-5-O6-MeG-TBA: (1) Positive control of MB-5-O6-MeG-TBA in the absence of hAGT; (2) Intensity in presence of the inactive mutant hAGT-C145S; (3) Decrease in fluorescence when adding 40 nM active hAGT; and (4) basal fluorescence of 5 nM of MB-TBA. (b) Decrease in intensity caused by the activity of hAGT at a range of concentrations (0, 20, 40, and 60 nM), in all of them the basal fluorescence of MB-TBA was subtracted. the removal of the methyl group. We obtained the same results when we used double-stranded 6-O6-MeG-TBA as a substrate (data not shown). As expected, hAGT activity was not affected by the position of the alkyl group within the sequence. We then tested hAGT activity over single-stranded methylated TBA in the previously optimized conditions and obtained similar results to those shown in Figure 4(b). The top right panel shows the HPLC chromatogram in the absence of hAGT. In this case, the HPLC analyses were performed at room temperature because the substrate was single-stranded TBA. Our results confirmed that hAGT has the capacity to removemethyl groups from single- or double- stranded TBA with the same efficacy. Therefore, we selected single-stranded TBA as substrate for the development of our fluorescence assay. 3.4. Fluorescence hAGT Activity Assay. On the basis of the results obtained in the melting temperature and the HPLC experiments, we tested the effectiveness of our proposed model to the DNA repair activity of hAGT by means of fluorescence. First of all, we determined the minimum amount of TBA required to achieve a detectable and reliable 84 Journal of Nucleic Acids 7 difference in intensity compared to the background. As expected, the negative control natural TBA quadruplex gave low background fluorescence, because of the proximity of the fluorophore and the quencher groups in the chair-like structure (Figure 5(a)). Although the fluorescence of MB- TBA was low, this fluorescence was subtracted from the fluorescence value in each experiment. The concentration of 5 nM of fluorescently labelled MB-O6-MeG-TBA was considered the optimal concentration as the fluorescence signal was intense and only a small amount of hAGT protein was required to achieve a substantial decrease in fluorescence in 20?40 min. In the optimal conditions, the presence of hAGT produced a remarkable decrease in fluorescence intensity, caused by the demethylation of the O6 position of guanines, thereby allowing the TBA to form its typical quadruplex structure, which brings together the fluorophore and the quencher groups, as occurred in the negative control. The rate of decrease in fluorescence intensity correlated directly with the amount of hAGT in the reaction mixture (Figure 5(b)). Moreover, the inactive mutant hAGT-C145S did not exhibit any decrease in fluorescence (Figure 5(a)). This result was expected because of the inability of this mutated protein to repair the modified TBA. Figure 5(b) shows the fluorescence intensities for the real time hAGT assay. All these observations are consistent with the hypothe- sis and design of our FRET method. 4. Conclusion Although radioactivity has been widely used in the search of potential inhibitors of hAGT [23, 24], this technique does not allow real-time data acquisition and, in addition, requires radioactive materials. Here, we developed a sensitive fluorescence method that allows the quantification of hAGT activity in a single step and in a straightforward manner. Although another fluorescence method has already been developed for this purpose [25], it requires the addition of a restriction enzyme, followed by the addition of an exonuclease after the hAGT activity reaction. Consequently, although it is a real-time assay, a three-step reaction is required before observing an increase in fluorescence. In contrast, in our assay, a change in fluorescence is detected in a single step (homogeneous), and this method does not depend on the activity of two additional enzymes. Our assay is based on the detection of conforma- tional changes of TBA in the presence or absence of O6- methylguanines in its structure. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methylguanine. Fluorophore groups can be added to the modified sequence in order to detect the conformational changes by fluorescence. The variation in fluorescence can be quantified as a direct measurement of hAGT activity. In addition, this technique requires lower amounts of substrate and does not call for the use of radioactive materials. Furthermore, this method can be easily transferred to a high throughput experiment for the evaluation of small molecules as potential hAGT inhibitors [36]. Research in this direction is currently underway in the laboratory. Abbreviations BME: 2-mercaptoethanol Dabsyl: 3-(N-4?-sulfonyl-4-(dimethylamino)- azobenzene)-3-aminopropyl dmf: Dimethylformamidino group DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid FAM: Fluoresceine AGT: O6-alkylguanine-DNA alkyltransferase hAGT: Human AGT HPLC: High performance liquid chromatography IPTG: Isopropyl ?-D-1-thiogalactopyranoside MB: Molecular Beacon O6-MeG: O6-methylguanine TBA: Thrombin-binding aptamer cTBA: Complementary TBA TEAA: Triethylammonium acetate TFA: Trifluoroacetic acid Tris: Tris(hydroxymethyl)aminomethane Tm: Melting temperature UV: Ultraviolet. Acknowledgments This work was supported by the Fondo de Investigaciones Sanitarias (Grant PI06/1250), by the Direccio?n General de Investigacio?n Cient??fica y Te?cnica (Grant BFU2007- 63287, CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208), the COST project (G4-net, MP0802), the Instituto de Salud Carlos III (CIBER-BNN, CB06 01 0019), and a SNS Miguel Servet contract from the Instituto de Salud Carlos III. The authors thank Tanya Yates for editing the paper and the reviewer for the detailed revision of the paper. This work is dedicated to thememory of A?ngel Ram??rez Ortiz who was a great mentor, colleague, and friend. References [1] M. R. Middleton and G. P. Margison, ?Improvement of chemotherapy efficacy by inactivation of a DNA-repair path- way,? Lancet Oncology, vol. 4, no. 1, pp. 37?44, 2003. [2] A. Sabharwal and M. R. Middleton, ?Exploiting the role of O6- methylguanine-DNA-methyltransferase (MGMT) in cancer therapy,? 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Fried, ?DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase: effects of protein and DNA alkylation on complex stability,? Journal of Biological Chemistry, vol. 278, no. 10, pp. 7973?7980, 2003. 87 88 Supplementary data Dev el op men t of a Nov el Fl u or esc en c e As say Ba s ed on th e Us e of th e Th ro mb in Bi n d in g Ap t a mer for th e D et e cti on of O 6 -alkyl gu an in e? DN A Al kylt ran sf er as e Acti vity Maria Tintor?, Anna Avi??, Federico M. Ruiz, Ram?n Eritja and Carme F?brega* CONTENTS: Figure S1 . UV spe c t ra o f M B -T B A at 2 0 and 8 0 ?C . Figure S2 . UV spe c t ra o f M B -5 -O 6 -Me G-T B A at 20 and 80 ?C . Figure S3. M el tin g cu rves o f M B -T B A an d M B -5 -O 6 -M e G-T B A re c o rded at 29 5 n m. Figure S4. M el tin g cu rves o f M B -T B A an d M B -5 -O 6 -M e G-T B A re c o rded at 49 5 n m. Figure S5. C D spe c t ra o f T B A, 5 -O 6 -M e G-T B A and 5 -O 6 -M e G-T B A. Figure S6. C D spe c t ra o f T B A, 3 HP-T B A, 6 HP-T B A an d 9 HP-T B A. 89 Figure S1 . UV s pe c t ra o f MB -T B A (5 ?-F AM -GG T TGG T GT GGT T GG-Da b s yl-3 ?) at 20 and 80 ?C . Figure S2 . UV spe ct ra o f M B -5 -O 6 -M e G-T B A (5 ?-F AM-G GT T MeG G T GT GGT T GG- Dab s yl-3 ?) at 2 0 an d 80 ?C . 20 0 2 5 0 3 0 0 3 5 0 4 0 0 45 0 5 0 0 5 5 0 6 0 0 0 ,0 0 0 ,0 5 0 ,1 0 0 ,1 5 0 ,2 0 0 ,2 5 0 ,3 0 0 ,3 5 0 ,4 0 A bs ??(nm) 2 0?C 8 0?C M B-TBA 20 0 2 5 0 3 0 0 3 5 0 4 0 0 45 0 5 0 0 5 5 0 6 0 0 0 ,0 0 0 ,0 5 0 ,1 0 0 ,1 5 0 ,2 0 0 ,2 5 0 ,3 0 0 ,3 5 0 ,4 0 A bs ( m) 2 0?C 8 0?C M B- B 20 0 2 5 0 3 0 0 35 0 4 0 0 4 5 0 50 0 55 0 6 0 0 0, 0 0, 5 1, 0 1, 5 2, 0 A bs ??(nm) 20?C 80?C M B-5 - O 6 - MeG-TBA 20 0 2 5 0 3 0 0 35 0 4 0 0 4 5 0 50 0 55 0 6 0 0 0, 0 0, 5 1, 0 1, 5 2, 0 A bs ( m ) 20?C 80?C M B-5 - O 6 - M - B 90 Figure S3. Me l tin g cu rves o f M B -T B A an d MB -5 -O 6 -M e G -T B A re c o rd ed at 295 n m. A qu ad rup le x-to -ran d o m -co il tra ns i t io n is ob se rve d fo r th e MB -T B A (T m = 4 6 ?C ). No tra n s i t io n was o bse rv ed fo r M B -5 -O 6 -M e G-T B A. Figure S4. Me l tin g cu rve s o f M B -T B A and MB -5 -O 6 -M e G-T B A re c o rd ed at 49 5 n m . A qu ad rup le x-to -ran d o m -co il tra ns i t io n is ob se rve d fo r th e MB -T B A (T m = 4 6 ?C ). No tra n s i t io n was o bse rv ed fo r M B -5 -O 6 -M e G-T B A. 20 3 0 40 5 0 6 0 7 0 8 0 0 ,8 7 5 0 ,9 0 0 0 ,9 2 5 0 ,9 5 0 0 ,9 7 5 1 ,0 0 0 1 ,0 2 5 A bs 295 n m T(?C ) MB-TBA MB-5-O 6 -MeG-TBA 2 0 3 0 4 0 5 0 6 0 7 0 8 0 0 , 95 1 , 00 1 , 05 1 , 10 1 , 15 1 , 20 1 , 25 1 , 30 1 , 35 A bs 495 n m T(?C ) MB-TBA MB- 5-O 6 - MeG-TBA 91 Figure S5. C D spe c t ra o f T B A, 5 -O 6 -Me G-T B A and 5 -O 6 -M e G-T B A re co rde d at 2 5 ?C . Con d i tion s 10 mM sod iu m c aco d yl at e p H 7 .0 an d 100 mM KC l ., samp le con c en trat io n 4 ?M . Figure S6. C D spe c t ra o f TB A, 3 HP-T B A, 6 HP -T B A and 9 HP-T B A re co rd e d at 2 5 ?C . Con d i tion s 10 mM sod iu m c aco d yl at e p H 7 .0 an d 100 mM KC l ., samp le con c en trat io n 4 ?M . 220 240 260 280 300 320 - 20 - 10 0 10 20 30 40 ? ? ?de g c m 2 dm ol -1 ) ???nm) TBA 5-O 6 -MeG-TBA 6-O 6 -MeG-TBA 220 240 2 60 280 300 320 - 20 - 10 0 10 20 30 40 ? ? ?de g c m 2 dm ol -1 ) ???nm) TBA 3-H P-TBA 6-H P-TBA 9-H P-TBA 92 Appendix 2 Thrombin binding aptamer, more than a simple aptamer: chemically modified derivatives and biomedical applications. 93 94 Thrombin binding aptamer, more than a simple aptamer: chemically modified derivatives and biomedical applications. Anna Avi??, 1 Carme F?brega, 1 Maria Tintor?, 1 and Ramon Eritja 1 Current Pharmaceutical Design (2012) Volume 18 , Issue 14, pages 2036- 2047 . DOI: 10.2174/138161212799958387 . Impact factor: 3.288 1 Institute for Research in Biomedicine of Barcelona, IQAC-CSIC, CIBER-BBN Networking, Centre on Bioengineering Biomaterials and Nanomedicine. Cluster Building, Baldiri i Reixach 10, E-08028 Barcelona 95 96 Current Pharmaceutical Design, 2012, 18, 000-000 1 1381-6128/12 $58.00+.00 ? 2012 Bentham Science Publishers Thrombin Binding Aptamer, More than a Simple Aptamer: Chemically Modified De- rivatives and Biomedical Applications An n a Av i ? ? , Car me F ?b reg a , Ma r? a Ti n t o r? an d Ra mo n E ri t j a* Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedi- cine, Edifici Helix, Baldiri Reixac 10, E-08028 Barcelona, Spain Abstract: Th e th ro m b in b in d in g a p ta m e r (TBA ) is a we ll c h a ra c te riz e d c h a ir- lik e , a n tip a ra lle l q u a d ru p le x stru c tu re th a t b in d s sp ec ific a ll y to th ro m b in a t n an o m o la r co n ce n tra tio n s a nd th e re fo re it h a s in te re s tin g a ntic o a g u la n t p rop e rtie s . In th is a rtic le we re v ie w th e re s e a rc h in v o lv e d in th e d e v e lop m e n t o f n e w TBA d e riv a tiv e s with im p ro v e d a n tic o a g u la n t p rop e rtie s a s we ll a s th e u se o f th e TBA a s a mo d e l c o m p ou n d fo r the stu d y o f q u ad ru p lex stru c tu re s . Sp e c ific a lly , we d e s c ribe th e im p a c t o f mod ifie d n u c leo s id e s an d no n -n a tu ra l b a c k - b o n e s in th e g ua n in e te tra d s o r in the lo op s an d th e in trod u c tio n o f p e n da n t g ro u p s a t th e 3 ? o r 5 ?-en d s . Th e m o d ifie d o lig on u c le o tid e s a re sh o w n to be ex c e lle n t too ls fo r th e u nd e rs ta n d ing o f th e mo le c u la r stru c tu re o f th e TBA a n d its fo ld in g p rop e rtie s . Fin a lly , we re v ie w th e u se o f th e TBA -Th r o m b in re c o g n itio n sy s te m fo r th e d e ve lo p m en t o f a n a ly tic a l to o ls b a s e d o n th e TBA fo ld in g . Keywords: Th ro m b in b in d in g ap t am er , T B A , G-q u ad ru p lex , th ro m b in , an t i co ag u lan t, o lig o n u cl eo tid e sy n th es is , DN A , ap t am e r. INTRODUCTION Ap tam er s a r e o lig o n u cl eo tid es th a t we re o rig in ally d er iv ed fro m an in vitro se le ct io n an d p o ly m era se ch a in re ac tio n p ro c es s k n o w n as S E L E X (sy ste m a ti c ev o lu t io n o f lig an d s b y ex p o n en tia l en ri ch m en t) [1 -4 ] wh ich s el e ct s th em o n th e b as is o f th ei r sp e ci fi c an d tig h t b in d in g af fin i ty to a ta rg et o f ch o ic e fro m a lib r a ry o f seq u en ce s in c lu d in g p ro te in s . T h ro u g h th i s ap p ro a ch , a la rg e n u m - b er o f ap t am e rs w ith v e ry h ig h af fin i ty h av e b een d ev elo p ed fo r d iag n o sti c s, th e r ap eu t ic s an d o th er t ech n i c al ap p li c at io n s [5 ], b u t th er e i s s til l ro o m fo r im p ro v em en t in t e rm s o f in cr e as in g th ei r b in d in g p ro p ert ie s an d th eir p h ar m a co k in e ti c p ro p e rt ie s [6 ]. Th e th ro m b in b in d in g ap tam er (T B A ) i s th e fir s t ex am p le o f a p o ten ti al n u c le i c a cid th er ap eu ti c ag en t, t arg e ted to a p ro te in th a t d o es n o t p h y sio lo g i ca lly b in d n u cl ei c a c id s wi th th e fo llo w in g co n - sen su s s eq u en c e: 5 ? -G 1 G 2 T 3 T 4 G 5 G 6 T 7 G 8 T 9 G 10 G 11 T 12 T 1 3 G 14 G 15 -3? [7 ]. T h i s 1 5 -b as e- lo n g o lig o n u cleo t id e b in d s sp e ci fi ca lly to th ro m - b in at 1 0 n M co n cen tr at io n s an d th e re fo r e, i t h a s in t er e stin g an ti co - ag u lan t p ro p er ti es . I t in h ib i ts sp ec if ic al ly c lo t-b o u n d th ro m b in an d red u c es a rt er ia l th ro m b u s fo rm at io n . In ad d itio n , it d o e s n o t co m - p ete w ith o th er k n o w n act iv e s it e in h ib ito rs o f th ro m b in [7 -1 0 ] . Nev e rth e le s s, T B A b in d in g to o th er se ru m p ro t ein s o r p ro teo ly ti c en zy m es i s es sen ti ally u n d et ec tab l e. In an e ffo rt to id en t ify th e r eg io n o f th ro m b in with wh i ch th e T B A ap t am er in t er a ct s, th e in h ib i tio n o f f ib rin o g en - clo ttin g a ctiv it y was stu d i ed u sin g r eco m b in an t mu t ag en es is o f an io n -b in d in g ex o si te o f th ro m b in (ex o s it e I ) [1 0 ]. T h e r e su lt s su g g e st ed th at th e sin g l e- st ran d ed DN A b in d in g sit e is lo ca ted in th e th ro m b in ex o s it e I an d o v er lap s th e th ro m b in p l at el et r e cep to r an d th ro m b o m o d u li n b in d in g sit e s. T h e T B A b in d in g si te o n th ro m b in wa s al so ex am in e d b y so lid -p h as e p l at e b in d in g as say s [1 1 ] an d b y ch em ic al mo d i fi ca - tio n s s tu d ie s [1 2 ]. T h es e s tu d ie s sh o w ed th at th e T B A ap t am e r b in d s sp e cif i ca lly to - th ro m b in b u t n o t to -th ro m b in , wh i ch is a p ro teo ly t ic cl eav ag e p ro d u ct o f - th ro m b in in th e fib r in o g en - b in d in g ex o site . B o th re su lt s su g g e st ag ain th at th e th ro m b i n ex o si te I is im p o r tan t fo r th e ap t am er- th ro m b in in t er ac tio n . Th e aw a r en e ss o f th e fo ld ed st ru ctu re o f th is ap ta m e r, b o th fr e e in so lu tio n o r b o u n d to th ro m b in , i s e s sen t ia l to u n d er st an d it s *Ad d re s s co rre s p on d en c e to th is au th o r a t th e In s titu te fo r Re s e a rc h in Bio- m e d ic in e , IQ A C-CSI C , CIB ER-B B N Ne tw o rk in g Ce n tre o n Bio e n g in e e r- in g , Bio m a te ria ls a n d Na n o m e d ic in e , Ed ific i He lix , Ba ld iri Re ix a c 1 0 , E - 0 8 0 28 Ba rc e lo n a , Sp a in ; Te l: + 3 4 (9 3 )4 0 39 9 42 ; Fa x : + 3 4 (9 3 )20 4 59 0 4 ; E-m a il: re c g m a @c id .c s ic .e s ; ra m o n .e r itja @i r b b a r c e lo n a .o r g b io lo g ic al ac tiv i ty an d u s efu l in th e fu tu r e d ev elo p m en t o f o lig o n u - cl eo tid e-b as ed th er ap eu ti cs o r d ru g d esig n . T h e T B A h as b e e n ch a ra ct er iz ed b y NMR sp ec tro sco p y [1 2 -1 5 ] an d X-r ay c ry st al lo g - rap h y [1 6 -1 8 ]. T h e s e s tu d ie s h av e l ed to th e d es c rip t io n o f it s co m - p act an d sy m m etr ic a l ch ai r- lik e , u n im o l e cu la r an tip ar al le l q u ad ru - p lex st ru ctu re . T h is st ru c tu re co n si st s o f tw o G-te tr ad s co n n e ct ed b y th re e ed g e-w i s e lo o p s: t w o T T lo o p s (T 3 T 4 an d T 1 2 T 1 3 ) at o n e en d an d a sin g l e T 7 G 8 T 9 lo op in th e o th er en d F ig . ( 1A ) . T h e co n fo rm a - tio n al d i str ib u tio n o f th e fo u r co -p l an a r 2 ? -d eo x y g u an o sin e s in th e G-q u ar te ts o f th e T B A ap t am er i s w el l d ef in ed an d th ey a re s tab i - liz ed b y cy cli c Ho o g st een h y d ro g en b o u n d in g F ig . ( 1B ). All su g a r p u ck er s ar e p r ed o m in an t ly South (S) w h ile th e g u an in es o n th e sam e G -q u a rt et p lan e d i sp l ay a lt ern atin g 5 ?- syn-anti-syn-anti-3? co n fo rm at io n s wi th r esp ec t to th e g ly co sy l to rs io n an g l e ( syn -G a t p o sitio n s G 1 , G 5 , G 1 0 an d G 1 4 ; anti-G a t p o si tio n s G 2 , G 6 , G 1 1 an d G 1 5 , F ig . ( 1 )), ex c ep t fo r th e G 8 an d th e th y m in es in th e lo o p s wh ic h ar e a ll anti . T h e tw o T T lo o p s, b o th at o n e en d o f th e q u ad ru p l ex , sp an a n a rro w g ro o v e, wh i l e th e T G T lo o p , p la ced at th e o th er en d , sp an s a wid e g ro o v e. It h a s b e en k n o w n fo r s ev e ra l y e ar s th at n o t o n ly th e p r im a ry n u cleo t id e seq u en c e, b u t al so en v iro n m en ta l co n d itio n s an d in p ar - ticu la r c at io n s, p l ay an im p o r tan t ro l e in th e fo rm at io n , to p o lo g y an d st ab il ity o f G -q u ad ru p l ex e s [1 9 -2 5 ] . In th e c a se o f th e th ro m b i n b in d in g ap tam er , i t w as b el iev ed th at th e p r e sen ce o f K + in th e me - d iu m was n ec e ss ary to sh if t th e eq u il ib riu m to w a rd th e q u ad ru p l e x co n fo rm at io n , su b seq u en tly fav o u rin g th ro m b in b in d in g , its io n i c si ze fit tin g in to th e fr e e sp a c e ex i st in g in th e c en t er o f ea ch q u art et . P rel im in ary stu d i es with Mn 2 + su g g es ted th at it c an b in d s tro n g ly i n tw o si te s wi th o n e in th e e ach n a rro w g ro o v e [2 6 ]. B o th Mn 2 + io n s ar e r el e as ed wh en th e ap t am er is co m p l ex ed wi th th ro m b in , in d ic at - in g th at b o th n ar ro w g ro o v es a re in v o lv ed in th e T B A - th ro m b i n in te ra ct io n s. S o m e au th o rs h av e u s ed a co m b in a tio n o f te m p er atu re - d ep en d en t UV sp e ct ro s co p y , ca lo ri m e try , NMR an d ele ct ro sp r a y io n iz atio n m as s sp e ct ro m e try t e ch n iq u es to ev a lu a te th e ef f ec t i n th e st ab i lity , h y d ra tio n an d th erm o d y n am i c s o f th e mo n o v al en t an d d iv al en t me ta l io n s in th e fo rm atio n o f 3 D stru c tu r es o f th e T B A co m p lex es [2 6 -3 4 ]. Div a len t io n s (P b 2 + , Ba 2 + an d S r 2 + ) an d NH 4 + b in d an d stab ili z e th e q u ad ru p lex st ru ctu re w ith ev en h ig h er ef fi - ci en cy th an K + wh il e Li + , N a + , C s + , Mg 2 + an d C a 2 + fo rm we ak e r co m p lex es o n ly a t v ery lo w t em p e r atu r es . T h es e r e su lt s h av e b e e n ra tio n a li zed in t erm s o f th e ir rad i i; ca tio n s wi th an io n i c rad iu s i n th e r an g e 1 .3 -1 .5  fi t we ll wi th in th e tw o G-q u ar te ts o f th e co m - p lex wh il e th e o th e r c a tio n s d o n o t. T h e d iv al en t c atio n s l ik e P b 2 + , Ba 2 + an d S r 2 + e ff ic i en tly o c cu p y th e reg io n b etw een th e tw o q u ar - 97 2 Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 Avi?? et al. tet s in th e T B A -io n co m p lex in a 1 :1 s to ich io m e try [2 7 , 2 9 , 3 3 ] . T h e ap t am er co m p l ex w ith mo n o v al en t an d d iv a len t io n s u n fo ld s i n a mo n o p h as ic tr an s it io n [3 0 ]. Ho n g et al. hav e d e te rm in ed th e alk a li me t al b in d in g si te an d co n st an t b y el e ct ro sp r ay io n i za tio n (E S I ) an d in f r ar ed mu lt ip h o to n d isso ci at io n (IR M P D ) r e sp e ctiv ely [3 5 ] . T h e b in d in g co n s tan t o f p o tas siu m i s 5 -8 tim es g r e at er th an th o se fo r o th e r alk al i me ta l io n s an d th e K + b in d in g sit e i s d if fe r en t f ro m o th er m et a l b in d in g sit es . In a 1 :1 T B A -m et al co m p lex , p o ta ss iu m co o rd in at ed b etw een th e b o tto m G-q u ar t et an d th e tw o ad j ac en t T T lo o p s o f th e T B A . In a 1 :2 ra tio T B A -m et al co m p l ex , th e s e co n d p o tas siu m io n b in d s a t th e d ist an t T G T lo o p . In th e o th e r h an d , Na + , Rb + an d C s + b in d at th e lat e ra l T G T lo o p in bo th 1 :1 an d 1 :2 co m p lex es , p re su m ab ly d u e to th e fo rm at io n o f io n -p ai r ad d u ct s. B y co n tr as t, so m e p u b li sh ed wo rk s p ro v id e ev id en c e th at th e T B A is ab l e to b in d th ro m b in in th e ab s en c e o f d iv a l en t an d mo n o v al en t io n s [2 4 , 2 5 , 3 6 -3 7 ], wh ich su g g e st s th at th e b in d in g to th ro m b i n p ro m o te s th e T B A fo ld in g to it s 3 D st ru ctu re , ev en in th e ab s en c e o f s al ts . S ev er al g ro u p s h av e stu d i ed an d su g g es ted th at mo l ecu la r cro w d in g c au s es a st ru ctu ra l tr an s it io n fro m an an t ip a ra ll el to a p ar all el DN A G -q u ad ru p l ex an d th ey ar e an i m p o rt an t f ac to r t o co n tro l th e fo rm at io n o f G -q u ad ru p l ex [3 8 -3 9 ]. Miy o sh i et al . h av e sh o w n th at d iff e ren t mo l e cu la r cro w d in g p ro m o te s an d st ab i li ze s th e G-q u ad ru p lex stru ctu r e o f th e T B A b y a fav o u r ab le en th alp i c co n trib u t io n th at ex ce ed s an u n fav o u r ab le en tro p i c co n tr ib u tio n . Mo reo v e r, th e th er m o d y n am ic e ff ec t co rr el at es with th e n u m b e r o f h y d ro x y l g ro u p s o f th e mo l e cu la r cro w d in g co so lu te [3 9 ]. It is wo r th n o tin g th at d e sp it e th e ro b u st s tab ili ty o f th e in - tr am o l ecu l ar q u ad ru p l ex st ru c tu re , al t ern a tiv e in te rm o l e cu la r q u ad - ru p lex e s ar e p o ss ib le at h ig h ap t am er co n c en tr at io n , a s d et e ct ed b y C D an d ele c tro p h o re si s mig r at io n ex p er im en ts [4 0 ]. T h e c ry s ta l stru ctu re o f th e T B A -th ro m b in co m p l ex so lv ed b y P ad m an ab h an et al. [1 6 ] a t 2 .9  reso lu tio n d if fe rs in th e ap ta m e r q u ad ru p l ex to p o l- o g y with th e N MR st ru ctu re . In d e ed , th e co r e o f th e tw o G -t et rad s is th e sa m e in th e tw o mo d els , a lth o u g h stru c tu r al d if f er en c es ex is t in th e w ay th e cen tr al b a se s ar e co n n e ct ed . A d if f er en c e co n c ern in g th e d i sp o si tio n o f th e tw o T T an d th e T G T lo o p s with re sp e ct to th e g ro o v es. In an e ffo r t to re so lv e th is am b ig u ity , th e st ru c tu r e o f th e T B A -th ro m b in co m p l ex h a s b een d e te rm in ed at 2 .8  , b u il t o n th e basi s o f th e NMR s tru ctu r e o f th e ap t am er [1 7 ] . T h e r esu l ts co n - fir m ed th at b o th st ru ctu re s f it th e cry st al lo g rap h i c d at a eq u ally w ell , th u s l e av in g th e d o u b t o n wh ich b in d in g mo d el i s th e co r re ct o n e. I n b o th mo d els, th e T B A i s s an d w ich ed b etw e en tw o sy m m et ry - re la ted th ro m b in mo l e cu le s an d in t er ac ts wi th th e ex o s it e I o f a th ro m b in mo l e cu le an d ex o s it e II o f th e s eco n d o n e . In p a rti cu l ar , th e tw o T T lo o p s in th e NMR st ru c tu re in t er ac t wi th th e fib r in o g en - re co g n itio n sit e (ex o sit e I) o f th e th ro m b in mo l ecu le an d th e T G T lo o p in te ra c ts with th e h ep ar in -b in d in g s it e (ex o sit e II ) o f th e n eig h b o u rin g th ro m b in , wh e re a s in th e X -r ay s tru ctu r e th e o p p o sit e o ccu r s [1 6 -1 7 ]. T h e s tru ctu r e o f th e co m p l ex b e tw e en th ro m b in an d T B A is sh o w n in F ig . ( 2 ). Th e u n cer ta in ty b etw een th e tw o mo d els w as cau sed b y th e ab s en c e o r p o o r el e ct ro n d en s ity in th e r eg io n o f th e T T lo o p s an d in th e G 1 0 fo r th e X-r ay st ru ctu re an d in th e G 1 4 an d in th e T G T lo o p fo r th e NMR stru ctu r e. In a mo re sy st em ati c an aly s is [1 8 ] , eig h t o ri en ta tio n s o f th e NMR ap t am er we r e ev alu at ed in an e ffo r t to re co n ci le th e NMR an d X-r ay d at a [1 6 -1 7 ]. T h e re su l tin g cry st al - lo g rap h i c R - fa cto rs an d th e an a ly si s o f th e ap t am er -p ro t ein co m - p lex e s cl e arly d is tin g u ish ed b e tw e en th e tw o p o s sib l e o l ig o n u cl eo - tid e s b ack b o n e d ir ec tio n a li ti es . Ho w ev e r, d u e to th e mis sin g d en s it y in th e co n n ec tin g lo o p s o f th e ap tam er , th e d et a il s o f th e l ig an d p ro tein in t er ac tio n s co u ld n o t b e p ro p e rly ad d re s sed . Mo r eo v er , ev en r e cen t p ap er s s ti ll d i s cu s s mo d if ied ap t am er -th ro m b in in t er ac - tio n s o n th e b as e s o f b o th mo d el s [3 9 ]. T h e re cen t so lv ed h ig h re so lu tio n s tru c tu r e o f th e co m p l ex o f th ro m b in wi th a mo d ifi ed T B A (m T B A ) , wh i ch co n t ain s a 5 ? -5 ? in v er sio n b et w e en T 3 an d T 4 , cl ar if ie s sev er al q u es tio n s reg ard in g th ro m b in - ap ta m e r in t er a ct io n [4 1 ]. T h e ap t am er tig h tly b in d s t o th ro m b in ex o s it e I b y i ts T T lo o p s, th ro u g h a m ix o f h y d ro p h o b ic an d p o lar in t er a ct io n s in ag r e em en t with th e r esu l ts o b t ain ed in th e sy st em at ic an a ly s is [1 8 ]. Ho w ev e r, th e in t e ra ct io n d et ai ls ar e d i ff er - en t fo r th e tw o ap tam er s d u e to th e ch a in in v e rs io n o f th e mT B A . T h is ch a in in v er sio n al lo w s th e fo rm at io n o f a g rea t n u m b er o f co n ta ct s wi th th e en zy m e an d le ad to an in cr e as e in sh ap e co m p le - m en t ar ity . In ad d itio n , th e q u ad ru p l ex st ru ctu re i s ef fi ci en tly s tab i - liz ed b y a p o tas siu m io n , wh i ch is s an d w i ch ed b etw e en th e tw o q u art et s. Th e an tip ar al le l q u ad ru p l ex st ru ctu re o f th e T B A h a s a d i st in c - tiv e d en atu ra tio n - ren atu r at io n p ro fi le th at is r ev er sib le an d o b se rv - Fig. (1). A ) Fo ld in g to p o lo g y o f th e in tra m o lec u la r qu a d rup le x ad o p ted b y th e d (G 1 G 2 T 3 T 4 G 5 G 6 T 7 G 8 T 9 G 1 0 G 1 1 T 1 2 T 13 G 1 4 G 1 5 ) th ro m b in - b in d in g DN A ap ta m e r c o n ta in in g th re e e dg e -w is e lo o p s . B) Stru c tu r e o f th e G-qu a rte t with c y c lic a rra y o f fo u r g u an in e s fo rm ed b y Ho o g s te en - ty p e H-bo n ds , M in d ic a te s a me ta l io n . 98 Chemically Mo dified Thrombin Binding Aptamers Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 3 Fig. (2). S tru c tu re o f th e c o mp le x fo rm ed b y th ro m b in an d th e th ro m b in b in d in g a p ta m e r (TBA ). Th ro m b in is c o lou re d in g re en with th e a m in o a c id re s id u e s th a t c on ta c t th e TBA in re d (c a rto o n rep re s e n ta tio n ). Th e TBA is c o lo u re d in b lu e e x ce p t th e re s idu e s inv o lv ed in th e TT a nd TGT lo o p s th at a re h ig h lig h te d in y e llo w (s tic k re p re s e n ta tio n ) . ab le b y d iff er en t t ech n iq u e s, p a rt icu l a rly b y NMR ex p erim en ts , wh ich su g g est th at th e d en a tu r atio n o f th e q u ad ru p l ex o c cu r s b y th e o p en in g o f th e G-G b as e p ai rs th a t ar e n o t p ro t e ct ed b y a lo o p , fo l - lo w ed b y th e o p en in g o f th e T G T lo o p [4 2 ]. 1. CLINICAL TRIALS WITH THE THROMBIN-BINDING APTAMER Th e an ti co ag u l an t p ro p e rti e s o f T B A wer e f ir st ev alu at ed o n cy n o m o lg u s mo n k ey s, an d sh e ep s [8 ]. T h e rap id o n s et an d th e sh o r t h alf -l if e o f TB A (t 1 / 2 : 2 min ) in vivo le ad to an in t er e st fo r th e u s e o f T B A fo r c er ta in acu t e cl in i ca l se tt in g s su ch a s su rg i ca l in te rv en t io n s wh er e reg io n al an ti co ag u l atio n i s req u i red . T B A was ab le to in h ib i t clo t -b o u n d th ro m b in an d p la te le t th ro m b u s fo rm at io n in an ex v iv o wh o le- ar te ry an g io p l as ty mo d el [9 ] . Mo r eo v e r, wh en T B A wa s ad m in i st er ed b y in fu sio n in a sh o rt -t erm c an in e c ard io p u lm o n ar y b y p ass mo d e l i t wa s sh o w n th at T B A co u ld b e u s ed a s an t ico ag u - lan t sa f ely an d a s e ff ic i en tly a s h ep ar in e [4 3 ] . Clin ic al tr ia ls ev a lu - atin g T B A (A R C -1 8 3 , HD 1 , Ar ch e m ix C o rp o r atio n ) a s an t ico ag u - lan t d u rin g co ro n a ry art e ry b y p ass g ra ft su rg ery we re h a lt ed af te r p h ase I d u e to su b o p tim a l d o sin g p ro fil e s, p ri m a ri ly cau sed b y th e re st ri ct ed b in d in g a ffin ity o f th e ap t am er [4 4 ]. A mo re p o ten t s ec - o n d -g en er atio n D N A ap t am e r (N U 1 7 2 , A rch em ix C o o rp ., N u v el o In c. ) w as d ev elo p ed sh o w in g c le a r in h ib i tio n o f c lo t fo rm at io n [4 5 ]. 2. MODIFICATIONS ON THE THROMBIN-BINDING AP- TAMER In re cen t y e a rs, sev er al a tt em p t s to im p ro v e p h ar m a co lo g i ca l p ro p ert ie s o f th e T B A h av e b een d es cr ib ed , su ch a s s tab ili ty , h ig h e r th ro m b in af fin i ty , lo n g er l if e ti m e in vivo etc . T h es e mo d if ic at io n s h av e in clu d ed su b st itu t io n s in th e n u cl eo sid es [4 6 ], L N A [4 7 -4 8 ] , UN A [4 9 ], RN A [5 0 -5 1 ] o r 2 ?- O -m e th y l-R N A [5 0 , 5 2 ], meth y l - p h o sp h o n ate o r p h o sp h o ro th io ate in te rn u cl eo s id e l in k ag e s [5 0 , 5 2 ], p art ia l in v er sio n o f th e T B A p o lar ity with an d 5 ?-5 ? in t ern u cl eo sid e lin k ag e o r ch an g e in th e lo o p si z e an d s eq u en c e [2 8 ]. In so m e c as es , th e mo d i fi ca tio n s in tro d u c ed a r e ev alu at ed in d if f er en t p o si tio n s o f th e ap t am er in o rd er to in c re a se th e k n o w led g e o f th e in t e ra ct io n s b etw een th ro m b in an d th e T B A wh ich ar e c ri ti ca l fo r th e b io lo g i ca l ac tiv i ty . B e sid es th e se mo d ifi c at io n s, th ro m b in b in d in g ap t am e r h a s b een fu n ct io n al iz ed with d if f er en t d er iv a tiv e s su ch as f lu o re s ce in , b io tin o r th io l g ro u p s to b e in co rp o r at ed in b io s en so rs fo r th e d et ec - tio n o f th ro m b in . T h e s e d e riv ativ es wi ll b e d e s crib ed in se c tio n 7 . Her ein , w e h av e cl as si fi ed th e mo d if ic at io n s o f th e T B A d ep en d in g o n th e lo c atio n : G -t et rad s, lo o p s o r ch an g es o f th e o v e ra ll q u ad ru - p lex stru ctu r e. 2.1. Modifications of the Guanine Tetrad Sev er al mo d if ic at io n s h av e b e en in t ro d u c ed in th e G- te tr ad s , so m e o f wh i ch ar e an a lo g u es o f th e g u an in e b a s e. O th e r mo d i fi ca - tio n s ar e r el at ed wi th th e su g ar st ru ctu re o r with th e in te rn u cl eo tid e p h o sp h ate b o n d s in th e g u an in e t et rad . T h e g u an in e an a lo g u es th a t h av e b e en in t ro d u ced in th e T B A a r e su m m ar iz ed in F ig . ( 3 ). Hy - p o x an th in e, 7 -d e a zag u an in e an d C 8 -m e th y lg u an in e w er e th e fi rs t g u an in e an alo g u e s to b e in tro d u c ed in to th e T B A to u n d er st an d it s stru ctu re b y NMR . T h e se g u an in e d e riv a tiv es a re u n ab l e to fo r m th e h y d ro g en b o n d req u ired fo r th e fo rm at io n o f th e G- te tr ad an d co n - seq u en tly c au se sig n if i can t d i sru p tio n to th e ch a ir -l ik e s tru ctu r e [1 2 ]. He an d co -w o rk er s stu d ied th e N 2 an d C 8 -alk y l su b st itu t ed o f th e G re sid u es fo rm in g G-t et r ad s [5 3 ]. T h e s e p o sit io n s a re n o t fo rm in g th e H-b o n d in g o f th e t et rad s an d ar e av a il ab le fo r at ta ch in g o n e o r mo re g ro u p s p o in t in g aw ay fro m th e ch ai r- lik e st ru c tu re . T h is i s th e m ain re aso n wh y th e se su b s ti tu tio n s c au s ed re la tiv el y sm a ll p ertu rb at io n o n th e q u ad ru p lex st ru ctu re . Ho w ev e r, th ey c a n p ro d u ce d i ff e ren t e ff e ct s o n th e th ro m b in a ctiv ity . T h e in c re as e d ac tiv i ti es fo r th e su b s ti tu tio n s o n C 8 po sitio n s m ay b e ex p la in ed b y th e st ab i li za tio n o f syn co n fo rm a tio n o f th e G re sid u es, wh i le th e in cr e as ed a ct iv it ie s fo r th e su b sti tu tio n s o n N 2 p o sitio n s may b e d u e to th e in t er a ct io n with th ro m b in . 6-T h io g u an in e r ed u c ed th e q u ad ru p lex fo rm at io n d u e to th e in cr e as ed r ad iu s an d d e cr ea s ed e le c tro n eg ativ ity o f th e su lp h u r [5 4 ] . T h is mo d i fi ca tio n cau sed a d es tab ili z at io n o f th e Ho o g st e en h y d ro - g en b o n d in g o f g u an in e tetr ad s . Mo r eo v er , th e th io l g ro u p at p o si - tio n 6 d is ru p ted th e in te r ac tio n s w ith w at er mo l ecu l e s an d w it h ca tio n s , b e co m in g a w eak er h y d ro g en b o n d acc ep to r th an th e o x o g ro u p . 8 -A m in o g u an in e d id n o t sig n i fi can t ly alt e r th e st ru ctu re o f th e T B A q u ad ru p lex b u t i t h as a s m a ll d e st ab i li za tio n e ff ec t o n th e T B A q u ad ru p lex . A d e ta il ed s tu d y o f th i s mo d i fi ca tio n in th e G 2 p o sitio n wa s c ar ri ed o u t b y mo lecu l ar d y n am i c s sim u la tio n s, NMR , UV sp e ct ro s co p y an d c ir cu l ar d ich ro ism . T h e p r es en c e o f 8 - am in o g u an in e d id n o t a ff e ct h y d ro g en b o n d in g o r p u rin e -io n in t er - ac tio n , b u t c le a rly red u c ed th e st r en g th o f st a ck in g in t er a ctio n s [5 5 ] . Nal lag atl a et al. pr ep a red a lib r a ry o f al l p o s sib le su b s titu tio n s o f g u an in e b y iso g u an in e in th e T B A b y sp l it an d mix sy n th e si s [5 6 ] . T h e lib r a ry wa s sc re en ed fo r b in d in g to h u m an th ro m b in an d se - le ct ed seq u en c es w er e in d iv id u a lly r esy n th e si z ed an d th ei r a ff in it ie s wer e as s ay ed b y iso th erm al t it ra tio n ca lo ri m e try . T h re e mo d ifi e d ap ta m e rs c ar ry in g o n e sin g l e i so g u an in e we r e fo u n d to h av e h ig h e r b in d in g af fin i ty fo r th ro m b in th an th e u n m o d if ied T B A . T h e th er - m al s tab i li ty o f th e s e mo d i fi ed T B A s wa s n o t an aly sed al th o u g h it is p re su m ed th a t th e ef f ec t o f th e mo d if ic a tio n wi ll d ep en d o n th e p o sitio n o f th e ap ta m e r. Fin ally , th e e ff ec t o f ad d in g a th ird t et r ad o n th e T B A ap ta m e r h as al so b e en ex p lo red [2 8 ]. T h is mo d if ied th ro m b in b in d in g ap - tam er i s mo r e st ab l e th an th e n at iv e T B A d u e to th e en th a lp i c co n - trib u t io n o f th e ex t ra g u an in e t et rad . 2.2. Modification of the 2?-deoxyribose of the Guanine Tetrad Sev er al mo d i fi c atio n s in th e T B A h av e b een ap p l ied to th e su g ar mo ie ty o f th e g u an in e t et rad . T h e st ru ctu re s o f th e mo d ifi e d 2 ?-d eo x y rib o s e in co rp o ra ted in th e T B A a re su m m a ri zed in F ig . ( 4 ) . 99 4 Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 Avi?? et al. In so m e o f th em , th e 2 ?-d eo x y rib o s e mo i ety wa s rep la c ed b y a n o n ca rb o h y d rat e s tru ctu r e. On th e co n t ra ry , o th e r mo d if i ca tio n s ar e b as ed o n th e ad d itio n o f d if fe ren t g ro u p s in th e 2 ? -p o si tio n . T h es e g ro u p s may in flu en ce su g ar p u ck e rin g an d g ly co sid ic b o n d co n fo r- m at io n o f th e G -t et rad . In a v e ry in t e re st in g wo rk , S h af er ?s g ro u p [5 1 ] ex am in ed th e in flu en c e o f in d iv id u al n u c leo sid e co n fo rm at io n o n th e o v era ll fo ld - in g to p o lo g y b y se le ct iv e r ep l ac em en t o f d eo x y G b y rib o G . T h e u n im o le cu la r an t ip a ra ll el T B A is rev er sed to b im o l ecu la r p ar al le l q u ad ru p lex b y a sp ec if ic rib o n u cl eo tid es su b st itu t io n s. T h e p ar al le l q u ad ru p lex co n fo rm at io n im p li es th at a ll n u cl eo s id es ar e in th e anti co n fo rm at io n . T h e s tro n g p r ef er en c e o f g u an in e rib o n u cl eo s id e s fo r th e anti co n fo rm atio n is th e d r iv in g fo r c e fo r th e ch an g e in to p o lo g y an d a lso im p a ct in q u ad ru p lex mo le cu l ar ity . T h e d en a tu r atio n b eh av io u r o f th e T B A d e riv at iv e s c ar ry in g rib o n u cl eo tid es w a s a lso d es cr ib ed b y M erg n y ? s g ro u p [5 0 ]. T h e au th o rs p rep ar ed a T B A an alo g u e wi th a ll th e G o f th e tw o t et rad s rep l a ced b y rib o G . In th i s c as e, th e mo d i fi ed T B A p re s en ted a co m - p lex b eh av io u r w ith a n o n -su p eri m p o sab l e an d mu lti -p h as ic re - sp o n se u p o n h eatin g an d co o lin g (h y s te re si s ). T h e s am e au th o r s al so d es c rib ed th e s am e T B A an alo g u e s wi th 2 ?-O -M e g u an o sin e su b st itu t io n s. T h i s an a lo g u e, in st ead , sh o w ed r ev e rs ib le tr an s it io n s with co n cen t r atio n in d ep en d en t o f th e m el tin g t em p e ra tu r e (T m ). I n fa ct , su b sti tu tio n o f th e r ib o se 2 ? -H wi th a m eth o x y g ro u p d es tab i - liz ed th e q u ad ru p lex st ru c tu r e. LN A are 2 ? -O -4 ? -C - m e th y len e-l in k ed rib o n u cl eo tid e n u cl ei c ac id s an a lo g u es th a t b in d w i th in c r ea sed a ffin ity to D N A an d R N A . Th e b icy c li c st ru c tu r e o f DN A fo rc e s th e su g ar to b e in th e C3 ? - endo co n fo rm a tio n , an d n u cleo t id e s with a C 3 ?-endo co n fo rm a tio n Fig. (3). Ch e m ic a l stru c tu re o f th e mo d ifie d g u a n in e s in th e TBA te tra d s . Fig. (4). Ch e m ic a l stru c tu re o f th e mo d ifie d c a rb o h yd ra te mo ie ty in th e g u an in e TBA te tra d s . LN A : lo c k e d n u c le ic a c id , UN A : u n lo c ke d n u c le ic a c id . NH NN N S NH 2 NH NN N NH 2 O NH NN N O NH NN H C O NH 2 NH NN N O NH 2 H 2 N NH NN N O NH 2 R R= CH 3CC CH 3 PhCC NH NN N O NHR R= CH 2 -Ph CH 2 -CH 2 -Ph CH 2 -Ch 2 - CH 2 -Ph CH 2 - ( 1-Nap h) CH 2 - (2-Na ph) CH 2 - (4'-Bip h) CH 2 - (3'-Bip h) CH 2 - (1-Ada m)Hyp oxa nt hin e 9 -de az ag ua nin e N 2 -al kyl gua ni ne C 8 -al kyl gua ni ne 6-th iogua nin e 8 -amin ogu ani ne iso gua ni ne O O O O O O O O O O O O O O O O O O O OH OCH 3 OH O F G G G G G G G H Rib o 2 '- O- me t hy l LN A 2 '- F- ar aN North-m e tha no car ba nu c le o side South- me t ha no c arba nu c l eo si de U N A 100 Chemically Mo dified Thrombin Binding Aptamers Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 5 p ref e r th e g ly co sid i c b o n d to b e in th e anti co n f ig u ra tio n . T h re e d iff er en t wo rk s w e re ad d r es s ed to stu d y th e ef fe ct o f L N A in th e T B A q u ad ru p lex [4 7 -4 8 , 5 7 ]. May o l?s g ro u p p rep a red fo u r d if f er en t T B A -b as ed o lig o n u cl eo tid es co n ta in in g L N A re sid u e s [4 7 , 5 7 ]. T h e fir st an a lo g u e was fu lly su b s titu ted b y LN A res id u es . T h is o lig o n u - cl eo tid e wa s u n stru c tu r ed mo st p ro b ab ly d u e to th e d ec re a sed f lex i - b ility o f th e o l ig o m er . Oli g o n u cl eo t id e s co n ta in in g G-L N A in th e eig h t p o si tio n s o f th e te tr ad s o r in th e fi r st G 1 p o sit io n ( syn co n f ig u - ra tio n ) g av e m ix tu r es o f sev er al s tru ctu r es . On th e o th er h an d , th e o lig o n u cleo t id e co n t ain in g G -L N A in th e l a st g u an in e G 1 5 ( anti co n fig u r at io n ) fo ld ed in th e sam e T B A ch ai r- lik e q u ad ru p lex . Bo n i - fa cio et al. also s tu d ied th e e ff ec t o f s in g le L N A su b st itu t io n s o n d iff er en t p o sit io n s o f th e T B A [4 8 ]. T h e L N A su b s ti tu tio n s h a d eith er a mo d e ra te s tab ili zin g o r d e st ab i li zin g ef fe ct o n th e fo ld e d stru ctu re , d ep en d in g o n th e p o sitio n o f th e L N A in th e T B A . T h e th erm a l st ab il ity o f th e su b st itu t ed ap t am er s d id n o t co rr el at e t o th ro m b in in h ib i tio n . Dam h a an d co -w o rk er s s tu d ied th e im p ac t o f 2 ?-d eo x y -2 - flu o ro a rab in o n u cleo sid e r es id u e s (2 ?-F - a raN ) o n th e th ro m b in b in d - in g ap ta m e r [5 8 ]. 2 ?-D eo x y -2 ?- flu o ro - D - ar ab in o n u cl ei c a cid s (2 ?F - ar aN ) co n f er DN A -l ik e ( South / East ) co n fo rm atio n to o l ig o n u cl eo - tid e s wh il e r en d er in g th em mo r e n u cl ea s e r es is tan t . It w a s fo u n d th at in co rp o r atio n o f 2 ?-F -a ra N G o r T res id u es in to th e T B A s tab i - liz e s th e co m p l ex (  T m + 3 ?C /2 ?-F - ar aN mo d if ic at io n ). O lig o n u - cl eo tid es with al l n u c leo t id e s r ep l a ced b y 2 ? -F -a r aN in th e G - syn po sitio n s o r in th e G -t et rad s sh o w ed a mo d er a ted in c re as e o f th e mel tin g t em p er atu r e co m p a r ed to th e u n m o d ifi ed T B A . T h e C D sp e ct ru m an d th e h y st er e si s o b se rv ed in th e h ea tin g an d co o lin g p ro ce ss e s o f th e se an alo g u es su p p o rt ed a p ar al l el st ru ctu re with al l anti-d G an d th e ex i st en c e o f mu ltim er ic G-q u ad ru p lex s tru ctu r es . On th e co n tra ry , wh en th e 2 ?-F -a ra N ar e rep la c ed in th e G- anti po sitio n s , in th e lo o p s o r in b o th th e re su l ted q u ad ru p l ex st ru ctu re s co rr e sp o n d to an tip ar al le l q u ad ru p l ex with al te rn a tin g syn - anti Gs . T h e la ck o f co n cen t r atio n d ep en d en c e in th e T m d at a an d th e la c k o f h y ster e si s in th e h e atin g / co o lin g p ro c es se s su p p o rt a u n im o l e cu - la r G-q u ad ru p l ex stru ctu r e. Mo reo v e r, n u cl e as e r es is tan ce o f th i s mo d ifi ed T B A w a s in cr e as ed u p to 4 8 -fo ld in 1 0 % f et al b o v in e se ru m (F B S ). Carb acy cli c b i cy clo [3 .1 .0 ] h ex an e lo ck ed n u cl eo s id e an alo g u e s ar e a d i ff er en t ? lo ck ed ? n u cl eo s id e f ro m th e p r ev io u s ly p re sen t e d L N A [5 2 ]. An ad v an tag e o f th is m eth an o ca rb a n u cl eo s id e sy st e m o v er L N A s i s th at b o th North (N)- an d South (S)-lo ck ed p lat fo rm s can b e p r ep ar ed b y sh ift in g th e p o sit io n o f th e fu sed cy clo p ro p an e rin g . I t h a s b een d es cr ib ed th e ef fe c ts o f r ep l ac in g a sin g l e 2 ? - d eo x y g u an o sin e re sid u e at th e 3 ?- en d o f th e T B A (p o s itio n s d G 1 4 an d d G 1 5 ) with meth an o ca rb a n u cl eo s id e s lo ck ed in ei th e r th e N - o r S -co n fo rm at io n [5 9 ]. T h es e p o si tio n s w e re s el ec ted to ex p lo r e th e co m b in ed e ff ec ts o f a co n st r ain ed su g a r p u ck e r ( N or S ) an d th e co rr e sp o n d in g b ias ed g ly co sy l to r sio n an g l e ( anti o r syn ) a s so ci at e d with a p ar ti cu l ar p s eu d o su g ar co n fo rm at io n . E x p erim en t al an d th eo r et ic al r e su lt s in d i ca ted th at a N -p s eu d o su g ar co n fo rm a tio n fav o u rs th e anti g ly co sy l o ri en t at io n , wh e r ea s th e S -p seu d o su g a r co n fo rm at io n fav o u r s th e syn di sp o sit io n o f th e b a se . T h e in tro d u c - tio n o f me th an o c arb a n u cl eo s id e s a t p o si tio n s G 1 4 an d G 1 5 with lo ck ed -N ( anti ) an d lo ck ed -S ( syn ) co n fo rm at io n s fix ed th e co n fo r - m at io n al s ta te o f th e se n u cl eo s id e s an d h elp ed to u n d e rs tan d th e im p a ct o f co n fo rm atio n al r e str i ctio n s o n th e an t ip ar al l el, G -q u ar te t DN A stru ctu r e o f th e TB A . Th e se r e su lt s in d ic a ted th a t th e g ly co sy l co n fo rm at io n is mo r e re st ri ct iv e fo r th e T B A st ab il ity th an th e su g ar p u ck e rin g . Wen g e l? s g ro u p ex am in ed th e in flu en c e o f u n lo ck ed n u cl ei c ac id (U N A ) o n th e th e rm o d y n am i c s tab ili ty , b in d in g a ffin ity an d b io lo g ic al ac tiv i ty o f th e q u ad ru p l ex TB A [4 9 ] . UN A i s an a cy c li c R N A mim i c, wh ich m is s es th e b o u n d b etw een th e C 2 ? an d C 3 ? ato m s o f th e r ib o se rin g . T h e mo d if ied v ari an t s ar e ap tam er s sin g l y su b st itu t ed w ith a UN A m o n o m e r in ev ery p o s sib l e p o sit io n . U N A mo d ifi ed T B A s in p o sit io n s U 3 , U 7 an d U 1 2 sh o w ed an an tip ar al le l fo ld in g to p o lo g y . In co n tr a st, mo d ifi c atio n s o f an y o f th e g u an in e mo n o m ers fo r m in g G- t etr ad s r e su lt ed in sig n i fi c an t d es tab i li za tio n o f th e q u ad ru p lex s tru ctu r e. Th e mo d i fi ed TB A with UN A in p o si- tio n 7 re su lt ed in th e h ig h e st th ro m b in b in d in g af fin i ty . R ec en t ly , th e sam e au th o rs h av e a lso ev a lu at ed th e e ff e ct s o f th e mo d i fi ca tio n o f 2 ?-C -p ip e ra zin o -U N A m o n o m er [ 6 0 ]. T h i s m o n o m er is ch ar ac - te ri zed b y mo re e ffi c ien t st ab il iz at io n o f q u ad ru p lex e s st ru c tu re s i n co m p ar iso n to reg u l ar U N A an d in cr e as es th e rm o d y n am i c st ab il it y o f T B A b y 0 .2 8 -0 .4 4 k c al /m o l in a p o si tio n d ep en d in g man n e r w it h re ta in ed q u ad ru p l ex to p o lo g y an d mo le cu l ar ity . 2.3. Modifications in the Internucleotide Phosphates of the Guanosine Tetrad Th e T B A h as b een mo d i fi ed wi th th r ee d if fe ren t p h o sp h at e lin k er s, p h o sp h o ro th io at e , m eth y lp h o sp h o n at e an d fo rm a ce ta l. T h e stru ctu re s o f th e se l in k ag e s a re su m m a ri zed in F ig . ( 5 ) . T h e mo d i- fi ed T B A o lig o n u cleo tid es co n t ain in g th io p h o sp h o ry l su b s titu t io n s at d i ff er en t in t e rn u cl eo tid e s it es w er e s tu d ied . It w as fo u n d th at th es e l in k ag e s d o n o t d isru p t th e an tip ar al le l n tr am o l ecu l a r q u ad ru - p lex [5 2 ]. T h e su b st itu t io n s p la ced b e tw e en p lan e s o f G-q u ar te ts l e d to a d ro p in fo rm a tio n fr e e en erg y , an d th e st ab il ity d e cr ea s es lin e - ar ly with th e n u m b er o f th es e mo d if ic at io n s. T h e T B A co n t ain in g p h o sp h o ro th io ate l in k ag e s h av e mo r e r es is t an c e to v ario u s n u c le - as es . In th i s way , th e in vivo hal f- li fe o f th e mo d if ied T B A s ar e in cr e as ed . M erg n y ? s g ro u p al so stu d i ed th e in tro d u c tio n o f p h o s- p h o ro th io at e b o n d s in all o f G- fo rm in g t et rad s [5 0 ]. T h e r esu l tin g mo d ifi ed T B A was l es s s tab l e th an th e u n m o d ifi ed T B A . It a lso h a d an in tr am o l ecu la r G-q u ad ru p l ex s tru c tu r e with co n cen tr atio n in d e - p en d en t me lt in g tem p er atu r e s sh o w in g a rev er sib l e q u ad ru p l ex e s t o ran d o m co i l t r an si tio n s . T h e sa m e g ro u p stu d ied th e mo d i fi ed T B A ca rry in g b a ck b o n e me th y lp h o sp h o n ate in th e tw o G- te tr ad s [5 0 ] . T h e me th y lp h o sp h o n ate T B A v ari an t su f fe red a lo ss o f n eg ativ e ch a rg e a t th e l ev e l o f th e p h o sp h at e b ack b o n e th a t l ed to a st ro n g d est ab i li za tio n . No n o b se rv ab l e m el tin g tr an s it io n wa s d et e ct ed . T h e n eg a tiv e ch arg e o f th e o x y g en ato m s in th e p h o sp h a te g ro u p s was fo u n d to b e in v o lv ed in a co m p lex p att e rn o f w at er b rid g e s with th e su g a r g ro u p an d th e ed g e s o f th e g u an in e u n its . A s er i es o f T B A an a lo g u es we r e sy n th es iz ed co n ta in in g o n e o r mo r e p h o s - p h o d iest er lin k ag es r ep l ac ed b y a n atu r al fo r m a c et al g ro u p [6 1 ] . T h e fo rm a ce ta l g ro u p is a ch ir al an d th e in co rp o r at io n o f th es e mo iet ie s in to o lig o n u cl eo tid es d e cr ea s ed th e ti ssu e u p t ak e an d in - cr ea s ed th e in vivo hal f-l if e. Un fo rtu n a te ly , n o stru c tu r al s tu d ie s wer e c arr i ed o u t wi th th es e T B A an alo g u e s. Fig. (5). Ch e m ic a l st ru c tu re o f th e mo d ifie d in te rn u c leo tid e p h o sp h a te b o n d s in th e g ua n in e TBA te tra d s . In su m m a ry , sev er al G -t etr ad mo d ifi c at io n s o f th e T B A h av e b een stu d i ed . S o m e o f th em h av e d e st ab i li zed o r d i sru p t ed th e q u ad ru p lex st ru c tu r e b ec au s e th e in t ro d u ced mo d if ic at io n h av e ch an g ed d i re ct ly th e H -b o n d in g tet rad ar r an g em en t, su g ar p u ck er - in g o r g ly co sid ic g u an in e o ri en t at io n . In ad d i tio n , so m e mo d i fi ca - tio n s h av e d e st ab il i zed th e an tip a r all e l q u ad ru p lex to fo rm a mo r e u n d efin ed mu lti m e r q u ad ru p lex st ru ctu re s. O CH 2 O O PS O O - O PO O CH 3 P ho s ph o ro t hi oa t e Meth ylp ho s ph o na t e Forma c e ta l 101 6 Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 Avi?? et al. 3. MODIFICATION OF THE LOOPS Th e T B A i s fo r m ed b y tw o g u an in e t et r ad s co n n ec ted b y th r e e ed g e-w is e- lo o p s: a c en tr al T G T lo o p an d tw o T T lo o p s as it i s sh o w n in th e F ig . ( 1A ) . T h e lo o p s a re im p o rt an t in th e fo ld in g o f th e ap t am er an d in th e in te r ac tio n s wi th th e th ro m b in . Mo st o f th e mo d ifi ca tio n s ar e b a sed in ch an g es o f th e co m p o s it io n o r in th e len g th o f th e lo o p s . T h y m in e b a se s in th e lo o p r eg io n ex clu siv el y p ref e r th e anti o r ien t at io n . T h e l it er atu re r ep o rt s th a t G 8 sh o w s b as e st ack in g in t er ac tio n s wi th th e f ir st t et rad , b u t th e co n fo rm at io n o f th e n u c leo sid e i s n o t men tio n ed . 3.1. Modifications of the Loop Composition by Natural Nucleo- sides Sh afe r? s g ro u p h a s u n d e rt ak en a sy st em at ic ex am in atio n o f th e th erm o d y n am ic st ab il ity o f th ro m b in ap t am er an alo g u e s co n t ain in g seq u en ce mo d i fi c atio n s in o n e o r mo re lo o p s [2 8 ]. T h e re su l ts in d i - ca ted th a t ch an g es in lo o p seq u en ce s h ad a sig n i fi c an t im p a ct o n th e ap ta m e r st ab i lity . Mo st o f th e ch an g e s in th e c en tr al l o o p led to a d ec re as e in th erm o d y n am ic st ab il ity , in d i c at in g th at, at le as t a m o n g th e s eq u en c es ex p lo red , th e T G T lo o p seq u en ce i s o p tim a l fo r s ta - b ility . T h es e ef fe ct s may i n v o lv e ch an g es in b o th sta ck in g in t er ac - tio n s an d ca tio n b in d in g . T h e im p a ct o f r ep la cin g sin g l e T s in th e ex te rn a l lo o p s with C s p ro v id e ev id en c e fo r h y d ro g en b o n d fo rm a- tio n b etw e en th es e lo o p s , as o b s e rv ed in th e NMR st ru ctu re s. T h e st ab il ity o f ap tam e rs co n t ain in g a C in p o s itio n 3 o r 1 2 w as s im i la r o r s lig h tly h ig h er th an th e u n m o d if ied T B A . A re cen t p ap er h a s ex am in ed th e s tab ili ty o f th e G -q u ad ru p l ex o f TB A in wh i c h th y m in e r es id u es w er e su b s titu ted b y ad en in e . T h e G-q u ad ru p l ex e s fo rm ed b y T 4 A an d T 1 3 A were mo r e s tab le an d T 3 A , T 7 A , T 9 A an d T 1 2 A were mo r e u n st ab l e th an th at o f th e wi ld -ty p e [6 2 ] . 3.2. Modifications of the Loop Composition by Non-natural Nucleosides Rep la c em en t o f anti th y m in e s in th e lo o p s with anti co n fo r m a - tio n al ly b ia sed 2 ? -F - ar aT in c r ea sed th e th e rm al st ab il ity to d if f er en t d eg re es d ep en d in g o n th e n u m b e r an d p o si tio n o f th e mo d if ic a tio n . In ad d it io n , th e mo d i fi ca tio n o f G 8 w ith 2 ? -F - ar aG re su lt ed in a n in cr e as e o f th e s tab ili ty . Ov er al l, 2 ? -F - ar aN mo d ifi c at io n s in th e lo o p s s tab ili z es th e fo rm a tio n o f a u n im o l e cu la r G -q u ad ru p l ex [5 8 ] . L N A su b sti tu tio n s in th e lo o p s d em o n s tr at e a p o s itio n d ep en d en t ef fe ct o n th e st ab il ity o f th e T B A . T h e su b sti tu tio n o f G 8 fo r G - L N A in cr e as ed th e st ab i li ty , b u t th e su b s titu tio n o f T 7 d ec re a sed th e st ab il ity . Nev er th el e ss , su b st itu t io n o f T 4 d i sru p t ed th e ap t am e r [4 8 ]. On th e o th er h an d , sin g l e UN A mo d if ic at io n o f th e T B A i n U 3 , U 7 an d U 1 2 p o sit io n s d id n o t a ff ec t th e st ab i lity o f th e u n i - m o le cu la r an t ip a ra ll el s tru ctu r e. Ho w ev er , th e se sam e mo d if ic at io n s in p o sitio n s U 4 , G 8 , U 9 an d U 13 resu lt ed in sig n if i can t d e st ab i li za - tio n o f th e q u ad ru p lex s tru c tu r e [4 9 ]. Bo rb o n e?s g ro u p mo d ifi ed th e d if fe ren t p o si tio n s o f th e T B A with f l ex ib le a cy c li c th y m id in e s [6 3 ]. T h ey o b ta in ed th e sa m e p at - te rn o f th e rm o d y n am i c s tab i li ti es th an U N A m o d if ic at io n s. T h es e an alo g u e s w er e ab l e to fo ld in to a b im o l ecu la r o r mo n o m o le cu la r q u ad ru p lex s tru ctu r e d ep en d in g o n th e n a tu re o f th e mo n o v al en t ca tio n s (so d iu m o r p o t a ss iu m ) co o rd in a ted in th e q u ad ru p lex co re . T h erm al st ab i lity w a s in ag r e em en t wi th th e s tru ctu r al mo d el i n wh ich T 9 , T 4 an d T 13 are s ta ck ed o n th e ad ja cen t G-q u a rt et. T h es e in te ra ct io n s we re to ta lly o r p ar ti al ly d isru p t ed b y th e in tro d u ctio n o f th e a cy c li c n u cl eo t id e at th e se p o s it io n s. T h e T B A an a lo g u es co n - tain in g an acy cl ic r es id u e at p o sit io n s T 3 , T 7 o r T 1 2 resu lt ed in a sim il ar st ab il ity th an th e o b s erv ed fo r th e u n m o d if ied T B A , th u s su g g est in g a ma rg in a l ro le o f th es e p o s it io n s o n th e s tru ctu r al s ta - b ility [6 3 ]. Mo d ifi ca tio n s o f th e T B A lo o p s b y th io p h o sp h o ry l in tern u c leo - tid e b o n d s we re ev alu at ed [5 2 ] . No d es tab i li z atio n w a s o b s erv ed i n ea ch o f th e lo o p r eg io n s, al th o u g h th e st ab i lity ag a in st n u cl ea se wa s in cr e as ed in co m p a ri so n to th a t o f th e n at iv e T B A . F in a lly , a n e w T B A ap tam er mo d i fi ed with 4 -th io -2 ?-d eo x y u rid in e s rep la cin g so m e T s in th e lo o p s wa s d es c rib ed [6 4 ] . T h is su b s ti tu tio n wa s b as ed o n p rev io u s ex p er im en ts sh o w in g th at o lig o n u cl eo tid es w it h 4 -th io -2 ? -d eo x y u rid in es sh o w ed h ig h -a f fin ity b in d in g to HI V - 1 rev er se t ran sc rip t a se [6 5 ]. No th erm o d y n am ic d a ta w er e p e rfo rm e d b u t T B A mo d ifi ed w ith 4 - th io -2 ? -d eo x y u rid in es h as an in c re as e d an ti co ag u l an t an d an tith ro m b o ti c p ro p e rt ie s [6 4 ]. 3.3. Modification of the Loop Length Lo o p len g th p lay s an im p o rtan t ro le in in t r am o l ecu l ar q u ad ru - p lex fo rm a tio n . Wh en th e c en tr al lo o p was rep la c ed b y fo u r n u cl eo - tid e s, th e r esu l tin g ap tam e r h ad a lo w er st ab i li ty co m p a r ed to u n - m o d ifi ed T B A [2 8 ]. T h e th e rm o d y n am i c an aly s is in d ic at ed th at th e cen t r al lo o p s eq u en c e in th e p ar en t ap t am er i s o p t im a l fo r s tab i li ty . R ed u ct io n o f th e tw o ex te rn al T T lo o p s to a sin g l e T led to a co m - p let e d i sru p t io n o f th e q u ad ru p lex st ru c tu r e. T h i s w as ex p e ct ed d u e to th e d iff icu lty o f fo rm in g a s in g le b a s e lo o p . On th e co n tra ry , ex ten sio n to T T T lo o p s h ad th e s am e st ab il ity as th e u n m o d ifi e d T B A . Ad d it io n o f a sin g l e G a t th e 5 ?- en d d e cr e as ed th e st ab i lity o f th e ap t am e r wh il e ad d itio n o f a G at th e 3 ? -en d in cr e as ed th e st ab il - ity [2 8 ] . 4. SYNTHESIS OF DIFFERENT CONSTRUCTS BASED ON THE TBA In an e ffo r t to s el e ct mo r e p o ten t an d s el e ctiv e DN A l ig an d s to th ro m b in , s ev e ra l au th o r s h av e sy n th es i zed d if fe r en t co n s tru c ts . Mo st o f th em h av e mo d ifi ed th e T B A s tru ctu r e it se lf o r o th er s h av e in co rp o r at ed ad d i tio n a l s tru ctu r e s o r mo le cu l es to th e T B A . T h e fir st ap p ro ach w as co m p r is ed o f an u n im o l ecu l a r q u ad ru p l ex mo ti f an d co m p l em en ta ry fl an k in g s eq u en c es c ap ab l e o f fo rm in g an ad d i - tio n al Wa t so n -C ri ck d u p lex mo t if [6 6 ]. A ft er th a t, fo llo w in g th e sam e ap p ro ach , a n ew 2 9 n u cleo t id e s in g le s tr an d ed o lig o n u cl eo tid e b as ed o n a q u ad ru p lex /d u p l ex s tru ctu r e w as d e sc rib ed to b in d th e h ep ar in -b in d in g ex o s it e o f th ro m b in [6 7 ]. S ee la ?s g ro u p p ro p o sed a n ew co n s tru ct ar is in g b y rep l ac em en t o f th e T G T lo o p o f th e T B A b y a min i-h a irp in 5 ? -G C G A A G C -3 ?. T h i s fu s ed o lig o n u cl eo tid e ex h ib it ed a tw o -p h as e th e rm al tr an s it io n in d ic at in g th e p r es en c e o f th e tw o u n al te r ed mo i eti e s [6 8 ]. A n ew in t er es tin g ar ch i te ctu re d em o n s tr at ed th at th e co m b in a - tio n o f b iv alen t T B A ap t am er s, wh i ch sim u l tan eo u sly ta rg e ted an d ac co rd in g ly in h ib i ted th e reg u l ato ry ex o si te s I an d I I o f th ro m b i n [6 9 ]. T h i s ap p ro ach tu rn ed o u t to b e a co m b in a tio n o f f e atu r e s o f th e in d iv id u al ap t am e rs in o n e mo lecu l e: h ig h a ffin ity b in d in g an d an ti co ag u l an t a ctiv ity . A n ew q u ad ru p l ex s tru ctu r e w a s stu d i ed i n th e d ( G 2 T 4 G 2 C A G 2 G T 4 G 2 T ) seq u en c e, wh i ch d i ff er s fro m th e T B A in h av in g lo n g er fi rs t (T 4 ) an d th ird (G T 4 ) lo o p an d a sh o r te r (C A ) mid d le lo o p . T h is o l ig o n u cl eo tid e h as d i ff er en t st ran d d ir e ctio n a li - tie s, lo o p co n n ect iv it ie s an d syn / anti G- te tr ad d is trib u t io n [7 0 ]. Cir cu la ri z atio n i s an a tt ra ct iv e al te rn a tiv e to ch em i c al mo d i fi ca tio n fo r im p ro v in g ap tam e r st ab il ity . T h is n ew ap p ro ach wa s u sed in th e d esig n an d co n st ru c tio n o f a T B A ap tam er . T h e n ew co n stru ct h a s in cr e as ed ta rg e t b in d in g affin ity an d mu ch im p ro v ed st ab il ity i n b io lo g ic al flu id s [7 1 ]. May o l an d co -w o rk er s d e s crib ed a n ew to p o lo g y o f th e T B A th at co n si st s o f a s er ie s o f o lig o n u cl eo t id e s co n ta in in g 3 ?-3 ? o r 5 ? -5 ? in v er sio n o f p o la ri ty si t es [7 2 ]. T h e o l ig o n u cl eo tid e d (3 ? -G G T -5 ? - 5 ?-T G G T G T G G T T G G -3 ? ) wa s ch a r ac te ri zed b y an u n u su al fo ld - in g , th re e str an d s p a r all el to e ach o th er an d o n ly o n e str an d o rien t e d in an o p p o si te m an n er . T h i s led to an anti-anti-anti-syn an d syn- syn-syn-anti arr an g em en t o f th e G s in th e tw o t etr ad s . T h e th e rm a l st ab il ity o f th e mo d i fi ed o l ig o n u cleo tid e w as h ig h er th an th e co r re - sp o n d in g fo r th e u n m o d if ied T B A . S ev er al in t er c al at in g ag en ts h av e b een co n ju g a ted to th e 3 ? -en d o f th e T B A an d th ey h av e b een fo u n d to stab i li z e th e ap t am er . Mo reo v e r, th e h y d ro p h o b icity an d flu o r e sc en t p ro p er ti es may b e u sed to en h an c e th e b io av ai l ab il ity o f th es e co n ju g at e s [7 3 ] . F in al ly , th e c ap p in g o f th e 3 ?- en d o f th e T B A with b rid g ed n u cl eo s id e s wa s d es cr ib ed . T h e b r id g ed n u cl eo s id e s in c re a sed th e n u cl ea s e r es is - 102 Chemically Mo dified Thrombin Binding Aptamers Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 7 tan c e 3 6 -2 7 fo ld an d th e st ab i lity in s eru m 1 .5 -4 fo ld wi th o u t af fe ct - in g th e b in d in g a ffin iti e s o f th e ap tam e rs to th ro m b in [7 4 ] . 5. BINDING ACTIVITY OF MODIFIED THROMBIN BIND- ING APTAMERS Th e r ep o rt ed b ib l io g rap h y co n ce rn in g th e mo d i fi ed T B A s mig h t g iv e an in sig h t in to th e v ar i ab le s in v o lv ed in th e mo d e o f ac tio n o f th e T B A . Nev e rth el es s, th e mo d e o f ac tio n o f th e T B A s ac tu a lly r eq u ir es a mo r e wid e r eco g n it io n p ro c es s th a t in v o lv e s ev en lo c a lly a sin g l e r es id u e. S ev e ra l a ss ay s ar e d e s crib ed to s tu d y th e b in d in g o r in t er a ct io n o f th e mo d i fi ed T B A s to th ro m b in su ch a s n itro c e llu lo s e fi lt er b in d in g as s ay [5 8 ], i so th e rm a l t ri ta tio n ca lo - rim et ry [5 3 , 5 6 , 6 2 , 7 5 ], su rfa c e p la sm o n re so n an c e [4 9 ] o r b y n on- eq u ilib riu m cap i ll ary el e ctr o p h o r es is o f eq u i lib r iu m mix tu re s (N E - C E E M ) [7 4 ]. R e cen tly , th e in t e ra ct io n o f th e T B A w ith th ro m b i n was ev a lu a ted b y d iff e ren t ia l p u ls e v o lt am m et ry a t a g l as sy c a rb o n el ec tro d e an d a to m ic fo r ce mi cro s co p y at a h ig h ly o ri en t ed p y ro - ly ti c g r ap h it e el e ctro d e [7 6 ] . Th ro m b in b in d in g affin ity o f th e mo d if ied T B A s in th e t et rad s was stu d i ed b y d if fe r en t r e se ar ch e rs . T h e th ro m b in b in d in g o f a T B A co n tain in g 2 ?-d eo x y in o sin e o r 7 -d e az a-2 ?-d eo x y g u an o sin e was sig n i fi can tly d e cr e as ed a s th e r es id u es w er e u n ab l e to fo rm th e h y d ro g en b o n d s req u ired fo r th e fo rm atio n o f th e G -t et rad [7 7 ]. O n th e co n t ra ry , th e T B A co n ta in in g iso g u an in e sh o w ed an en h an c e d b in d in g activ i ty to h u m an -th ro m b in co m p a r ed to th e u n m o d ifie d T B A d ete rm in ed b y iso th erm al tit ra tio n c alo r im et ry [5 6 ]. T h e e ff ec t o f 2 ?-F -a ra N mo d i fi ca tio n s wa s co n d u ct ed u s in g n it ro c ellu lo s e f il te r b in d in g ass ay . T h e b in d in g o f 2 ?-F -ar aN ap tam er s to th ro m b in wa s alw ay s ad v er se ly af fe ct ed wh en th e mo d if ic at io n was o n G tet rad s th em se lv e s. S o m e lo o p mo d ifi c atio n s wi th 2 ? -F - ar aN al so red u c e d th ro m b in b in d in g . Ho w ev e r, th e tw o lo o p mo d ifi ed ap ta m e rs i n p o sitio n s 7 , 9 , 1 2 , 1 3 o r 3 , 4 , 7 , 9 sh o w ed a 4 -5 fo ld en h an cem en t in th ro m b in b in d in g af fin ity [5 8 ]. On th e o th er h an d , re al -t im e me asu rem en t s o f th e in t er ac tio n b etw een th ro m b in an d th e TB A co n tain in g UN A mo d if ic at io n s wer e p e rfo rm ed b y su rf a ce p la sm o n r e so n an c e. T h e mo d if ic at io n o f th e G 1 p o sitio n sh o w ed sim il ar af fin i ti es to th e u n m o d ifi ed T B A . T h e re st o f th e Gs in v o lv ed in th e t etr ad s sh o w ed a h ig h er d is so c ia - tio n co n st an t o r we r e n o t mea su r ab l e, p r esu m ab ly d u e to a la ck o f sig n if ic an t af fin i ty to w ard s th ro m b in af te r th e in co rp o r atio n o f UN A in th es e p o si tio n s . Th e U 7 UN A mo d ific at io n lo ca t ed in th e cen t r al lo o p w as th e o n ly U N A m o d ified ap t am er th at sh o w ed sm al l b u t sig n ifi c an t im p ro v e m en t in af fin i ty [4 9 ]. S im i la r r esu l ts w er e o b tain ed w ith 2 ? -C -p ip er a zin o -U N A -U mo n o m e r, b u t in th i s c as e th e p r es en c e o f a p o si tiv e ly ch arg e d e cr e as ed th e th ro m b in a ffin it y [6 0 ]. An o th er in t er e stin g wo rk h as fo cu s ed o n th e r ep la c em en t o f th y m in e lo o p re sid u e s b y ad en in e . I so th e rm a l ti tr at io n ca lo ri m e tr y (IT C ) me asu rem en t s in d i c at ed th a t th e b in d in g co n st an t o f th e in - te ra ct io n s b etw e en T 1 3 A , T 7 A , T 9 A an d T 12 A ap tam e rs an d th ro m - b in was clo se to th a t o f th e u n m o d ifi ed T B A , wh e re a s T 1 3 A was sig n if ic an t ly lo w er an d T 4 A d id n o t ap p e ar to b in d th ro m b in [6 2 ] . T h e b in d in g en erg y o f th e mo d ified T B A co n t ain in g a 5 ?-5 ? -s it e o f p o lari ty in v e rs io n to th ro m b in w as ch a r ac te ri zed b y m ean s o f IT C . T h e eq u i lib r iu m co n s t an t fo r th e in te ra ct io n o f th e mo d i fi ed T B A was ab o u t o n e o rd e r o f m ag n itu d e h ig h er th an th a t fo r th e T B A . T h e b in d in g p ro c es s w as en th alp i c al ly d riv en wi th a l arg e r fav o r - ab le en th alp y fo r th e mo d i fi ed ap ta m e r [7 5 ]. T h e co n s tru ct fo rm e d b y a q u ad ru p lex co re o f th e T B A an d a d u p lex in te r ac ted wi th a 2 0 to 5 0 ? fo ld h ig h er af fin i ty to th e h ep a rin -b in d in g ex o si te th an th e u n m o d ified T B A b y n itro cel lu lo s e fi lt er [6 7 ] . F in al ly , th ro m b i n b in d in g aff in it ie s o f c ap p ed T B A s w ith 2 ? ,4 ?-b rid g ed n u c leo t id e s wer e me asu red u sin g n o n - eq u ilib riu m c ap il la ry el ec tr o p h o re si s o f eq u ilib riu m mix tu re s (N E C E E M ) [7 4 ]. T h e b in d in g ab ili ti es w er e alm o st th e s am e l ev e l th an th e n at iv e T B A . Acco rd in g to th e d i ff e ren t st r at eg ie s u s ed to m ea su r e th e th ro m b in b in d in g , we c an co n clu d e th at n o im p o rt an t b in d in g ch an g e s ar e o b s erv ed wh en th e mo d if ic a tio n d o es n o t d is ru p t th e q u ad ru p lex st ru c tu r e. In ad d it io n , so m e mo d ifi c atio n s in th e lo o p s o r in th e o v er al l co n s tru ct stru ctu r e d o n o t af fe ct o r in c re a se th e b in d in g af fin i ty . 6. THROMBIN INHIBITION BY MODIFIED THROMBIN BINDING APTAMERS Pro th ro m b in T im e (P T ) is th e mo r e u sed as s ay to stu d y th e in h ib itio n o f th ro m b in . P T as say i s a ro u tin e d iag n o st ic a s say th a t ev alu at es in vitro th e ac tiv atio n o f ex t rin si c p ath w ay o f th e co ag u la - tio n c a sc ad e . T h i s u l tim at e ly m ea su r es th e co n v er sio n o f f ib rin o g e n in fib r in b y th ro m b in , wi th th e co n seq u en t fo rm a tio n o f a so lid g e l clo t . Wh en th is as s ay is p er fo rm ed in p r es en c e o f th e ap tam er T B A , th e b in d in g o f fib rin o g en to th e th ro m b in i s in h ib it ed an d a lo n g er tim e is req u i red to fo rm a c lo t. Mo r eo v e r, o th e r im p o rt an t as say s ar e fib r in o p ep tid e A rel e as e a ss ay , p la t el et ag g r eg a tio n an d th ro m - b u s g ro w th . Th e e ff e ct s o f th e su b sti tu tio n s at N 2 an d C 8 o f th e G r es id u e s wh ich fo r m th e G- te tr ad o n th e th ro m b in in h ib i to ry a ct iv ity m e as - u red b y P T we re re l ativ ely sm a ll . T h e in tro d u c tio n o f a b en zy l g ro u p in to N 2 o f G 6 an d G 1 1 an d n ap h th y lm e th y l g ro u p s in to N 2 o f G 6 in cre as ed th e th ro m b in in h ib ito ry a ct iv ity , wh er ea s o th e r su b - sti tu en t s in th e se p o si tio n s h ad a lm o st n o ef fe ct o r d e cr ea s ed th e ac tiv i ty . P a rti cu l ar ly , th e o lig o n u cl eo tid es ca rry in g a 1 ? n ap h th y lm eth y l g ro u p in th e N2 p o si tio n o f G 6 sh o w ed an in cr e as e in a ct iv ity b y ab o u t 6 0 % in vitro an d in vivo . T h e in t ro d u ct io n o f a re la tiv e ly s m a ll g ro u p su ch as me th y l an d p ro p y n y l, in to th e C 8 p o sitio n s o f G 1 , G 5 , G 1 0 an d G 1 4 in crea sed th e a ctiv ity , p r esu m ab l y d u e to th e st ab il iz at io n o f th e q u ad ru p lex , wh e re a s th e in t ro d u ctio n o f a l arg e su b s ti tu en t g ro u p , d e c re as ed th e a ct iv ity , p ro b ab ly d u e t o st er ic h in d r an c e [5 3 ]. T h e 2 ? -C -p ip e ra zin o -U N A -U mo n o m er mo d i - fi ca tio n sh o w ed an u n f av o r ab le im p ac t o f th e p ip er a zin o mo i ety o n th e in h ib it io n with th ro m b in [6 0 ]. T h e b io lo g ic a l ef fe c t o f th e UN A -m o d if ied TB A s w a s t e st ed in a p ro th ro m b in t im e a ss ay . Th e TB A mo d ifi ed with UN A -U 7 sh o w ed an in cr ea s ed in h ib ito ry e ff ec t re la tiv e to th e u n m o d ifi ed T B A , wh il e in h ib i tio n o f co ag u la tio n b y G 1 , U 3 , G 8 , U 9 an d U 1 2 was n e ar tw o fo ld d ec re as ed , an d U 4 , U 13 an d G 1 5 sh o w ed n o in f lu en ce o n fib rin - clo t fo rm a tio n [4 9 ]. T h e T B A mo d if i ed wi th L N A sh o w ed a d if f er en t th ro m b in in h ib itio n a c co rd in g th e p o s it io n o f th e mo d i fi ca tio n . S t ab l e ap tam - er s w ith L N A in p o si tio n s G 5 , T 7 o r G 8 sh o w ed a d e cr ea s ed th ro m - b in in h ib it io n me a su red b y fib rin clo t tin g as s ay . N ev e rth el es s, a l es s st ab le ap t am er w ith L N A at G 2 wa s as a ctiv e a s th e u n m o d ifi e d ap ta m e r [4 8 ] . In ad d i tio n , M ay o l? s g ro u p d es cr ib ed th at th e T B A mo d ifi ed b y L N A in th e G 1 5 p o sitio n d isp lay ed a p ro lo n g ed P T [4 7 ]. T h e mo d if ic at io n o f th e p h o sp h at e lin k ag es b y fo rm ac e ta l [6 1 ] o r th io p h o sp h o ry l [5 2 ] g ro u p s ex h ib it ed a sim il ar p ro th ro m b in tim e to th e o n e fo u n d fo r th e u n m od ified T B A . T h e ef f ec t o f th e mo d i- fi ed lo o p s o n th e th ro m b in in h ib i to ry ac tiv i ty w as al so s tu d ied u sin g acy cl ic n u c leo sid e s. In th i s ca se , th e an aly s is o f P T a ss ay s co n - fir m ed th at th e h ig h es t P T v alu e wa s o b tain ed fo r a mo d if ied T B A co n ta in in g an acy cl ic th y m id in e in p o s it io n 7 [6 3 ]. On th e o th e r h an d , th e T B A mo d if ied wi th fo u r 4 -th io d eo x y u rid in e sh o w ed a 2 - fo ld in cr e as ed in h ib itio n o f th ro m b in ca ta ly z ed fib r in clo t fo rm a - tio n , fib r in o p ep tid e A re l ea se an d th ro m b u s fo rm at io n [4 6 ]. T h e st ru c tu r al ch an g e s in th e o v e ra ll st ru c tu r e th a t h av e b e en d es cr ib ed d o n o t se em to a ff e ct to o mu ch th e th ro m b in in h ib i tio n [7 5 ]. T h e T B A co n t ain in g a 5 ?-5 ? in v er sio n o f p o la ri ty si te af fe ct e d sen sib ly th e b io lo g i c al in h ib i tio n . C o o k an d co -w o rk e rs p r es en t ed a se ri es o f co n st ra in ed u n im o le cu l ar q u ad ru p l ex /d u p lex mo l ecu le s with in c r ea sed th ro m b in in h ib it io n u sin g c lo t fo r m a tio n a ss ay an d re le as e o f f ib r in o p ep tid e A [6 6 ]. Mo r eo v er , S t ein er al so d es cr ib ed a q u ad ru p lex /d u p lex mo l ecu le co n s tru ct th at b in d s th e h ep ar in - b in d in g ex o site w ith 2 0 -5 0 fo ld h ig h er aff in ity m ea su r ed b y clo ttin g tim e [6 7 ] . Fin ally , th e n ew co n st ru c t as s em b l ed b y tw o d is tin c t ap t am er s th at t a rg et s th ro m b in co m b in e s fe a tu re s o f th e in d iv id u al ap t am e r 103 8 Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 Avi?? et al. su b d o m ain s w ith en h an c ed a ctiv iti e s r eg a rd in g b o th fu n ctio n a li ti es ; th es e a re p ro b ab ly d u e to an en h an ced a ff in ity o f th e b iv a len t fu sio n ap ta m e r. T h i s s tru ctu r e d i sp l ay ed en h an c ed an t ico ag u lan t a ctiv it y wh en co m p ar ed to th e T B A , h o w ev er , af fin i ti es w er e im p ro v e d o n ly tw o to th re e fo ld co m p a red to th o se o f th e in d iv id u a l p re cu r - so rs [6 9 ] . Sim il ar co n clu sio n s co u ld b e o b tain ed fo r th e th ro m b in in h ib i - tio n o f th e mo d ifi ed T B A . It is im p o rt an t to m en t io n th at T 7 p o si - tio n se em s v ery s en s itiv e to d iff er en t mo d i fi ca tio n s in t erm s o f in cr e as in g th e th ro m b in in h ib it io n . Mo r eo v er , th e ad d it io n o f d if - fe ren t co n s tru ct s wi th im p ro v ed p h arm a co k in et ic p ro p er ti es to th e T B A co u ld b e a re a so n ab l e id ea th a t p ro b ab ly wo u ld n o t co m p ro - m is e th e in h ib ito ry a ct iv ity . 7. NOVEL APPLICATIONS USING THROMBIN BINDING APTAMER. In ad d it io n to th e an ti co ag u l an t p ro p e rt ie s, a l arg e n u m b er o f an aly ti ca l to o l s b as ed o n th e fo ld in g an d re fo ld in g o f th e T B A h av e b een d ev e lo p ed . In th e fo llo w in g s ec tio n so m e o f th e se n ew d ev el - o p m en ts a re r ev i ew ed . 7.1. The TBA as Model for the Analysis of Binding Mode of Drugs with Affinity to G-Quadruplex G-q u ad ru p lex es h av e b e co m e s tru ctu r e s o f sp e ci al in t e re st fo r d ru g d ev elo p m en t d u e to th e ir p o s sib l e im p li ca tio n s in an ti can ce r re se ar ch . T h e p o t en t ia l ro l e o f G -q u ad ru p lex es h as b een h ig h lig h t e d with th e d ev elo p m en t o f s tr at eg i es d e sig n ed to st ab il iz e t elo m er e en d s as G-q u ad ru p lex s tru ctu r es u s in g sp ec if ic sm a ll mo le cu l es , wh ich c an d e st ab i li ze te lo m e r e m ain t en an ce in tu m o u r ce ll s [7 8 ] . G-q u ad ru p lex es a re al so fo u n d in tr an s cr ip tio n al r eg u l ato ry se - q u en ce s o f cr it ic al o n co g en e s su ch a s c-myc an d c-kit [7 9 , 8 0 ]. L ig an d s th at s el ec tiv e ly b in d an d s tab ili z e th es e st ru ctu re s w er e stu d i ed a s p o ten t ia l an ti can ce r d ru g s o f in t er e st [8 1 ]. T h e T B A wa s u sed a s a mo d e l fo r th e an a ly s is o f th e in t er ac tio n o f sev er al d ru g s with G -q u ad ru p l ex s tru ctu r e s. In o n e o f th e f ir st s tu d ie s, Jo ach im i et al. d es cr ib ed th e p o ten t ia l ro l e o f p o rp h y rin s in th e mo d u latio n o f th e an ti co ag u l an t p ro p e rti e s o f th e T B A [8 2 ] . L a te r, D el T o ro et al . co n fi rm ed th e fo rm atio n o f an in t er ac tio n co m p l ex with a sto i ch io m etry 1 :1 b e tw een th e p o rp h y rin (T m P y P 4 ) an d th e T B A [8 3 ]. U lt rav io le t m el tin g an d ci rcu la r d i ch ro i sm d at a re fl ec t ed th a t th e in iti al G-q u ad ru p lex st ru ctu re o f th e T B A w a s s tab ili z ed in th e in te ra ct io n co m p lex : b e in g slig h tly d i so rd e red b y th e p res en c e o f th e l ig an d . T h e in te ra ct io n b etw een th e p o rp h y rin (T m P y P 4 ) an d th e T B A wa s al so stu d i ed b y t im e -r e so lv ed flu o r es c en c e an i so tro p y . B as ed o n th e an i so t ro p ic d ec ay cu rv e s, a s an d w ich - ty p e b in d in g mo d e wa s p ro p o sed in wh i ch b o th te rm in al G -q u ar te t an d T -T b as e p air s st ack o n th e p o rp h y rin r in g [8 4 ]. T h e in t er a ctio n b e tw e en th e T B A an d th e b ip y rid in iu m s al ts w a s stu d ied b y cy c li c v o lt am m et ry . A st ro n g in t er a ctio n b etw een G -q u ad ru p lex fo rm in g D N A se - q u en ce s an d v io lo g en s w as o b se rv ed [8 5 ]. 7.2. TBA as Sensing Element for Thrombin and Metal Ions Th e co n fo rm at io n al ch an g e o f th e T B A d u rin g th e fo ld in g /u n fo ld in g p ro ce s s wa s ex p lo i ted fo r b u ild in g s en so r s fo r m et al io n s an d fo r d et ec tio n o f th ro m b in . T h is wo rk to g e th e r with th e u s e o f o th er ap t am er s as s en so rs h a s b een su m m ar iz ed in s ev e ra l re c en t rev i ew s [8 6 -9 0 ]. On e o f th e mo s t r el ev an t s tu d ie s i s th e d ev e lo p - m en t o f p ro b e s fo r th e d et e ctio n o f in tr ac el lu l ar p o t as siu m co n c en - tr atio n [9 1 -9 2 ] . Olig o n u cl eo t id e s co n t ain in g th e T B A s eq u en c e fu n ctio n ali z ed wi th a flu o r e sc ein e d e riv ativ e as flu o ro p h o r e an d a rh o d am in e d y e a s q u en ch er a t th e 3 ? an d 5 ?-en d s we r e p rep ar ed . Up o n b in d in g o f p o tassiu m , th e T B A p ro b es fo ld ed in th e in - tr am o l ecu l ar q u ad ru p l ex . T h e q u ad ru p lex fo ld in g in d u c ed b y p o t as - siu m wa s o b se rv ed b y a d ecr ea s e o f flu o r es c en c e d u e to flu o ro - p h o re-q u en ch er in te ra ct io n [9 1 -9 2 ]. T h e d ev elo p m en t o f q u ad ru - p lex DN A -b a sed F R E T p ro b es with sp ec ia l em p h a si s o n th e T B A q u ad ru p lex e s w er e r ev ie w ed [9 3 ]. A sim i la r F R E T ex p er im en t wa s ad ap ted r ec en t ly fo r th e d e t ec - tio n o f th e a ct iv ity o f h u m an O 6 -a lk y lg u an in e -D N A alk y l tr an s fe r as e (h A G T ) [9 4 ] . T h e mo d ifi ed T B A p ro b e co n t ain ed o n e O 6 - m eth y lg u an in e re sid u e th a t p r ev en t ed q u ad ru p l ex fo rm atio n . Up o n rem o v a l o f th e O 6 -m eth y l g ro u p in th e g u an in e b y h A G T , th e n a tu - ra l T B A s eq u en c e is fo rm ed an d i t fo ld s in to th e q u ad ru p l ex , in d u c - in g a d ecr e as e o f flu o r es c en c e d u e to flu o ro p h o re-q u en ch e r in t er ac - tio n [9 4 ]. A co lo rim et ri c a s say fo r th e d e te rm in at io n o f mer cu ry ( II ) u sin g th e T B A wa s al so rep o r ted [9 5 ]. T h e b in d in g o f m e rcu ry t o th e T B A in d u c ed th e fo ld in g o f th e mo le cu l e th at t rig g e r ed sa lt - in d u ced g o ld n an o p a rt ic le ag g r eg a tio n [9 5 ]. Th e fo ld in g /u n fo ld in g o f th e T B A c an al so b e reg u l at ed b y lig h t. T h e in co rp o ra tio n o f o -n itro b en zy l th y m id in e d er iv at iv e s (c ag ed n u cl eo sid es ) in th e T B A seq u en c e d id n o t allo w th e fo ld in g o f th e T B A , p r ev en t in g th ro m b in b in d in g . P h o to r em o v al o f th e n itro b en zy l g ro u p s o n th y m id in es g en e r at ed th e n a tiv e ap t am e r wh ich n o w i s cap ab l e o f b in d in g th ro m b in , wh ich p rev en ted b lo o d clo t tin g [9 6 ]. Al so , th e e ff ec t o f a p h o to a ctiv e n it ro b en zy l g ro u p o n a g u an in e re sid u e o f T B A h a s b e en stu d i ed u s in g c la ss i ca l mo l e cu - la r si m u la tio n s [9 7 ]. T h eo re ti ca l c al cu l at io n s a re ab l e to d e s crib e th e ch an g e in th e st ru ctu re wh en th e mo d i fi ed r e sid u e is in co rp o - ra ted in th e T B A a s we ll a s th e fo rm a tio n o f th e q u ad ru p l ex af te r p h o to ly sis [9 7 ]. T h e p h o to d ep ro t ec tio n o f th e n i tro b en zy l g ro u p s i s irr ev e rs ib l e an d fo r th i s re a so n , Og az aw ar a et al. [9 8 ] d ev e lo p ed a g u an in e d e riv a tiv e c ar ry in g a f lu o r en y lv in y l g ro u p at p o si tio n 8 . T h e flu o r en y lv in y l g u an in e d eriv at iv e m ay u n d erg o to cis - trans ph o to iso m er i za tio n th a t i s r ev e rs ib l e. T h e cis-trans iso m e ri za tio n af fe ct ed th e fo rm at io n o f th e q u ad ru p l ex st ru ctu re an d su b seq u en tl y th e b in d in g o f th ro m b in . In th i s w ay , th e b in d in g o f th ro m b in to th e T B A d eriv ativ es c ar ry in g g u an in e s with th e f lu o ren y lv in y l g ro u p can b e rev er sib ly mo d u lat ed b y l ig h t [9 7 ]. Th e co n ju g at io n o f s ev e ra l d er iv a tiv e s o f th e T B A to g o ld n an o p art ic le s w a s s tu d ied [9 9 ]. S o m e o f th e T B A -g o ld n an o p ar ti - cl es ar e h ig h ly ef fi ci en t as an ti co ag u l an t s [9 9 ]. Mo reo v e r, th e fu n c - tio n al iz a tio n o f iro n o x id e n an o p ar ti cl es w ith T B A h as d es cr ib e d [1 0 0 ]. T h e T B A m ag n eti c n an o p ar ti cl es co n ju g a te s sh o w ed a cl ea r mag n et ic re so n an c e im ag in g (MR I ) s ig n al wh en b in d in g to th ro m - b in [1 0 0 ]. S ev er al el ec tro ch em i c al s en s in g p la tfo r m s b a sed o n th e T B A q u ad ru p lex w er e d ev elo p ed fo r th e d et e ct io n o f th ro m b in . A l ab el - fr ee e l ec tro n i c d et ec tio n sy s tem fo r th e d ir e ct d et e ctio n o f th ro m b i n b as ed o n elec tro ch em ic al im p ed an c e sp ec tro s co p y was d ev e lo p e d [1 0 1 ]. T h e T B A ca rry in g an a m in o g ro u p was co v a len tly l in k ed t o mu lti w all ed c arb o n n an o tu b e d isp o s ab l e s cr e en -p rin ted c a rb o n el ec tro d e s b y am id e fo rm at io n an d th e re su lt in g e le ct ro d e s w er e ab le to s en s e th ro m b in at a d et e ctio n lim it o f 1 0 5 p M [1 0 1 ]. T h e in co rp o r atio n o f fe rro cen e to th e T B A in cr e as ed th e s en si tiv i ty o f th e d et e ctio n r ea ch in g a d e te ct io n lim i t fo r th ro m b in to 0 .5 pM [1 0 2 ]. C o n ju g at io n o f th e T B A to si lv e r n an o p a rt ic le s an d to g o l d n an o sh el ls allo w ed th e d et e ctio n o f th ro m b in b y su rfa c e- en h an c e d R am an sp ec tro sco p y [1 0 3 -1 0 6 ]. T h e in ter a ctio n o f th ro m b in wit h th e T B A wa s a lso stu d i ed o n q u an tu m d o ts an d in su rf a ce p l asm o n re so n an c e [1 0 7 ]. Th e ab so rp t io n an d red o x b eh av io r o f th e T B A an d th e co m p l ex th ro m b in -T B A wa s ev alu at ed b y d iffe r en ti al p u ls e v o lt am m et ry at a g las sy ca rb o n el ec tro d e s [7 6 , 1 0 8 ]. T h e T B A g u an in e o x id a tio n p eak w as fo u n d to b e s en s itiv e to G-q u ad ru p l ex fo rm atio n an d t o th ro m b in b in d in g , sh o w in g a h ig h er o x id atio n p o t en ti al [7 6 , 1 0 8 ]. R ec en t ly , th e ex c ell en t b in d in g p ro p er ti es o f a 2 9 -b a se -lo n g th ro m - b in -b in d in g ap ta m e r lin k ed to g o ld n an o p art ic le s w er e u s ed fo r th e d ev elo p m en t o f a s en si tiv e d et ec tio n o f DN A th at r el ied o n th e mo d u latio n o f th e th ro m b in a ct iv ity o n th e su r fa c e o f th e n an o p ar ti - cl es [1 0 9 ]. 7.3. Single-molecule Experiments on the TBA On e o f th e fi rs t s in g le -m o l ecu l e ex p er im en ts u sin g th e T B A - th ro m b in in t er a ctio n w as p er fo rm ed b y a to m i c fo r c e mi c ro s co p y 104 Chemically Mo dified Thrombin Binding Aptamers Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 9 (A F M ) [1 1 0 ]. An AF M g o ld -co at ed tip w as fu n ct io n al iz ed with th e th io la ted T B A . T h e th ro m b in w as lin k ed co v al en tly to a g o ld - co at ed g l as s slid e. T h e ru p tu r e fo rc e fo r a s in g le ap t am e r/ th ro m b i n co m p lex wa s d et er m in ed a s 4 .4 5 p N . T h e an aly si s o f th e sy st e m rev ea led th a t th e ru p tu re fo rc es co rr e sp o n d ed to th e mel tin g o f th e G-q u ad ru p lex o f th e ap t am er b o u n d to th e th ro m b in an d su b seq u en t d isso ci at io n o f th e co m p l ex [1 1 0 ]. Rec en t ly , th e T B A fo ld in g an d u n fo ld in g in d u ced b y io n s wa s stu d i ed u sin g n an o p o res en c ap su l at ed with sin g l e mo le cu l es . T h e T B A q u ad ru p lex w as fo rm ed r ap id ly in th e p r e sen ce o f p o ta ss iu m io n s an d h ad a s lo w u n fo ld in g re a ctio n . T h e so d iu m an d li th iu m co m p lex o f th e T B A w e re sim il ar b u t th e fo ld in g an d u n fo ld in g o f th e so d iu m co m p l ex wa s f as te r th an th e fo ld in g an d u n fo ld in g of th e lith iu m co m p lex [ 1 1 1 ]. Th e ex ce ll en t mo le cu l ar r e co g n itio n p ro p e rt ie s o f DN A w er e ex p lo it ed to in co rp o r at e fu n c tio n a li ti es in mo l ecu l ar co n s tru ct s an d fo r th e d e sig n o f 2 -d im en sio n a l ar ray s wi th we ll d ef in ed st ru ctu re s [1 1 2 -1 1 3 ]. A rem ark ab l e d ev e lo p m en t in th i s f ie ld was th e u s e o f st ab le DN A Ho ll id ay ju n ctio n s wi th ad d re ss ab l e st ick y en d s to fo r m tw o -d im en sio n al D N A c ry s ta ls [1 1 3 ]. T h e so - c al led D N A t il e sy s - tem w as u sed fo r th e a ss em b ly o f b id im en s io n al DN A ar r ay s, co n - tain in g th ro m b in b in d in g ap tam er s eq u en ce s [1 1 4 -1 1 5 ]. T h e DN A ar ray s tem p la ted th e fo rm at io n o f o rd ered th ro m b in a rr ay s th at w er e v isu a li zed b y A F M [1 1 4 -1 1 7 ]. O rig a m i D N A i s a new m eth o d fo r th e ra tio n a l o rg an i sa tio n o f s tru ctu r es th at u se s a c ir cu l ar v ir al s in - g le st r an d ed D N A (M1 3 D N A ) an d ab o u t tw o h u n d red o lig o n u cleo - tid e s (s t ap le s tr an d s ) th at ar e d es ig n ed to fo ld th e v ira l DN A in to a ra tio n a lly d e sig n ed sh ap e [1 1 8 ]. T h e T B A s eq u en c es we re al s o in tro d u c ed in D N A o rig am i s, sh o w in g a n an o m et ri c co n t ro l o f th e d ep o sit io n o f th ro m b in mo l e cu le s o n th e o rig am i [1 1 9 -1 2 0 ]. CONCLUSIONS Ap tam er s ar e a n o v el cl a ss o f n u cl ei c ac id s with a ff in ity to p ro - tein s th at may b e u s ed fo r th e rap eu ti c o r d iag n o st i c p u rp o se s. T h e th ro m b in -b in d in g ap ta m e r w as o n e o f th e fi rs t ap ta m e rs d ev e lo p e d b y S E L E X an d p ro b ab ly o n e o f th e m o st stu d i ed ap t am e r. T h e T B A is a r el ativ ely sh o rt s eq u en ce , ea sy to sy n th es iz e w ith a w el l-d ef in e d stru ctu re an d h as a g o o d aff in ity fo r th ro m b in . F o r all th e se r e aso n s , it c an b e co n sid er ed a p a rad ig m o f th e p o ten ti a l ap p li ca tio n s o f th e ap ta m e rs . Du r in g th e l as t 2 0 y e a rs , s ev e r al au th o r s h av e d ef in ed th e stru ctu ra l fa c et s o f th e T B A mo lecu le ex p lo r in g sev er al p o ten t ia l v ari ab l es su ch a s su g a r p u ck e rin g , g ly co s id i c b o n d co n fo rm a tio n , H-b o n d in g g ro u p s, b ack b o n e mo d if ic at io n s, et c? S o m e o f th e mo d ifi ed T B A d e riv a tiv es h av e a g o o d af fin i ty fo r th ro m b in , a s wel l a s a l arg e st ab il ity in p h y sio lo g ic al co n d itio n s, wh i ch h a s l e d to th e s et tin g o f so m e cl in i ca l a ss ay s . T h e l es so n s l ea rn ed in th i s p ro ce ss ar e im p o r tan t n o t o n ly fo r th e an ti co ag u l an t p ro p e rt ie s o f th e T B A b u t al so to im p ro v e th e u n d e rs tan d in g o f G-q u ad ru p l e x stru ctu re s p re s en t in t elo m er es an d so m e p ro m o t er reg io n s o f o n co - g en es . An im p o rt an t d ev elo p m en t in th e l as t y e ar s h a s b e en th e co n ju - g atio n o f th e T B A to n an o m at er ia ls su ch a s g o ld an d iro n o x id e n an o p art ic le s, wh i ch may in c re a se th e st ab i lity in p la sm a a s wel l a s it m ay o p en th e o p p o rtu n ity o f ad d in g re c ep to r -m ed ia ted sy st em s fo r ef fi ci en t in vivo t arg et in g . Mo r eo v er , th e T B A - th ro m b in re co g - n itio n sy s te m i s a lr ead y b e in g u sed fo r th e d ev e lo p m en t o f s en so r s b as ed o n b o th elec tr ic al an d o p tic al m eth o d s , an d mo r e r ec en tly fo r th e DN A - tem p la ted d ir e ct ed as s em b ly o f n an o m at e ri al s. As th e tim e g o e s o n , th e p o ten t ia l ap p li ca tio n s o f th is r el at iv e ly s im p l e D N A m o le cu l e ar e in cr e as in g ex p o n en ti al ly . T h i s in ten s e a ctiv it y will h elp to fu rth e r d ev elo p th e ap t am e r fi eld an d it may al so sp a n th e k n o w led g e ab o u t o th e r u se fu l n u c le ic a cid s fo r th er ap eu ti c o r d iag n o sti c p u rp o se s. ACKNOWLEDGEMENT Th is s tu d y was su p p o rted b y th e E u ro p e an C o m m u n iti e s (F U N M O L , F P 7 -N M P -2 1 3 3 8 2 -2 ), S p an i sh Min i st ry o f E d u ca tio n (MO L 2 M E D , C T Q 2 0 1 0 -2 0 5 4 1 ), th e Gen e r ali ta t d e C a ta lu n y a (2 0 0 9 /S G R /2 0 8 ), IR B B ar ce lo n a, C O S T (G 4 n et , MP 0 8 0 2 ) an d C IB E R -B B N ( V I N at io n al R & D & i P l an 2 0 0 8 -2 0 1 1 , In ici at iv a In - g en io 2 0 1 0 , C o n so lid e r P ro g ram , C IB E R Ac tio n s , In st itu to d e S alu d C ar lo s I II wi th a ss is tan ce f ro m th e E u ro p e an R eg io n a l De - v elo p m en t F u n d . REFERENCES [1 ] Irv in e D, Tu e rk C, Go ld L. SE LEX IO N . Sy s te m a tic e v o lu tio n o f lig a n d s b y e x po n en tia l e n ric h m en t with in te g ra te d op tim iz a tio n b y n o n -lin e a r a n a ly s is . J Mo l Bio l 1 9 9 1 ; 2 2 2 : 7 3 9 -6 1 . [2 ] Ellin g to n AD , Sz o s ta k JW. In vitro se le c tio n o f RNA mo le c u le s th a t b in d sp e c ific lig a n d s . Na tu re 1 9 90 ; 3 4 6 : 8 1 8 -2 2 . [3 ] Tu e rk C, Go ld L. Sy s te m a tic e v o lu tio n o f lig a nd s by e xp o ne n tia l e n ric h m e n t: RNA lig a nd s to b a c te rio ph a ge T4 DN A p o ly m e r a se . Sc ie n c e 1 9 9 0 ; 2 49 : 5 0 5 -10 . [4 ] Sz o s ta k JW. In vitro ge n e tic s . Tre n d s Bio c h e m Sc i, 1 9 9 2 ; 1 7 : 8 9 - 9 3 . [5 ] Bro d y EN , Go ld L. Ap ta m e r s a s th e ra p e u tic an d d ia gn o s tic a ge n ts . J Bio te c h n o l 2 0 0 0 ; 74 : 5 -1 3 . [6 ] Ku s s e r W. Ch e m ic a lly m o d ifie d n u c le ic ac id a p ta m e rs fo r in vitro se le c tio n s : e v o lv in g ev o lu tio n . J Bio te c h n o l 2 00 0 ; 7 4 : 2 7 -38 . [7 ] Bo c k LC, Griffin LC, La th a m JA , Ve rm a a s EH , To o le JJ . Se le c tio n o f sin g le stra n d e d - D N A mo le c u le s th a t b in d a n d in h ib it h u m a n th ro m b in . Na tu re 1 9 9 2 ; 35 5 : 5 6 4 -5 6 6 . [8 ] Griffin LC , Tid m a rs h GF, Bo c k LC, To o le JJ, Le u n g LL. In vivo an tic o a g u lan t p ro p e r tie s o f a n o v e l n uc le o tid e - b a se d th ro m b in in h ib ito r a n d d e mo n s tra tio n o f reg io n a l a n tic o a gu la tio n in e x tra c o rpo re a l c irc u its . Blo o d 1 9 9 3 ; 8 1 : 3 2 71 -6 . [9 ] Li WX, Ka p la n AV , Gra n t GW , To o le JJ , Le u n g LL. A n o v e l n u c le o tid e -ba s e d th ro mb in in h ib ito r in h ib its c lo t-b o u nd th ro m b in a n d re d uc e s a rte ria l p la te le t th ro m b u s fo rm a tio n . Blo o d 19 9 4 ; 83 : 6 7 7 -82 . [1 0 ] Wu Q, Tsia n g M, Sa d le r JE. Lo c a liz a tio n o f th e sin g le -s tr a nd e d DN A b in d in g site in th e th ro m b in a n io n -b in d in g e xo s ite . J Bio l Ch e m , 1 9 9 2 ; 2 6 7 : 24 4 08 - 1 2 . [1 1 ] Pa b o rs k y LR, Mc Cu rd y SN , Griffin LC, To o le JJ, Le u ng LLK . 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A do u b le c h a in re v e rs a l lo o p a nd tw o d ia g on a l lo o p s de fin e th e a rc h ite c tu re o f a u n im o le c u la r DN A q ua d ru p lex co n ta in in g a p a ir o f sta c k ed G(s yn )- G (s y n )-G (a n ti)-G (a n ti) te tra d s fla n ke d b y a G-(T-T) Tria d a nd a T- T-T t rip le . J Mo l Bio l 2 0 0 1 ; 3 1 0 : 1 8 1 -94 . [7 1 ] Di Giu s to DA , Kin g GC. Co n s tr u c tio n , sta b ility , a n d a c tiv ity o f mu ltiv a le n t c irc u la r a n tic o ag u la n t a p ta m e rs . J Bio l Ch e m 2 0 04 ; 2 7 9 : 4 6 48 3 -9 . [7 2 ] Ma rtin o L , Virn o A , Ra n d a z z o A, et al . A n e w mo d ifie d th ro m b in b in d in g a p ta me r c o n ta in in g a 5 '-5 ' in v e rs ion o f p o la rity site . Nu c le ic Ac id s Re s 2 0 0 6 ; 3 4 : 6 6 53 -6 2 . [7 3 ] Av i? o A, Ma z z in i S, Fe rre ir a R , Eri tja R. 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An a l Bio a n a l Ch e m 2 0 0 8 ; 3 90 : 1 0 23 -3 2 . [8 9 ] Bin i A, Min u n n i M, To m b e lli S, Ce n ti S, Ma s c in i M . An a ly tic a l p e rfo rm a n ce s o f a p ta m e r - b a s ed se n s in g fo r th ro m b in d e te c tion . An a l Ch e m 2 0 0 7 ; 7 9 : 3 01 6 -9 . [9 0 ] Li N, Ho CM. Ap ta m e r - b a s e d o p tic a l p rob e s with sep a ra te d m o le c u la r rec o gn itio n a nd sign a l tra n sd u c tio n m o du le s . J Am Ch e m So c 2 0 0 8 ; 1 3 0 : 23 8 0 -1 . [9 1 ] Na g a to is h i S, No jim a T, Ju s k o w ia k B, Ta k e n ak a S. A p y re n e - la b e le d G-q ua d r u p lex o lig on u c le o tid e a s a flu o re sc e n t p ro b e fo r p o ta s s iu m io n d e te c tio n in b io lo g ic a l a p p lic a tio n s . An g e w Ch e m In t Ed En g l 2 0 0 5 ; 4 4 : 5 0 6 7 -70 . [9 2 ] Na g a to is h i S, No jim a T, Ga le z o w sk a E, Ju sk o w ia k B, Ta ke n ak a S. G q u a d rup le x -b a s ed FRET p ro b e s with th e th ro m b in -b in d in g a p ta m e r (TBA) se q u e nc e d e sign e d fo r th e e ffic ie n t flu o rom e tric d e te c tio n o f th e p o ta s s iu m io n . Ch e m b ioc h e m 20 0 6 ; 7 : 1 7 30 -7 . [9 3 ] Ju s k o w ia k B. An a ly tic a l p o te n tia l o f th e q u a d ru p le x DNA - b a s e d FRET p ro b e s . An a l Ch im Ac ta 2 0 0 6 ; 5 6 8 : 1 7 1 -80 . [9 4 ] Tin to r ? M, Av i? ? A, Ru iz F M, Eri tja R , F? b re g a C. De v e lo p m e n t o f a n ov e l flu o re s ce n c e a ss a y b a s ed o n th e u se o f th e th ro m b in - b in d in g a p ta m e r fo r te h d e te c tion o f O6 - a lk y lgu a n ine - D N A a lk y ltr a n s f e r a s e a c tiv ity . J Nu c l Ac id s 2 0 1 0 ; 2 0 10 : 1 -9 . [9 5 ] Wa n g Y, Ya n g F, Ya n g XR. Co lo rim e tr ic b io s e n s in g o f me rc u ry (II) io n u s in g un m o d ifie d go ld n a no p a rtic le p ro be s a n d th ro m b in - b in d in g a p ta m e r . Bio s e n Bio e le c tr o n 2 01 0 ; 2 5 : 1 99 4 -8 . [9 6 ] He c k e l A, Ma y e r G. Lig h t re gu la tio n o f ap ta m e r ac tiv ity : a n a n ti- th ro m b in ap ta m e r with c a ge d th y m id in e n uc le o b as e s . J Am Ch e m So c 2 0 0 5 ; 1 2 7 : 82 2 -3 . [9 7 ] Ja y a p a l P, Ma y e r G, He c k e l A, We n n m o h s F. Stru c tu re -a c tiv ity re la tio s h ip s o f a ca g e d th ro m b in DN A a p ta m e r : In s ig h t g a in e d fro m m o le c u la r dy n a m ic s sim u la tio n stud ie s . J Stru c t Bio l 2 0 0 9 ; 1 6 6 : 2 4 1 -5 0 . [9 8 ] O g a s a w a ra S, Ma e d a M. Re v e rs ib le p h o to s w itc h in g o f a G- q u a d r up le x . An g e w Ch e m In t Ed En g l 2 0 0 9 ; 4 8 : 6 67 1 -4 . [9 9 ] Sh ia n g YC, Hu a ng CC, Wa n g TH , Ch ie n CW, Ch a ng HT. Ap ta m e r - c o n jug a te d n a n op a r tic le s b a s ed e ffic ie n tly p ro b e s a n d th ro m b in - b in d in g a p ta m e r . Bio s e n Bio e le c tr o n 2 01 0 ; 2 5 : 1 99 4 -8 . [1 0 0 ] Yig it MV, Ma z u m d a r D, Lu Y. MRI d e te c tio n o f th ro m b in with a p ta m e r fu n c tio n a liz ed su p e rp a ra m a gn e tic iro n o x id e n a no p a rtic le s . Bio c o n ju g Ch e m 20 0 8 ; 1 9 : 4 12 -7 . [1 0 1 ] Ka ra P, d e la Esc o s u r a - Mu n iz A, Ma lte z - d a Co sta M, Gu ix M , Oz s o z M, Me rk o c i A. Ap ta me rs ba s e d e le c tro c he m ic a l b io se n s o r fo r p ro te in d e te c tio n u s in g c a rbo n n a no tu b e s p la tfo r m s . Bio s e n Bio e le c tr o n 2 0 10 ; 2 6 : 1 7 1 5 -8 . [1 0 2 ] Liu X, Li Y, Zh e n g J, Zh an g J, Sh en g Q. Ca rb o n n a no tu b e - e n h a nc e d e le c tr o c he m ic a l a p ta s en s o r fo r th e d e te c tio n o f th ro mb in . Ta la n ta 2 0 1 0 ; 8 1 : 1 61 9 -2 4 . [1 0 3 ] Pa g d a CV, La n e SM, Ch o H, Wa c h s ma n n - H og iu S. Dire c t d e te c tio n o f a p ta m e r - th r o mb in b ind in g via su rfa c e - en h an c e d Ra m a n sp e c tro s co p y . J Bio m e d Op tic s 2 0 1 0 ; 15 : 0 4 7 00 6 /1 - 8 . [1 0 4 ] Oc h s e n ku e hn MA , Ca m p b e ll CJ. Pro b in g b io m o le cu la r in te ra c tio n s u s in g e n ha n c ed Ra m a n sp e c tro sc o py : la b e l-f r e e p ro te in d e te c tio n u sin g a G-q u a d ru p le x DN A a p ta m e r . Ch e m Co m m 2 0 1 0 ; 4 6 : 27 9 9 - 8 0 1 . [1 0 5 ] Ch o H, Ba k e r BR , Wa c h sm a n n - H og iu S, et al. Ap ta m e r - B a s e d SERR S Se n s o r fo r Th ro m b in De te c tio n . Nan o Le tt 2 00 8 ; 8 : 43 8 6 - 9 0 . [1 0 6 ] Pa g d a CV , La n e SM, Wa c h s m a n n - H o g iu S. Ra m a n a n d su rfa c e - e n h a nc e d Ra m an sp ec tro s c op ic stu d ie s o f the 1 5 -me r DN A th ro m b in b in d ing a p ta m e r. J. Ra m a n Sp e c 2 0 0 9 ; 4 1 : 2 41 -7 . [1 0 7 ] Ba la m u ru g a n S, Ob u bu a fo A, Mc Ca rle y RL, So p e r SA, Sp iv a k DA . Effe c t o f Lin k e r Stru c tu r e o n Su rfa c e De n s ity o f Ap ta m e r Mo n o la y e rs a nd Th e ir Co rre s po n d ing Pro te in Bin d in g Effic ie n cy . An a l Ch e m 2 0 0 8 ; 8 0 : 9 63 0 -4 . [1 0 8 ] Dic u le s c u VC, Ch io rc e a - Pa q u im AM, Eritja R, Oliv e ira - Bre tt AM . Th ro m b in -b in d ing a p ta m e r qu a d rup le x fo rm a tio n : AFM a n d v o lta m m e tr ic c h a ra c te r iz a tio n . J Nu c l Ac id s 2 0 1 0 ; 2 01 0 : 1 -8 . [1 0 9 ] Jia n JW, Hu a n g CC. Co lo rim e tric De te c tio n o f DN A b y Mo d u la tio n o f Th ro m b in Ac tiv ity o n Go ld Nan o p a rtic le s . Ch e m - Eu r J 1 7 : 2 3 7 4 -8 0 . [1 1 0 ] Ba s n a r B, Eln a th a n R, Willn e r I . Fo l lo w in g a p ta m e r-th ro m b in b in d in g by fo rc e m e a s u re me n ts . An a l Ch e m 2 0 0 6 ; 7 8 : 36 3 8 -42 . [1 1 1 ] Sh im JW, Ta n Q, Gu L Q. Sin g le - m o le c u le d e te c tio n o f fo ld ing a n d u n fo ld in g o f th e G-q ua d rup le x a p ta me r in a n an o po re n an o c av ity . Nu c le ic Ac id s Re s 2 0 0 9 ; 3 7 : 9 7 2 -8 2 . [1 1 2 ] Ald a y e FA , Pa lm e r AL, Sle im a n HF . As s e m b lin g m a te ria ls with DN A a s th e g u id e . Sc ie n c e 2 0 0 8 ; 3 2 1 : 1 79 5 -9 . [1 1 3 ] Win fre e E, Liu F, We n z le r LA , Se e m a n NC. De s ig n a n d se lf- a s s e m b ly o f tw o -d im e n s io na l DN A c ry s ta ls . Na tu re 1 9 9 8 ; 3 9 4 : 5 3 9 -44 . [1 1 4 ] Lin C, Ka tiliu s E, L iu Y, Zh a n g J, Yan H. Se lf-a s s e m b le d sig n a lin g a p ta m e r DN A a rra y s fo r p ro te in d e tec tio n . Ang e w Ch e m In t Ed En g l 2 0 0 6 ; 4 5 : 5 29 6 -3 01 . [1 1 5 ] Liu Y, Lin C , Li H , Ya n H. Ap ta m e r-d ire c te d se lf-a s s e m b ly o f p ro te in a rra y s o n a DNA n a no s tru c tu re . Ang e w Ch e m In t Ed Eng l 2 0 0 5 ; 4 4 : 43 3 3 -8 . [1 1 6 ] Li H, La Be a n TH , Ke n a n DJ . Sin g le -c h a in a n tib o d ie s ag a in s t DN A a p ta m e rs fo r u s e a s a d a p te r mo le cu le s on DN A tile a rray s in n a n o s ca le m a te ria ls o rg a n iza tio n . Org Bio mo l Ch e m 2 00 6 ; 4 : 3 4 2 0 -6 . [1 1 7 ] Ha n s e n MN , Zh a n g AM, Ra n g n e k a r A, et al. We a v e tile a rc h ite c tu re c on s tru c tio n stra te gy fo r DN A n a n o tec h no lo g y . J Am Ch e m So c 2 0 1 0 ; 1 3 2 : 14 4 81 -6 . 107 12 Current Pharmaceutical Design, 2012, Vol. 18, No . 0 0 Avi?? et al. [1 1 8 ] Ro th e m u n d PW. Fo ld in g DN A to c re a te n a n o sc a le sha p e s a n d p a tte rn s . Na tu re 2 0 0 6 ; 4 40 : 2 9 7 -30 2 . [1 1 9 ] Ch h a b ra R, Sh a rm a J, Ke Y, Liu Y, Rin k e r S , L in d s a y S, Ya n H . Sp a tia lly a d d re s s a b le m u ltip ro te in n an o a rra y s te mp la te d b y a p ta m e r - ta g g ed DN A n a n o a rch ite c tu r e s . J Am Ch e m So c 2 0 0 7 ; 1 2 9 : 1 0 30 4 -5 . [1 2 0 ] Rin k e r S, Ke Y, Liu Y, Ch h a b ra R, Ya n H. Se lf - a s s e m b le d DN A n a n o s tru c tu re s fo r d is ta n c e -d e p en d e n t m u ltiv a le n t lig a nd -p ro te in b in d in g . Na t Na n o te c hn o l 2 0 08 ; 3 : 4 1 8 -22 . Received: October 19, 2011 Accepted: November 28, 2011 108 Chapter 3 DNA Origami as DNA Repair Nanosensor at the Single- Molecule Level. 109 110 DNA Origami as DNA Repair Nanosensor at the Single- Molecule Level. Maria Tintor?,*1 Isaac G?llego,*2 Brendan Manning,3 Ramon Eritja1 and Carme F?brega1 Angewandte Chemie International Edition, (2013), Volume 52, Issue 30, pages 7747?7750 DOI: 10.1002/anie.201301293. Impact factor (2013): 11.336 1 IRB Barcelona, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine. c/ Jordi Girona 18-26. 08034 Barcelona (Spain). 2 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400 (USA). 3 Biokit, S.A. 08186 Lli?? d?Amunt, Barcelona (Spain). * These authors contributed equally to this work. 111 The self-assembly of DNA molecules provides an attractive route towards the formation of complex structures at the nanoscale. In particular, the DNA origami uses hundreds of nucleotides ?staples? to fold a long single-stranded DNA scaffold of 7-kilobase, the M13 phage genome, in a rational and desired shape. It represents a versatile tool for the self-assembly of other molecular species and constitutes an excellent platform to create a variety of new nanoscale devices with great potential and applications. In this chapter, we describe the use of the DNA origami as a nanosensor to analyze the enzymatic DNA repair activity of hAGT via TBA con?ormational changes that condition ?-thrombin interaction with DNA aptamers. As this structural change can be caused by a single methylation in the central guanines of the tetrads of the aptamers, it can be utilised to detect the DNA repair activity of hAGT, given that methylguanine is the substrate of this protein. These findings illustrate the potential use of the DNA origami as a protein recognition biosensor, and open the door to the development of a method for the evaluation of potential inhibitors of hAGT. As a result of the high impact achieved by the publication of this work, we were invited to write a review on the state of the art of DNA nanotechnology, and this work is included as an annex of this chapter. In this review we examine recent progresses towards the potential use of DNA nanostructures for molecular and cellular biology. 112 Biosensors DOI: 10.1002/anie.201301293 DNA Origami as a DNA Repair Nanosensor at the Single-Molecule Level** Maria Tintor, Isaac Gllego, Brendan Manning, Ramon Eritja, and Carme Fbrega* The development of DNA origami[1] has been one of the most important advances for structural DNA nanotechnology.[2] This method uses hundreds of nucleotide staples to fold a long single-stranded DNA scaffold of 7-kilobase, the M13 phage genome in a rational and desired shape. DNA origami is a versatile method for the self-assembly of other molecular species[3] and is an excellent way to create a variety of new nanoscale devices[4] with great potential and applications.[5] Yan and co-workers have used the DNA origami as an addressable support for label-free detection of RNA hybrid- ization;[6] more recently, Seeman and co-workers have developed a nanosensor to detect single nucleotide poly- morphism (SNP).[7] Both strategies are an innovative way to use the DNA origami method to create a nanosensor for biomedical applications at the single-molecule level using atomic force microscopy (AFM). Advances in the molecular biology of cancer have identified key mechanisms involved in the DNA repair pathways induced by chemotherapeutic drugs, as for example alkylating agents. Adducts formed at the O6 position of guanine are of major importance in both the initiation of mutations and in the cytotoxic effects of these agents. Human O6-alkylguanine-DNA alkyltransferase (hAGT) is a DNA- binding protein responsible for the repair of the O6-methyl- guanine, contributing to the resistance to chemotherapeutic agents. For these reasons, hAGT is considered relevant as a prognosis marker of cancer and is a potential therapeutic target.[8] Intense research efforts have been devoted to the identification of small molecules capable of inhibiting hAGT activity and enhancing the cytotoxic effect of the alkylating agents in tumor cells.[9] Several methods are available to characterize the mechanism of action of hAGT, its activity, and its inhibition by small molecules. However, most of these methods are based on radioactivity assays, while others are based on multiple-step enzymatic reactions.[10] Our research group developed a new fluorescence method using a DNAG- quadruplex, the thrombin binding aptamer (TBA), as amolec- ular beacon for the detection of hAGT activity and the development of new inhibitor compounds.[11] G-quadruplexes are a family of four-stranded DNA structures stabilized by the stacking of guanine tetrads in which four planar guanines form a cyclic array of hydrogen bonds stabilized by the presence of monovalent cations.[12] Modifications in the base composition of the tetrads are poorly tolerated by these structures. As an example, O6- methylguanine can form a smaller number of hydrogen bonds and consequently destabilize the G-quadruplex, provoking the loss of its conformation (Supporting Information, Fig- ure S1).[13] Herein, we exploit the spatial addressability of DNA origami in combination with the change of conformation of a DNA G-quadruplex to visually detect by AFM the change in its binding affinity to a-thrombin. As this structural change is caused by a single methylation in the central guanines, it can be utilized to detect the DNA repair activity of hAGT, given that methylguanine is the substrate of this protein. To attain this goal, we have used the specific binding properties of the TBAs (aptamers with anticoagulant properties) to a-throm- bin, their natural substrate.[14] The two TBA sequences used in this work (see the Supporting Information, Table S1, for sequences) are known to bind specifically and cooperatively to two specific and almost opposite epitopes of a-thrombin.[15] TBA1 (primarily fibrinogen-recognition exosite binding)[14] is a 15 mer nucleotide composed of two G-tetrads that are connected by three edge-wise loops, forming a well-charac- terized intramolecular chair-like, antiparallel quadruplex. In contrast, TBA2 (29mer nucleotide, heparin-binding exosite) forms a combined quadruplex/duplex structure.[15] Previous studies provide evidence that the TBAs are able to bind a-thrombin in the absence of monovalent cations promoting the TBA folding to its 3D structure, following the typical chaperone-macromolecule system.[16] Previously, to study the interaction between TBA1 and a- thrombin, fluorescence quenching experiments and electro- phoresis mobility shift assays were performed (Supporting Information, Figures S2 and S3). Both results confirmed that the introduction of a methylated guanine prevented a- thrombin interaction, and furthermore that a 10-fold concen- [*] M. Tintor,[+] Dr. I. Gllego,[$] [+] Dr. B. Manning, Dr. R. Eritja, Dr. C. Fbrega IRB Barcelona, IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine c/Jordi Girona 18-26, 08034 Barcelona (Spain) E-mail: carme.fabrega@irbbarcelona.org Dr. B. Manning Biokit, S.A. 08186 Lli? d?Amunt, Barcelona (Spain) [$] Present address: School of Chemistry and Biochemistry Georgia Institute of Technology Atlanta, GA 30332-0400 (USA) [+] These authors contributed equally to this work. [**] This work was supported by European Communities FUNMOL, ?Fondo de Investigaciones Sanitarias? (grant PI06/1250) and by ?Ministerio Ciencia e Innovacin? (grant CTQ-2010-20541-C03-03). C.F. is grateful to Generalitat de Catalunya and Instituto de Salud Carlos III for a SNS Miguel Servet contract. We acknowledge Gerard Oncins from Scientific and Technological Centers of the University of Barcelona (CCiTUB) for his help and advice regarding AFM. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201301293. Angewandte Chemie 7747Angew. Chem. Int. Ed. 2013, 52, 7747 ?7750  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 113 tration of a-thrombin can be sufficient to appreciate a differ- ent pattern of union of this protein to the TBAs and methyl- TBA-containing origami. Based on these preliminary studies, we designed a DNA origami in which some of the staple strands were modified by the insertion of TBA1 and TBA2 in the middle, protruding from the DNA origami surface[17] (see the Supporting Information, Table S1, for sequences). The staple strands were arranged asymmetrically along the length of the origami in a way that allowed the differentiation between methylated/ non-methylated, to enable the observation and quantification of a-thrombin interaction with the aptamers. For this purpose, we built a dual-aptamer system composed of two lines of five TBA1 and TBA2 doublets placed at a distance of about 5.8 nm from each other, increasing the recognition probabil- ities by at least 10-fold as reported by Rinker et al.[17] The right double line corresponds to the unmodified dual system, and the left dual-aptamer line consists of 15 mer methylated- TBAs and non-modified 29 mer TBAs (Supporting Informa- tion, Scheme S1). The formation of the DNA origami with the modified TBA staple strands was performed successfully, confirming that the addition of a methylated sequence does not affect its assembly (Supporting Information, Figure S4). Afterwards, the complex formation between a-thrombin and the non-methylated/methylated TBA modified origami was studied. In Figure 1, the asymmetric interaction can be observed. As expected (Scheme 1a), the complex was only formed with the native TBAs, whereas in the left line no interaction was reported, confirming that a-thrombin is not able to bind the disrupted quadruplex. The study of the profiles and pixel distribution (Figure 3b) confirms that the height of the dots in the dual-aptamer is in agreement with the expected size of a-thrombin (ca. 4 nm in diameter) in comparison with the height of the origami control (Fig- ure 3a). We observed that more than 95% of the chemically modified origami tiles faced pointing towards the solution, in agreement with data reported by Voigt et al.[4c] This result is a clear confirmation that the complex between the dual-aptamer system and a-thrombin is only formed with the non-methylated TBA, and confirms the ability of our design to discern between the methylated and non-methylated state. To explore the efficiency of our design, we performed a quantitative study of the binding location of a-thrombin. From the 160 well-formed DNA origami studied, around 20% of them contained all five a-thrombin molecules in positions coinciding with unmodified TBA and almost none in the methylated line. In all, more than 93% of the DNA arrays contained at least 1 a-thrombin attached to the unmodified TBAs (see the Supporting Information). We then intended to repair the O6-methylguanine of the TBA-containing staple strands by hAGT. For this purpose, the methyl-TBA-staple strands were incubated with hAGT. hAGT was removed and the resulting strands were used to assemble the DNA origami (seeMaterials andMethods in the Supporting Information). The recovery of the chair-like structure of the now demethylated 15mer was expected, leading to the binding of a-thrombin to both dual-aptamers, as the two of them contain the native 15 and 29mer TBAs. The binding of a-thrombin in both dual-aptamers is shown in Figure 2, confirming the repair of the alkylated guanine by hAGT. Upon quantitative exploration of the binding, we can conclude that a-thrombin binds with equal contingency in both lines of the origami, with no significant tendency (p< 0.5) for any of the dual systems composed by TBA1 and TBA2. The study of the height profiles corroborated the theoretical height of the a-thrombin on both dual-aptamers (Figure 3c). Furthermore, we titrated hAGT (0 to 10-fold origami:hAGT; see the Supporting Information) against methyl-TBA staples and incorporated these staples into the DNA origami. The results showed that a-thrombin binding to the methylated/repair side was clearly dependent on hAGT concentration (Supporting Information, Figures S10 and S11). In summary, we have developed a new method to study the DNA repair activity of hAGT. To the best of our Figure 1. a),b) AFM images (scale bars 200nm (a), 100 nm (b)) of the interaction of a-thrombin with the origami. The interaction can be observed as white aligned dots deposited over the origami surfaces. The complex was only formed with the native TBAs (right line; see inserts in (a) for more details), whereas in the left line, in which the 15mer TBA carried an O6-methylguanine, no interaction was observed. Scheme 1. a) Representation of the asymmetric binding of a-thrombin to TBA aptamers of methylated DNA origami. b) Methyl-TBA repair by hAGT, thus allowing G-quadruplex formation. c) Representation of the symmetric binding of a-thrombin to the repaired DNA origami quad- ruplexes. .Angewandte Communications 7748 www.angewandte.org  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2013, 52, 7747 ?7750 114 knowledge, this is the first time the enzymatic activity of hAGT has been visualized on an origami platform. This study combines the capabilities of the a-thrombin recognition/ binding to TBA and the single-molecule features of the DNA origami applied to the detection of DNA repair. The system appears to be extremely effective and reliable, and the results are clearly visualized by AFM. Their consistency suggests that our system could be further evolved to design hAGT activity assays for the identification of potential inhibitors as chemo- therapy enhancers and for the study of other DNA repair enzymes. The application of the DNA origami as a platform for single-molecule recognition opens the door for the development of new biosensors for the detection of a variety of complexes and the activity of other proteins. Finally, it can also contribute to the study of other DNA lesions that affect G-quadruplexes. This in turn would increase our knowledge on the effect of DNA damage in biologically relevant G- quadruplex structures.[18] Experimental Section Standard oligonucleotides were purchased from Sigma. Modified staple strands were synthesized on a DNA synthesizer following standard methods. All of the oligonucleotides sequences are detailed in the Supporting Information. Full-length hAGTwas overexpressed and purified as previously described.[10e] A mixture of the modified staple strands containing the methylated 15-mer TBA sequence were left to react with hAGT. DNA origami tiles were assembled following the method devel- oped by Rothemund.[1] A sufficient amount of a-thrombin was added and left to equilibrate before imaging. Images were acquired in tapping mode in liquid environment using triangular-shaped AFM probes and their anal- ysis was performed using NanoScope Analysis Version 1.40. All of the experiments were performed in triplicate. Statistical compari- sons of the binding performance were done according to Students t distribution. Received: February 13, 2013 Revised: April 12, 2013 Published online: June 13, 2013 .Keywords: biosensors ? DNA origami ? DNA repair ? G-quadruplexes ? hAGT [1] P. W. Rothemund, Nature 2006, 440, 297. [2] N. C. Seeman, Annu. Rev. 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Figure 2. Symmetric binding of a-thrombin to the origami after the repair of the methylation in the TBA1 (left line). The bottom-right panel shows the 3D profile of an origami with all its binding positions occupied by a-thrombin. Figure 3. Distribution of heights, corresponding AFM images, and their cross-sections. a) TBA- origami. b) a-thrombin methyl-TBA origami complex. c) a-thrombin complex with demethylated TBA origami. Angewandte Chemie 7749Angew. Chem. Int. Ed. 2013, 52, 7747 ?7750  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 115 [6] Y. Ke, S. Lindsay, Y. Chang, Y. Liu, H. Yan, Science 2008, 319, 180. [7] H. K. Subramanian, B. Chakraborty, R. Sha, N. C. Seeman,Nano Lett. 2011, 11, 910. [8] R. Pepponi, G. Marra, M. P. Fuggetta, S. Falcinelli, E. Pagani, E. Bonmassar, J. Jiricny, S. DAtri, J. Pharmacol. Exp. Ther. 2003, 304, 661. [9] A. E. Pegg, Chem. Res. Toxicol. 2011, 24, 618. [10] a) B. D. Wilson, M. Strauss, B. J. Stickells, E. G. Hoal-van Hel- den, P. van Helden, Carcinogenesis 1994, 15, 2143; b) M. E. Dolan, A. E. Pegg, N. K. Hora, L. C. Erickson,Cancer Res. 1988, 48, 3603; c) A. M. Moser, M. Patel, H. Yoo, F. M. Balis, M. E. Hawkins, Anal. Biochem. 2000, 281, 216; d) R. S. Wu, S. Hurst- Calderone, K. W. Kohn, Cancer Res. 1987, 47, 6229; e) F. M. Ruiz, R. Gil-Redondo, A. Morreale, A. R. Ortiz, C. Fabrega, J. Bravo, J. Chem. Inf. Model. 2008, 48, 844. [11] M. Tintore, A. Avino, F. M. Ruiz, R. Eritja, C. Fabrega, J. Nucleic Acids 2010, DOI: 10.4061/2010/632041. [12] a) I. Smirnov, R. H. Shafer, Biochemistry 2000, 39, 1462; b) B. I. Kankia, L. A. Marky, J. Am. Chem. Soc. 2001, 123, 10799. [13] M. Trajkovski, P. Sket, J. Plavec, Org. Biomol. Chem. 2009, 7, 4677. [14] L. C. Bock, L. C. Griffin, J. A. Latham, E. H. Vermaas, J. J. Toole, Nature 1992, 355, 564. [15] D. M. Tasset, M. F. Kubik, W. Steiner, J. Mol. Biol. 1997, 272, 688. [16] a) H. A. Ho,M. Leclerc, J. Am. Chem. Soc. 2004, 126, 1384; b) E. Baldrich, A. Restrepo, C. K. OSullivan, Anal. Chem. 2004, 76, 7053. [17] S. Rinker, Y. Ke, Y. Liu, R. Chhabra, H. Yan, Nat. Nanotechnol. 2008, 3, 418. [18] G. Biffi, D. Tannahill, J. McCafferty, S. Balasubramanian, Nat. Chem. 2013, 5, 182. .Angewandte Communications 7750 www.angewandte.org  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2013, 52, 7747 ?7750 116 Supporting Information DNA Ori ga mi as DN A r ep air nano sen so r at th e sin gle - mo lecu le level. Maria Tintor??, Isaac G?llego?, Brendan Manning, Ramon Eritja and Carme F?brega* Table of contents: 1. Ma t er ia ls a nd met hods Pa ge 1 2. T BA st a ple st r a nds, help er st ra nd s s equ enc es a nd DN A or i ga mi des i gn. Pa ge 6 3. Disr upt ion of the G - qua dr upl ex. Figure S1. Pa ge 1 3 4. F luor esc enc e a ss a ys to st udy ?- t hr omb i n - T B A bindi ng . Figures S2. Pa ge 1 4 5. EMSA assays to explore ? - t hr omb i n - T B A bindi ng . Figure S3. Pa ge 1 6 6. Or iga mi for ma t i on c ont r ol. Figure S4. Pa ge 1 7 7. Additional AFM images of ? - t hr omb in - T BA int er a ct i on on DNA or i ga mi : Figures S5, S6, S7 and S8. Pa ge 1 8 8. S ect i on a na l ys is of the AF M ima ges . Figure S9. Pa ge 20 9. T it ra t ion of the hAGT r epa ir a ct ivit y. Figures S10 and S11. Pa ge 21 10. Quantitative and statistic studies of ? - t hr omb i n - T B A binding on DN A or i ga mi . Pa ge 2 2 11. R ef er enc es Pa ge 2 3 117 1. Materials and Methods 1. 1. Abbr evia t i ons. 3?-Da bsyl CP G: 1-Di met hoxyt r it ylox y -3- [O - (N -4'-su lf onyl -4-( di met hyla mi no)- azob enz ene)-3 -a mi nopr op yl]-pr op yl-2-O -succ i noyl- l ong c ha i n a lkyla mi no-C P G 6?-F AM: 6- [(3?,6?- dipi va l oylf lu or esc ei nyl) -ca r boxa mi do]- hex yl -1-O-[ (2 -c ya noet hyl)- ( N,N -di is o pr opyl)]-p hosp hor a mi dit e AF M: a tomic f or c e micr osc op y BC NU: bis - c hl or oet hyl nit r osour ea or car must i ne CP G: cont r ol l ed por e gla ss dmf : N, N-di met hyla mino met hyl i dene hAGT : hu ma n O 6 -a l kyl gua ni ne-D N A a lkylt r a nsfer a se MALDI : ma t r ix -a ss ist ed la s er dis or pt ion/ ioniza t i on O 6 -M eG : O 6 - met hylgua ni ne PAGE: Pol ya cr yla mi de gel elect r op hor es is RP -HP LC : r ever s e pha s e high pr ess ur e liqui d c hr oma t ogr a phy T EAA: tr iet hyla mmoniu m a c et a t e T HAP : tr ihydr ox ya c et op henone monohydr a t e T OF : time of fli ght UV: ult r a vi ol et 118 1. 2. C hemi ca ls T he st a nda r d p hosp hor a mi dit es a nd a nc i l la r y r ea gent s used dur i ng oli gonuc l eot i de s ynt hes i s wer e obt a ined fr om Appl i ed Bios ys t ems a nd Link T echnol ogi es Lt d. 5?-F luor esc ei n CE phosphoamidite (6? - FAM) was acquired from Link Technologies, 3? -Da bsyl CP G, O 6 - met hyl gua ni ne (O 6 -M eG) a nd G d m f p hosphor a mi dit es wer e fr om Glen R es ea r ch. Ma t r ix for MALDI- TOF experiments were 2?,4?,6? -t r ihydr ox ia c et op henone monohydr a t e (T HAP , Aldr ic h) a nd a mmoniu m c it r a t e diba sic (F lu ka ). Solvent s f or HP LC a na lysis wer e pr epa r ed usi ng tr iet hyla mmoniu m a c et a t e (T EAA ) a nd a c et onit r i l e ( Mer c k) a s mob i l e p ha se. T he r est of t h e chemi ca ls a r e a na l yt ica l r ea gent gr a de fr om c ommer c ia l sour c es a s speci fi ed. Ult r a pur e wa t er (Mil l ip or e) wa s used in a ll exp er i ment s. T he 10 % non dena t ur i ng P AGE wer e pr epa r ed fr o m the st oc k solut i on a cr yla mi de/ b is -a cr yla mi de s olut i on 4 0% (S igma ). 1. 3. Inst r u ment a t ion Modi fi ed st a pl e st r a nds wer e synt hes iz ed on a n ABI 3400 DNA Synt hes iz er (App l i e d Bios yst ems ). Semipr epa r a t ive RP -HP LC wa s per for med on a Wa t er s chr oma t ogr a phy s yst e m using S ymmet r y Nuc l eos i l Semipr epa r a t ive 120 C18 (250x8mm) c olu mn. Ana l yt ica l RP -HP LC wa s per f or med usi ng a n X Br i dge OS T C18 2. 5 ? m c olu mn. Ma ss spect r a wer e r ec or ded on a MALDI Voya ger DE RP time - of - f li ght (T OF ) spect r omet er ( Appl i ed Biosyst ems). Mol ecula r a bsor pt ion sp ect r a bet ween 220 a nd 55 0 nm wer e r ec or ded wit h a Ja sco V650 spect r op hot omet er . T he t emp er a t ur e wa s c ont r ol l ed wit h a n 89090 A Agil ent P elt i er devic e. Hel l ma qua r t z cuvet t es (0. 5 a nd 1. 0 c m pa t h lengt h, 500 or 1000 ? l volu me) wer e us ed. T he C D spect r a wer e r ec or ded on a Ja sco J - 810 sp ect r opol a r imet er a tt a ched to a Jula bo F/25HD cir cula t i ng wa t er ba t h in 1 cm pa t h - l engt h qua r t z cyl i ndr ica l c ells. Flu or omet r i c mea sur ement s wer e p er for med on a spect r oflu or omet er Ja sco FP 6200 a t 25?C . Set temp er a t ur e wa s c ont r ol l e d wit h a n 89090 A Agi l ent P elt i er devic e a nd H el l ma qua r t z cuvet t es wer e us ed (100 ? L volu me). Non - dena t ur ing P AGE wa s r un on a n AA H oef er SE 600 st a nda r d ver t ica l elect r op hor es is devic e, us i ng a n Ap el ex E lect r ophor es is Power Suppl y PS 3002. Gels wer e ima ged wit h a G en e Genius Bioi ma gi ng syst em (S yngene). AF M ima gene s wer e obt a ined b y liqu i d ta pping on a Na nosc op e 3 A Mult i mode 4 AF M, using Br uker SNL - 10 sili c on tips. 1. 4. Ol igonu cl eot i des S ynt hesis Sta nda r d st apl e st ra nd oli gonuc l eot i des wer e pur cha sed to Sigma . Modif i ed st a pl e st r a nds wer e synt hes i z ed on a n AB I 3400 DN A S ynt hes iz er ( App l i ed Bios yst ems ), usi ng t he 200 - nmol sca l e synt hes is a nd the st a nda r d pr ot oc ols. T he mor e la b i l e dmf gr up wa s used for the pr ot ect i on of a ll gua ni nes . For stra nds c ont a i ni ng O 6 -M eG or FAM, thes e phosp hor a mi dit es wer e sit e - spec if ica ll y ins er t ed int o the oli gonuc l eot ide. I n the ca s e of the f lu or esc ent pr ob es, the qu enc her group was introduced at the 3? end using the controlled pore glass functionalized with a 3? - Da bsyl der iva t i ve CP G. O 6 -M eG- c ont a ini ng oli gonuc l eot i des wer e deb l oc ked a cc or di ng to t he manufacturer?s instructions (ammonia deprotect i on wa s p er for med over ni ght a t r oo m temp er a t ur e a nd f oll owed by 1 h a t 55?C ). T he r esult i ng pr oduct s wer e desa lt ed by S ep ha dex G - 25 (NAP -10, Amer s ha m Biosc i enc es) a nd pur if i ed b y RP -HP LC using Nuc l eosi l c olu mns. T he lengt h a nd homogeneit y of t he oligonu cl eot i des wer e c hec ked b y M ALDI -T OF . T he DN A - st ra nd c onc ent r a t ion wa s det er mi ned b y a bsor ba nc e mea sur ement s (260 nm) a nd their ext i nct i o n coeff ic i ent . Oli gonu cl eot i de sa mp l es wer e kept a t 4 ?C unt i l fur t her use. All t he oli gonuc l eot i des sequ enc es a r e det a il ed in T abl e 1. 119 1. 5. C D spect r a. Mea sur ement s wer e c onduct ed in 10 mM s odiu m ca codyla t e pH 7. 0 a nd 100 mM KC l. Sa mp l e conc ent r a t ion wa s bet ween 1 - 4 ?M. Each sample was allowed to equilibrate at the initial t emp er a t ur e wit hout a ny ext er na l c ont r ol of t emp er a t ur e f or 5 min b ef or e t he melt i ng exp er i ment b ega n. Spect r a wer e r ec or ded a t r oom t emper a t ur e usi ng a 100 nm/ mi n sca n r a t e, a spect r a l ba nd widt h of 1 nm a nd a time c onst a nt of 4 s . All the sp ect r a wer e c or r ect ed wit h th e buff er bla nk a nd plot t ed using Mi cr os oft Offi c e Exc el 2007 soft wa r e. 1. 6. F luor esc enc e a ss a y Bindi ng b et ween hu ma n ?- t hr omb i n ( Ha ema t ologic T ec hnol ogi es I nc. ) a nd T B A, 5 - O 6 - M eG - T BA a nd 6 - O 6 - M eG - T BA mol ecu la r bea cons (MB) wa s monit or ed b y mea sur i ng c ha nges i n flu or es c enc e int ens it y of t he M B solut ion over t ime. 1 50 ? L of a 1 ? M solut i on of M B - T BA i n MB buff er (20 mM T r is pH 7. 4, 140 mM Na C l, 5 mM KC l, 1 mM CaC l 2 , 1 mM MgC l 2 , 5% v/ v). Ea ch time, 1. 5 ? L of ?- t hr omb i n wer e a dded a nd a ft er a quic k ma nua l mix, th e flu or es c enc e int ensit y wa s mea sur ed a t exc it a t i on wa vel engt h of 485 nm a nd emiss i o n wa vel engt h of 520 nm. T he mea s ur ement s wer e p er for med u nt i l r ea c hing a mola r ra t io of 3:1 bet ween ?- t hr ombi n a nd the mol ecu la r bea con M B - T B A. T his sa me exp er i ment wa s r ep ea t ed using t he modi fi ed M B - 5- O 6 - M eG - T BA a nd M B - 6- O 6 - M eG - T B A s equ enc es. All th e exp er i ment s wer e p er f or med in tr ipli ca t es. 1. 7. EMS A a ssa y In t hes e exp er i ment s T B A s equ enc es wit h a n over ha ng of 5 thymi nes wer e used, to ma ke t h e DNA longer a nd ea si er to ma nipula t e. T BA( T ) 5 and 5-O 6 -M eG -T B A(T ) 5 (1 ? M) wer e incuba t e d wit h sp ec if i ed a mount s of ?- t hr ombi n f or 1 hour a t r oom t emp er a t ur e. T he r ea ct i on buf f er wa s 250 mM T r is pH 7. 4 , 150 mM a cet ic a ci d, 15 mM ED T A a nd 37. 5 mM ma gnes iu m a c et a t e. T he r ea ct i on wa s st opp ed b y a ddi ng t he loa di ng buff er a nd a non - dena t ur i ng 10% P AGE wa s r un a t const a nt 160 V f or 3 hour s at 20 ?C . T he gels we r e st a i ned wit h S ybr Gol d ( I nvit r ogen, 1? dye in 100 mL wa t er ) or Sybr Gr een (I nvit r ogen, 1? dye in 100 mL wa t er ). 1. 8. hAGT expr ess i on a nd pur ifi ca t ion Full- l engt h hu ma n AGT wa s over expr ess ed a nd pur i fi ed a s pr evi ousl y descr ib ed. [1 ] Br ief l y, hAGT pr ot ei n (p et -21a (+) vect or , Nova gen) wa s over expr ess ed in t he E. c ol i st r a in Ros et t a a nd onc e t he cu lt ur e r ea c hed a n OD 6 00 va lu e of 0. 8, it wa s induc ed b y a ddi ng 1 mM IP T G dur ing 4 h at 30 ?C . T he p el l et fr om a 1 L cu lt ur e wa s disr upt ed b y s oni ca t i on a nd c ent r ifu ged. T h e super na t a nt wa s filt er ed, loa ded int o a HiT ra pT M FF colu mn (GE H ea lt hca r e) a nd elut ed wit h a n imi da z ol e gr a di ent . Fina l l y the pr ot ei n wa s loa ded int o a Super dex 75 16/ 60 colu mn (GE Hea lt hca r e), b ei ng t he buf f er 200 mM Na C l, 20 mM Tr is pH 8. 0, 10 mM DT T a nd 0, 1 mM EDT A. T he pr ot ei n wa s c onc ent r a t ed to 2 mg/ ml in hAGT r ea ct i on buff er (200 mM Na C l, 50 mM T r is pH 8. 0, 1 mM DTT a nd 5 mM EDT A) a nd kept a t -20 ?C in pr es enc e of 40 % glyc er ol. 120 1. 9. hAGT r epa ir of the met hyla t ed T BA st ra nds 1. 9. 1 Repa ir of the met hyla t ed T BAs in t he or i ga mi hAGT wa s incuba t ed dir ect l y wit h t he met hyla t ed or i ga mi a dsor b ed over the mica sur fa ce, (for or i ga mi for ma t i on det a ils , s ee AF M ima gi ng s ect i on1. 10) . Suffici ent a mou nt of hAGT to r ea ch a conc ent r a t ion 10 f ol d b igger tha n t he or i ga mi wa s a dded to the s olut i on a nd left t o r ea ct f or one hour in hAGT r ea ct ion buff er . T hen, the mica sur fac e wa s wa shed t hr ee t imes wit h 40 ? L of T AE - M g + 2 buff er (40 mM Tr is pH 8. 0, 12. 5 ma gnesi u m a cet a t e, 2. 5 mM EDT A a nd 20 mM a cet i c a ci d). Aft er wa r ds, 10 fol d conc ent r a t ion of ?- t hr omb i n wa s a dded to the solut i on a nd left to equ i libr a t e f or some minut es bef or e ima gi ng. 1. 9. 2 Rea ct ion of hAGT wit h the met hyla t ed T BA st a ple st r a nds A mixt ur e of t he f ive modif i ed st a pl e st r a nds c ont a i ni ng t he met hyla t ed 15 - mer T B A s equ enc e wa s dr i ed u nder va cuu m a nd r esus p ended in hAGT r ea ct ion buff er to r ea c h a conc ent r a t ion of 35 ? M. T hen, incr ea si ng c onc ent r a t ion s (0, 70, 175 a nd 350 ? M) of hAGT w er e a dded, a nd t h e r esult ing mixt ur e s w er e incuba t ed dur i ng one hour a t 37 ?C . In or der to pur if y t he demet hyla t ed st a ple st r a nds, the r ea ct i on mixt ur e s w er e loa ded int o a Niqu el H is Spi nT ra p colu mn (GE Hea lt hca r e) a nd wa shed out wit h wa t er thr ee t imes to a ll ow t he oli gonu cl eot i des t o elut e. Rec over ed sa mp l es wer e qua nt if i ed, conc ent r a t ed to 10 ? M a nd st or ed unt il fur t her use. 1. 10. AF M ima gi ng Ta ll r ect a ngl e DN A or i ga mi til es wer e a ss embl ed foll owi ng the met hod devel op ed b y Rot hemu nd [2 ] . H elp er st r a nds modif i ed wit h dif f er en t T BA s equ enc es a nd u nmodif i ed help er st ra nds wer e mix ed wit h the vir a l DN A M13 a t mola r r a t io of 10 :5:1. I n the ca s e of th e demet hyla t ed or i ga mi, help er st r a nds modi f i ed wit h T B A s equ enc es, hAGT -demet hyla t ed T B A st a ple st r a nds a nd unmodi f i ed help er st ra nds wer e mix ed wit h t he vir a l DNA M13 a t mola r r at i o of 10:50 :5:1. T he or i ga mi wa s a ss emb l ed us i ng a Bior a d T er moc yc l er st a r t ing a t 90?C a nd decr ea s ed to 20?C a t -1?C per minut e. T wo met hodol ogi es wer e us ed to b ind ?- t hr omb i n to t h e or i ga mi sur fa ce wit h equa l suc c ess . Fir st ly, a rra ys wer e mix ed wit h ?- t hr omb i n (1 :10 r a t io or i ga mi:t hr ombi n) a nd incuba t ed dur i ng 1 hour b ef or e ima gi ng. 1 ? L of sa mp l e wa s dep os it e d on a mica sur fa ce a nd left t o a ds or b f or 1 minut e b ef or e 40 ? L of T AE - M g + 2 buf f er wa s a dded. In the s ec ond ca se, 1 ? L of pla in or iga mi wa s dep osit ed on a mica sur fa ce a nd left to a ds or b for 1 minut e, a nd then 60 ? L of T AE - M g +2 buff er (40 mM tr is pH 8. 0, 12. 5 ma gnes iu m a c et a t e, 2. 5 mM EDT A a nd 20 mM a c et i c a ci d) wer e a dded. Suffic i ent a mou nt of ?- t hr omb in to r ea c h a conc ent r a t ion 10 f ol d bigger t ha n t he or iga mi wa s a dded to t he solut ion a nd left t o equ i l ibr a t e for s ome minut es b ef or e ima gi ng. T o p er for m the mea sur ement s shown in t his st udy, a n Extender Mult i mode AF M a tta ched t o a Na nosc op e II I elect r oni cs (Br uker ) wa s used. I ma ges wer e a c qu ir ed in ta ppi ng mode in liqu i d envir onme nt usi ng tr ia ngu la r - sha ped AF M pr ob es (S NL - 10, Br uker ) wit h a nomi na l spr ing c onst a nt of 0. 35nN/ nm. 30 minut es wer e la ged b ef or e a cquir i n g top ogr a phic ima ges s o a s t o t her mica l l y equ il ibr a t e t he sa mp l e a nd the pr ob e a nd t o ensur e a sta ble envir onment . T he ca nt il ever s a r e ma de of si l ic on nit r i de, whi l e t he tip p yr a mi d is ma de of si l ic on for ma xi mu m sha r pness a nd r esolut i on. In or der to ma i nt a i n the sa mp l e int egr it y whil e sca nni ng, fr ee a mp lit u de wa s ma i nt a ined a s low a s poss ib l e (t yp ica l l y b et ween 0. 2 a nd 0. 4V) a nd a mpl it u de s et p oi nt wa s ma xi mi z ed f or thes e c ondit i ons. Sca n r a t e wa s s et b et we e n 1. 5 a nd 3 Hz dep endi ng on t he s oft ness of the sa mp l e a nd ima ge r esolut i on wa s set to 512x512 pix els. 121 C ompa r ison of a ver a ges of t he t wo t yp es of bindi ng (r ight line a nd left line) wa s per f or me d using Student?s t model. 122 1. TBA staple strands, helper strands sequences and DNA origami design. ODN Sequence (5? to 3?) TBA1 * GGTTG G TG TG G TTG G 5 - O 6 - M eG - TBA 1 * GGTT G M e G TG TG G TTG G 6 - O 6 - MeG - TBA 1 * GGTTG G M e TG TG G TTG G TBA2 AGTCCG TG G TAG G G CAG G TTG G G G TG ACT Fluorescence assays MB - TBA1 * FAM - G G TTG G TG TG G TTG G - D absyl MB - 5 - O 6 - MeG - TBA1 * FAM - G G TT G M e G TG TG G TTG G - Da bsyl MB - 6 - O 6 - MeG - TBA1 * FAM - G G TTG G M e TG TG G TTG G - Da bsyl EMSA assays TBA1 ( T) 5 * GGTTG G TG TG G TTG G TT TTT 5 - O 6 - MeG - TBA1 ( T ) 5 * GGTT G M e G TG TG G TTG G TT TTT Origami assembly t - 5r 4e - 5 - O 6 - MeG - TBA1 * AAAG G CCG CTCC AA A ATT TT GGTTGMeGTGTGGTTGGTTTT G G AG CCTTAG CG G AG T t - 5r 8e - 5 - O 6 - MeG - TBA1 * CCA AA TC ATT A C TTAG TT TT GGTTGMeGTGTGGTTGGTTTTC CG G AACG TACC AAG C t - 5r 12e - 5 - O 6 - MeG - TBA1 * TAAA TA TTG AG G CA TATT TT GGTTGMeGTGTGGTTGGTTTTG TA AG AG CA CAG G TAG t - 5r 16e - 5 - O 6 - MeG - TBA1 * TTTC A TTTC TG TAG CTT TTT GGTTGMeGTGTGGTTGGTTT TC AAC A TG TTTAG AG AG t - 5r 20e - 5 - O 6 - MeG - TBA1 * CATG TC AA AA A TCA CCTT TT GGTTGMeGTGTGGTTGGTTTT A TCA A TA TAAC CC TCA t3r 4e - TBA1 GTTTG CCAC CTCAG AG TTT T GGTTGGTGTGGTTGGTTT TCC G CCACCG CCAG AA T t3r 8e - TBA1 ATACCC A AAC ACC ACG TT TT GGTTGGTGTGGTTGGTTT TG A ATAAG TG ACG G A AA t3r 12e - TBA1 AGG TTT TG G CCAG TTA TT TT GGTTGGTGTGGTTGGTTT TC A AA ATA AAC AG G G AA t3r 16e - TBA1 A CG CTC A ACG AC AA A ATT TT GGTTGGTGTGGTTGGTTT TG G TA AAG TA TCC CA TC t3r 20e - TBA1 TTG AA TTA TTG AA A AC TTT T GGTTGGTGTGGTTGGTTT TAT AG CG ATT ATAA CTA t5r 2f - TBA2 AATG CCCC A TA AA TCC TT TT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TTC A TTA AA AG A ACC AC t5r 6f - TBA2 AGCACCG TAG G G AAG G TTTT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TTAA A TA TTTT A TTT TG t5r 10f - TBA2 GCAA TAG C AG AG A ATA TTT T AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TAC A TA AA A ACAG CC A T t5r 14f - TBA2 TCA TT ACCG A ACA AG A TTTT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TA AA A TA A TA ATTC TG T t5r 18f - TBA2 AGG CG TTAG G CTTAG G TTTT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TTTG G G TTAAG C TTAG A t - 3r 2f - TBA2 TGTAG CA TA AC TTTC A TTT T AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TAC AG TT TC TA ATTG TA t - 3r 6f - TBA2 TTTC A TG A TG ACCCC CTT TT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TAG CG A TTA AG G CG CAG t - 3r 10f - TBA2 TTTC A ACTACG G A ACA TTTT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTT TTAC A TTA TTA ACAC TA T t - 3r 14f - TBA2 TCAG AAG CC TCC AA CA TTTT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TG G TCAG G A TTT AA A TA t - 3r 18f - TBA2 CAA A ATT AG G ATAA AA TT TT AGTCCGTGGTAGGGCAGGTT GGGGTGACTTTT TA TTTT TAG G A TA TTC A 123 Tabl e 1. Modifi ed oli gonucl eoti des used in this wor k. All the st r and s used for the G- quadr upl ex di sr upti on studies and for ?- t hr om bi n i nt er act ion studi es wer e synt hesi zed i n the l aborat or y, as wel l as the st apl e st r ands cont ai ning m et hyl at ed - TBA (*) . Bol d rem ark s the sequence of TBA1 and TBA2 and r ed i dent i fi es the positi o n of the O 6 -m ethyl guani ne. The rest of the st aple st r ands wit h TBA1 and TBA2 sequences i nser t ed wer e purchased fr om comm er ci al sour ces. Scheme S1 . Sket c h of the DN A or i ga mi desi gn, showing the r ight a nd left dua l -a pt a mer syst e m for med by T BA1 (b lu e), T B A2 ( gr een) a nd 5-O 6 -Met hyl gua ni ne-T B A1 (r ed). T he loop indi ca t es the dir ect i on of the or i ga mi. List of the helper strands used to build the DNA origami. Al l t hes e oli gonuc l eot ides wer e pur cha sed fr om c omm er c ia l sour c es : t 1r 0g AGGGT T GAT AT AAGT AT AGC C C GGAAT AG GT G t 1r 2e T AAGC GT C GGT AAT AAGT TTT AAC C C GT C GAG t 1r 2f AGT GT AC T AT AC AT GGCTTTT GAT CTTT CC AG t 1r 4e AAC C AG AG AC C CT C AGAAC C GC C AC GT TC C AG t 1r 4f GAGC C GC C C C AC C AC C GGAAC C GCT GC GCC GA t 1r 6e GAC T T AC GT AAAGGT C GC AAC AT AC C GT C AC C t 1r 6f AAT C AC C AC C AT TT GGGAAT T AGAC C AAC C T A t 1r 8e TT AT T AC GT AAAGGT C GC AAC AT AC C GT C AC C t 1r 8f T AC AT AC AC AGT AT GTT AGC AAAC T GT AC AG A t 1r 10e T GAAC AAAG AT AAC C C AC AAG AAT AAG AC T CC t 1r 10f AT C AG AG AGT C AG AGGGT AAT T GAAC C AGT C A 124 t 1r 12e T ATTTT GC AC GCT AAC G AGC GT CT GAAC AC C C t 1r 12f T CTT AC C AAC C C AGC T AC AAT TTT AAAG AAGT t 1r 14e AT C GGGCT GAC C AAG T AC C GC AC T CTT AGTT GC t 1r 14f GGT ATT AAT C TTT CCTT AT C ATTC AT AT C GC G t 1r 16e C AT AT TT ATTTC GAGC C AGT AAT AAAT C AAT A t 1r 16f AG AGGC AT AC AAC GC C AAC AT GT ATCT GC GAA t 1r 18e AC AAAG AAAAT TT C AT CTTCT GAC AG AAT C GC t 1r 18f TTTT AGTT C GC GAG AAAAC T TTTTTT AT GAC C t 1r 20e AAAT C AAT C GTC GCT ATT AAT T AA AT C GC AAG t 1r 20f C T GT AAAT AT AT GT GAGT GAAT AAAAAGGC T A t 1r 22e TTT AAC GT TC GGGAG AAAC AAT AAC AGT AC AT t 1r 22f C TTTT AC AC AG AT GAAT AT AC AGT GC C AT C AA t 1r 24e TT ATT AAT GAAC AAAG AAAC C AC C TTTT C AGG t 1r 24f AT TTT GC GTTT AAAAGT TT GAGT AC C GGC AC C t1r 26e C T AAAGC AAAT C AAT AT CT GGT C AC C C GAAC G t 1r 26f AAAC C C T CTC AC CTT GCT GAAC C T AG AGG AT C t 1r 28e C T AAAAGC AAAT C AAT AT CT GGT C AC C C GAAC G t 1r 28f GC GT AAG AAG AT AG AAC C CTT CT GAAC GC GC G t 1r 30e GTT GT AGC C CT GAGT AG AAG AAC T AC TT CT G t 1r 30f AT C AC TT GAAT AC TT CTTT GATT AGTT GTT CC t 1r 32h T AC AGGGC GC GT AC T AT GGTT GCT AAT T AAC C t 3r 0g T GCTC AGT AC C AGGC GG AT AAGT GGGGGT C AG t 3r 2e GGAAAGC GGT AAC AGT GC C C GT AT C GGGGTTT t 3r 2f T GCCTT GAC AGT C TCT GAAT TT AC CC CTC AG A t 3r 4f GC C AC C AC T CTTTTC AT AAT C AAAT AGC AAGG t 3r 6e TT ATT C AT GTC AC C AAT GAAAC C AT T ATT AGC t 3r 6f C C GGAAAC T AAAGGT GAAT T AT C AT AAAAG AA t 3r 8f AC GC AAAG AAG AAC T GG C AT GAT TT GAGTT AA t 3r 10e GC GC ATT AAT AAG AGC AAG AAAC AAT AAC GG A t 3r 10f GC C C AAT AG AC GGG AG AAT T AAC TTT CC AG AG t 3r 12f C CT AAT TT AAGC C TT AAAT C AAG AAT C GAG AA t 3r 14e C T AAT TT AC C GTTTTT ATTTT C ATCTT GC GGG t 3r 14 f C AAGC AAGC GAGC AT GT AG AAAC C AG AG AAT A t 3r 16f T AAAGT AC C AGT AGGGC T T AAT T GCT A AAT TT t 3r 18e T AT GT AAAG AAAT AC C GAC C GT GTT AAAGC C A t 3r 18f AAT GGTTTT GCT GAT GC AAAT C C ATTTT CC CT t 3r 20f T AGAAT C C C CTTTTTT AAT GGAAAC GG AT T C G t 3r 22e AC AG AAAT C TTT GAAT AC C AAGT T AAT TTC AT t 3r 22f C CT GATT GAAAG AAAT T GC GT AGAAG AAGG AG t 3r 24e C GAC AAC T T C ATC AT ATT C CT GATC AC GT AAA 125 t 3r 24f C GGAAT T AC GT ATT AAAT C CTTT GGTT GGC AA t 3r 26e GC C AC GC TTT GAAAGG AAT T GAGG AAAC AAT T t 3r 26f AT C AAC AGG AG AGC C AGC AGC AAAAT AT TTTT t 3r 28e GT C AC AC GAT T AGT CTTT AAT GC GGC AAC AGT t 3r 28f GAAT GGC T AC C AGT AAT AAAAGGGC AAAC T AT t 3r 30e GT AAAAG AC T GGT AAT AT C C AGAAAT T C AC C A t 3r 30f C GGC CT T GGT CT GTC C ATC AC GC AT T GAC GAG t 3r 32h C AC GT AT AAC GT GCTTTC CT C GTT GC C AC C GA t 5r 0g C CT C AAG AG AAG G AT T AGGAT T AGAAAC AGT T t 5r 2e AC AAAC AAC T GCCT AT TTC GGAAC C T GAG AC T t 5r 4e T C GGC ATT C C GC C GC C AGC AT T GAT GAT ATT C t 5r 4f C AC C AG AGT T C GGTC AT AGC C C C CTC GAT AGC t 5r 6e AT T GAGGG AAT C AGT AGC GAC AG AC GT TTTC A t 5r 8e GAAGG AAAAAT AG AAAAT T C AT ATTT C AAC C G t 5r 8f T C AC A AT C C C GAGGA AAC GC AAT AA T GAAAT A t 5r 10e C TTT AC AGT AT CTT AC C GAAGC C C AGTT AC C A t 5r 12e GAGGC GT TT CC C AAT C C AAAT AAG AT AGC AGC t 5r 12f AT T ATTT ATT AGC GAAC C T C CC GAC GT AGGAA t 5r 14e T AAGT C CT GC GCC C AAT AGC AAGC A AG AAC GC t 5r 16e GC GTT AT AC GAC AAT AAAC AAC AT AC AAT AG A t 5r 16f C C AGAC G AC AAAT T CTT AC C AGT AGAT AAAT A t 5r 18e T AAC C T CC AAT AAG AAT AAAC AC C T AT C AT AT t 5r 20e AAAAC AAAC T GAG AAG AGT C AAT AT AC C T TTT t 5r 2 0f TT AAG AC G AT T AAT T AC AT TT AAC AC AAAAT C t 5r 22 e AAC C T AC C GC GAAT T AT TC ATTT C AC AT C AAG t 5r 22 f GC GC AG AG AT AT C AAAAT T ATTT GT AT C AGAT t 5r 24 e GGAT TT AGTT C AT C AAT AT AAT C C AGGGT T AG t 5r 24f GAT GGC AAAAGT AT T AG AC TTT AC AAGGT T AT t 5r 26e AGGC GGT C TCTTT AGG AGC AC T AAAC AT TT GA t 5r 26f C T AAAAT AAGT AT T AAC AC C GC CT C GAAC T GA t 5r 28 e GAAAT GG AAAAC AT C GCC ATT AAAC AG AGGT G t 5r 28 f T AGC C CT ATT ATTT AC AT T GGC AGC AAT AT T A t 5r 30 e AG AAGT GT C ATT GC AAC AGG AAAAAAT C GT CT t 5r 30f C C GCC AGC TTTT AT AAT C AGT GAG AG AAT C AG t 5r 32h AGC GGG AGC T AAAC AGG AGGC C G AG AAT C CTG t - 1r 0g T AT C AC C GT AC TC AGG AGGT TT AGAT AGT T AG t - 1r 2e AC GT T AGTTCT AAAGT TTT GTC GT GAT AC AGG t - 1r 2f C GT AAC G AAAA T GAAT TTT CT GT AGT GAAT TT t - 1r 4e C AAT GAC AGC TT GAT AC C GAT AGT C TC CCT C A 126 t - 1r 4f C TT AAAC AAC AAC C AT C GCC C AC GC GGGT AAA t - 1r 6e AAAC G AAAT GC C AC T AC GAAGGC AGC C AGC AA t - 1r 6f AT AC GT AAG AGGC AAAAG AAT AC AC T GAC C AA t - 1r 8e C C AGGC GC GAGG AC AG AT GAAC GGGT AG AAA A t - 1r 8f C TTT GAAAAT AGGC T GGC T GA C CT AC CTT AT G t - 1r 10e GGAC GT T GAGAAC T GGC TC ATT AT GC GCT AAT t - 1r 10f C GAT TTT AGGAAG AAAAAT C T AC GGAT AAAAA t - 1r 12e TTT GCC AGGC GAG AGGC TTTT GC AAT C CT GAA t - 1r 12f C C AAAAT AAGGGGGT AAT AGT AAAAAAAG AT T t - 1r 14e TTTT AAT T GCC C GAAAG AC T T C AAC AAG AAC G t - 1r 14f AAG AG G AAC G AGC TT C AAAGC G AAA GT TT C AT t - 1r 16e C GAGT AG AAC AGT T GATT CC C AAT AT TT AGGC t - 1r 16f T CC AT AT ATTT AGT TT GAC C ATT AAGC AT AAA t - 1r 18e C T GT AAT AGGT T GT AC C AAAAAC AC AAAT AT A t - 1r 18f GC T AAAT C CTTTT GC GGGAGAAGC C C GGAG AG t - 1r 20e T C AGGTC ATTTTT GAG AG AT C T AC CCTT GCTT t - 1r 20f GGT AGC T ATT GCCT GAG AGT CT GGTT AAA T C A t - 1r 22e AAAT AAT TTTT AAC C AAT AGG AAC AAC AGT AC t - 1r 22f GC TC ATTT C GC GTCT GGCCTT C CT GGC CTC AG t - 1r 24e GC TTCT GGC AC TC C AGC C AGC TTT AC AT T ATC t - 1r 24f GAAG AT C GT GCC GGAAAC C AGGC AGT GC C AAG t - 1r 26e C C C GGGT AC CT GC AGGT C GACT CT C AAAT AT C t - 1r 26f C TT GC AT GC C GAGCT C GAAT T C GTC CT GT C GT t - 1r 28e GGGAG AGGC AT T AAT GAAT C GGC C AC CT GAAA t - 1r 28f GC C AGC T GC GGTTT GC GT ATT GGGAAT C AAAA t - 1r 30e AGT TT GGAC GAG AT AGGGT T GAGT GT AAT AAC t - 1r 30f GAAT AGC C AC AAG AGT C C AC T ATT AAGC C GGC t - 1r 32h GAAC GT GGC GAG AAAGG AAGGG AAT GC GC C GC t - 3r 0g C C CTC AG AAC C GC C AC C CTC AG AAAC AAC GC C t - 3r 2e T GCT AAAC T C C AC AG AC AGC C C T CT AC C GC C A t - 3r 4e AT AT AT T CTC AGC TT GCTTTC GAGT GGGAT TT t - 3r 4f T C GGTTT AGGT C GCT GAGGC TT GC AAAG AC T T t - 3r 6e C TC AT CTT GGAAGT TT CC ATT AAAC AT AAC C G t - 3r 8e AGT AAT C TTC AT AAGGG AAC C G AAC T AAAAC A t - 3r 8f AC GGT C AAT GAC AAG AAC C GGAT AT GGTTT AA t - 3r 10e AC G AAC T AT T AAT C ATT GT GAAT TTC AT C AAG t - 3r 1 2e AC T GGAT AT C GTTT AC C AGAC G AC TT AAT AAA t - 3r 12f C AT AAC C C GC GT CC AAT AC T GC GGT ATT AT AG t - 3r 14e GAAGC AAAAAAGC GGAT T GC AT C AAT GTTT AG t - 3r 1 6e T C GC AAAT AAGT AC GGT GTCT GGAC C AG AC C G 127 t - 3r 16f T GC AAC T AGGT C AAT AAC C T GTTT AGAAT T AG t - 3r 18e C AAC GC A AAGC AAT AAAGC C T C AGG AT AC AT T t - 3r 20e AG AG AAT C AGC T GAT AAAT T AAT GCTTT ATTT t - 3r 20f AC C GTT CT GAT GAAC GGT AAT C GT AAT AT TTT t - 3r 22e C TTTC AT CTC GC ATT AAAT TTTT GAGC AAAC A t - 3r 22f GTT AAAAT AAC AT T AAAT GT GAGC AT CT GCC A t - 3r 24e TT C GC C AT GGAC GAC G AC AGT AT C GT AGC C AG t - 3r 24f GTTT GAGGT C A GGC T GC GC AAC T GTT CC C AGT t - 3r 26e T C AT AGCTT GT AAAAC G AC GGC C AAAGC GC C A t - 3r 26f C AC GAC GT GTTT CCT GT GT GAAAT TT GC GCT C t - 3r 28e T GGTTTTT CTTTC C AGT C GGGAAAAAT C AT GG t - 3r 28f AC T GC CC GCTTTT C AC C AGT GAGAT GGT GGTT t - 3r 30e T GGAC TC C GGC AAAAT C C CTT AT AC GC C AGGG t - 3r 30f C C GAAAT C AAC GT C A AAGGGC G AAAAGGG AG C t - 3r 32h C C CC GATTT AG AGC TT GAC GGGG AAAAG AAC G t - 5r 0g C TC AGAGC C AC C AC C C TC ATTTT CC GT AAC AC t - 5r 2e GAG AAT AGGT C AC C AGT AC AAAC T C C GC C AC C t - 5r 2f T GAGTTT C AAAGG AAC AAC T AAAG AT C TC C AA t - 5r 4f AAAAAAGGC TTTT GC GGGAT C GT C GGGT AGC A t - 5r 6e GC GAAAC AAG AGGC T TT GAGG AC T AGGG AGT T t - 5r 6f AC GGC T AC AAGT AC AAC GG AG AT T C GC GAC C T t - 5r 8f GC TC C AT GAC GT AAC AAAGC T GCT AC AC C AG A t - 5r 10e AAAG AT T CT AAAT T GGGCTT GAGAT T C ATT AC t - 5r 1 0f AC G AGT AG AT C AGT T GAGAT TT AGC GC C AAAA t - 5r 1 2f GGAAT T AC C AT T GAAT C C CC CTC AC C AT AAAT t - 5r 14 e T AC CTTT AAG GT CTTT AC C CT GAC AAT C GT C A t - 5r 1 4f C AAAAAT C AT T GCT CCTTTT GAT AAT T GCT GA t - 5r 16f AT AT AAT GGGGGC GC GAGC T GAAAT T AAC AT C t - 5r 18e T AT ATTTT C AT AC AGGC AAGGC AAAGC T AT AT t - 5r 18f C AAT AAAT AAAT GC AAT GC CT GAG AAGGC C GG t - 5r 20f AG AC AGT C TC AT AT GT AC C C C GGTTT GT AT AA t - 5r 22e AC C C GTC GTT AAAT T GT AAAC GT T AAAAC T A G t - 5r 22f GC AAAT AT GAT T CTC C GT GGGAAC C GT T GGT G t - 5r 24e GGC GAT C GC GC ATC GT AAC C GT GC GAGT AAC A t - 5r 24f T AGAT GGGGT GC GGGC CTCTT C GC GC AAG GC G t - 5r 26e GC TC AC AAGGGT AAC GC C AGGGT TTT GGGAAG t - 5r 26f AT T AAGT TTT CC AC AC AAC AT AC GC C T AAT GA t - 5r 28e AGC T GAT T AC TC AC AT T AAT T GC GT GTT AT CC t - 5r 28f GT GAGC T AGC C CTTC AC C GC CT GGGGTTT GCC t - 5r 30e T AT C AGGGC GAAAAT C CT GTTT GAC GGGC AAC 128 t - 5r 30f C C AGC AGGC G AT GGC C C ACT AC GT GAGGT GC C t - 5r 32h GT AAAGC AC T AAAT C GGAAC C C T AAAAC C GT C 129 3. Disruption of the G-quadruplex G-qua dr upl ex es a r e a fa mi l y of f our -st r a nded DN A st r uct ur es st a bili z ed b y the st a cki ng of gua ni ne t et r a ds, in whic h f our pla na r gua ni nes f or m a cycl ic a r r a y of hydr ogen b onds. [ 3 ] Modi fi ca t ions in the ba se c omp osit i on of t he t et r a ds a re p oor l y t ol er a t ed b y t hes e st r uct ur es. As a n exa mp l e, O 6 - met hyl gua ni ne [4 ] , a non- na t ura l ba se, ca n f or m a s ma l l er nu mb er of hydr oge n bonds a nd c ons equ ent l y dest a bil iz e t he G - qua dr upl ex. Schem e S2 . a) Schem ati c repr esent ati on of a G -t et r ad and a G - quadr upl ex. b) Chem ical st r uct ur e of guani ne and i t vari ant, O 6 -m et hy l guani ne. T o det er mi ne t he disr upt ion of the qua dr upl ex st r uct ur e, its c ir cula r dichr ois m sp ect r u m wa s r ec or ded. As s hown in Figur e S 1, the c ir cula r dic hr oi sm sp ect r a of met hyla t ed der i va t i ves of T BA1 did not s how t he pr es enc e of t he ma x i mu m a t 295 nm, cha r a ct er ist ic of a nt ipa r a ll el qua dr upl ex es a s T BA. Fi gur e S1. Cir cul ar di chroism of the m et hyl at ed der i vati ves of TBA1 (i n pi nk and gr een) , cont r ast ed wit h the pr ofi l e of the unm odi fi ed TBA1 (i n blue ) . 130 4. Fluorescence assays to study ?-thrombin-TBA binding. A f lu or esc enc e qu enc hi ng a ss a y wa s int ended t o eva l ua t e the va r ia t ion in t he b indi ng b et we e n ?-t hr ombi n a nd na t i ve or modi fi ed T BA. T his exp er i ment wa s des igned on t he ba sis of our pr evi ous wor k in t he devel op ment of a flu or esc ent T B A pr ob e to det ect the a ct i vit y of t he DN A r epa ir pr ot ei n hAGT . [5] Ther e, we demonst r a t ed tha t the int r odu ct i on of a met hyla t ed gua ni ne i n the t et r a ds of the T B A pr event s its f ol di ng, lea vi ng it in a n ext ended c onf or ma t i on. Given t ha t the two c onf or ma t i ons br ing the t wo en ds of T B A toget her or ta ke them fur t her a par t, we inc or por a t ed flu or es c enc e pr ob es a t ea ch ends, to be a bl e to mea sur e the cha nges in int ens it y. A T BA1 s equ enc e a nd its equ i va l ent s wit h the 5t h a nd 6th gua ni ne r epla c ed b y O 6 - met hyl gua ni ne wer e s ynt hes iz ed , containing a fluorophore (6? -F AM) a nd a qu encher gr ou p (Dabsyl) attached to their 5? or 3? ends respectively. A conformational change in the MB -T B A occurs when ? -t hr omb i n binds t he a pt a mer , du e t o its ca pa bilit y of f ol di ng t he r a ndom c oi l e d oli gonuc l eot i de int o its cha ir -l i ke qua dr upl ex st r uct ur e. T his r ea rr a ngement br ings c los e th e flu or op hor e a nd the qu enc her gr oup a tt a ched t o t he ends, a nd t hus r edu c es the int ens it y of its emit t ed f lu or es c enc e. It is exp ect ed tha t O 6 - met hyl gua ni ne- modi f i ed MB-T B As will not b e recognized nor folded by ? -t hr ombi n a nd t he flu or ophor e a nd the qu enc her wil l r ema i n physi ca ll y s epa r a t ed b eyond the F ?r st er dist a nc e, not a ll owi ng a si gnif ica nt decr ea se in t h e flu or es c enc e int ensit y. Schem e S 3 . Schem at i c repr esent ati on of the bases of the fl uor escence assay. A. Fl uor escence i s bl ocked when TBA1 i s i ncubat ed wi t h thr om bi n, which i s abl e to fol d it int o a quadr upl ex and thus reduce it s em i ssi on. B. By cont r ast, m et hyl at ed TBA1 cannot be folded and ther ef or e i t i s not abl e to adop t it s quadr upl ex st ructur e and the fl uor escence em i tt ed rem ai ns alm ost unchanged. Titra t ion of the modif i ed MB -T BAs wa s ca r r ied out in p hysi ol ogi ca l buff er a t 25 ?C . Figur e S 2 shows the tit r a t ion cur ves for the unmodi f i ed mol ecu la r bea con T B A a nd the t wo modif i ed T B As, eit her in p osit i on 5 or 6. As pr edi ct ed, the f lu or es c enc e of the M B -T B A wa s decreased almost in a continuous way when an excess of ? -t hr ombi n wa s a dded, u nt i l r ea c hi ng stabilization, indicating that ? -t hr omb i n wa s fol di ng the MB -T BA ba c k int o its qua dr upl ex st r uct ur e. T he st oi c hi omet r ic b indi ng r a t io obt a i ned fr om the tit r a t ion cur ve wa s 1:1 -1:1. 5, a s descr ib ed pr evi ous l y [ 6 ] . By contrast, the interaction between ? -t hr ombi n a nd the met hyla t ed M B-T BAs wa s clea r l y s ma l l er . Alt hough the f luor esc enc e int ensit y decr ea s ed s moot hl y a t the b egi nni ng, it r ea ched r a pidl y a pla t ea u wit hout r educi n g fluorescence?s intensity significantly. These 131 observations indicated that ? -t hr omb i n is a lmost u na bl e t o f ol d t he T B A typ ica l st r uct ur e. T he variation in the slope of the schuss proved that the interaction of ? -t hr omb i n wit h T BA dep ends on the ca pa bilit y of the a pt a mer in a dopt ing its qua dr upl ex st r uct ur e. In a ddit i on, this dif f er enc e seemed t o a pply f or bot h met hyla t ed T B As, indica t i ng t ha t modi fi ca t ions in b ot h t et r a ds a r e evenl y eff ic i ent in disr upt ing t he c ha ir -l ike st r uct ur e [ 5] and hence, making it impossible for ? - t hr omb i n to int er a ct . Fi gur e S2. Tit rati on of MB- TBAs i n physi ological buff er at 25?C. The di am ond red gr aph repr esent s the tit r ati on of the unm odi fi ed MB - TBA, triangle green plot shows the interaction of ? -t hr om bi n wi t h the 5- O 6 - m ethyl at ed der i vat ive of MB - TBA and the squar ed bl ue on e t he i nt er acti on wit h 6 -O 6 - Me G - TBA m ol ecul ar beacon. In the three cases, the concentration of MB is 1?M. The experiments were performed in t ri pl icat es. 132 5. EMSA assay to explore ?-thrombin-TBA binding. T o c onfir m the f lu or es c enc e r esult s, gel - mob i lit y s hif t a ssa ys wer e p er f or med t o a na l yz e t h e interaction between ? - thrombin and the modified TBAs. Increasing concentrations of human ? - t hr omb i n wer e incuba t ed wit h a const a nt T BA c onc ent r a t ion , a s shown in figur e S3. When non- modif i ed T BA(T ) 5 s equ enc e wa s incubated with ? -t hr omb i n, a clea r migration of the bands could be appreciated from 10 ?M concentration, representing that a population of the DNA was binding ? -t hr ombi n a t 1:10 r a t io. However , when incr ea si n g concentrations of ? -t hr omb i n wer e incuba t ed wit h 5 - O 6 -M eG-T B A(T ) 5 , no c omp l ex f or ma t io n cou l d b e obs er ved, even a t a mola r r at io of 1:15 a nd 1:20. T hes e r esult s c onf ir med t ha t the met hyla t i on of the a pt a mer pr event s its qua dr upl ex st r uct ur e f or ma t i on a nd thus ma kes it impossible for ? -t hr ombi n to r ec ogni z e a nd b ind t o it, a t lea st a t thes e c onc ent r a t ion r a t ios. Fr om this EMS A a ss a y, the a ppa r ent K D value for TBA: ? -t hr ombi n is est i ma t ed to b e 15 nM a nd f or 5-O 6 -M eG -T BA, >120 nM. Cons i der i ng tha t 6-O 6 -M eG -T BA a nd 5-O 6 -M eG- T BA b eha ved si mi la r l y in t h e flu or es c enc e a ss a ys, EMS A exp er i ment s wer e dec i ded to b e done onl y f or 5 -O 6 -M eG-T B A, a s only one of the s equ enc es wil l b e r equir ed to desi gn the or iga mi modif i ed st a ple st r a nds. Fr om t hes e r esu lts, we could presume that a concentration of 10 fold of ? -t hr ombi n ca n be suf fi c i ent to a ppr ecia t e a dif f er ent pa t t er n of u ni on of t his pr ot ei n to the T BA a nd O 6 -M eG- T BA-c ont a i ning or i ga mi. Fi gur e S3. Gel -m obil it y shi ft assays. Non- denat ur i ng (10% pol yacr yl am ide) PAG E im age of the ti tr ati on exper im ent for TBA(T) 5 (l anes 1 to 5) and 5-O 6 - Me G - TBA(T) 5 (l ines 6 to 9) . Lane M cor r esponds to a 10 bp doubl e st r anded DNA l adder . TBA( T) 5 (1) and 5-O 6 - MeG- TBA( T) 5 (6) ser ved as cont rol s, and wer e l oaded at 1 ?M con centr ati on, whi ch was the sam e for all the sam ples. Lanes 1, 2, 3, 4 and 5 cor r espond to TBA with increasing concentrations of ?- thrombin. TBA: ? -t hr ombi n rati os wer e the fol lowi ng: 1: 0, 1: 5, 1: 10, 1: 15 and 1: 20. Lanes 6, 7, 8 and 9 cor r espond to the sam e increasing concentration of ? -t hrom bi n i ncubat ed wi t h the m et hyl at ed TBA (1:0, 1:10, 1: 15 and 1: 20). 133 6. Origami formation control T he f or ma t i on of the ta l l r ect a ngl e or i ga mi wher e s ever a l st a ple st r a nds wer e modif i ed b y th e ins er t ion of T B A1, 5-O 6 -M eG-T B A1 a nd T BA2 wa s per f or med succ ess fu l l y, a s it is shown i n figur e S4. T he pr ot r udi ng T B As ca n b e c lea r l y s een b y mea ns of AF M a s two pa r a ll el lines a s it wa s int ended in the desi gn of the a r ra y. Fi gur e S4. AFM im age of the DNA or i gam i carr yi ng the modif i ed TBA st apl e st r ands. The or i gam i was form ed corr ectl y, showi ng that the i ntr oducti on of the m odif i ed til es di d not aff ect to i t s assem bl y. The pr otr udi ng TBA sequences can be obser ved as whit e li nes of up to 3 nm hei ght above mica, whi l e the pl annar unm odi fi ed ori gam i pr ot r udes 1. 5 nm fr om mica. [2 ] Image hei ght : -2. 0 nm to 2. 7 nm . 134 7. Additional AFM images of ?-thrombin-DNA origami interaction Fi gur e S5. AFM im ages of the or i gam i cont aini ng m et hyl at ed TBA st apl e st rands in the l ef t li ne, i ncubat ed with ?- thrombin. Image size: 3 ?m x 3 ?m. In this case, the height represents an increas e up to 3-4 nm , due to the ?- t hr om bi n mol ecul es at tached to the ori gam i surf ace. Im age hei ght : -2. 0 nm to 2.7 nm . Fi gur e S6. AFM im ages of the or i gam i cont aini ng m et hyl at ed TBA st apl e st rands in the l ef t li ne, i ncubat ed with ?- thrombin. Image size: 1 ?m x 1 ?m. Image height: - 2. 0 nm to 2. 7 nm . 135 Fi gur e S7. AFM images of the origami containing demethylated TBA staple strands, incubated with ? - thrombin. Images size: 3 ?m x 3 ?m. Image height: - 2.0 nm to 2. 7 nm . Fi gur e S8. AFM im ages of the or i gam i cont ai ning demethylated TBA staple strands, incubated with ? - thrombin. Images size: 1 ?m x 1 ?m. Images height: - 2. 0 nm to 2.7 nm . 136 8. Section analysis of the AFM images. Figure S9. Line section of the methylated origami: a) AFM images of the methylated origami binding Dthrombin. b) Horizontal section of the origami, in yellow, origami surface; in green, protruding methylated TBAs; in blue five Dthrombin molecules interacting with the natives TBAs. C) Cross-section of the origami. 137 9. Titration of the hAGT repair activity. Figure S10. Titration of hAGT activity in the origami. Repair of the methylTBAs with increasing concentrations of hAGT (0x, 2x, 5x and 10x). Figure S11. AFM images of the demethylated origami binding Dthrombin, showing improving occupation due to the increase in TBA repair by hAGT. 138 10. Quantitative and statistic studies of ?-thrombin-TBA binding on DNA origami 10. 1. Qua nt it a t ive st udi es of T BA- ?-t hr omb i n bindi ng on the met hyla t ed or i ga mi. T o exp l or e t he bindi ng r ea ct i ons qua nt it a t i vel y, all ?- t hr omb i n mol ecul es on t he wel l-s ha pe d two- di mens i ona l or i ga mi t emp la t es wer e c ount ed a cc or di ng to their posit i on, a nd of the 160 wel l- formed DNA origami structures investigated, around 20% of them contained all five ? - t hr omb i n mol ecu l es in p osit i ons c oi nci di ng wit h the unmodif i ed T B As. If we c onsi der t hos e origamis that were found to contain more than 4 ? -t hr ombi n mol ecu l es a tt a ched to t he T B A over ha ngs, mor e tha n t he 40% a r e to fit in t his gr oup . Acc or di ngl y, the c ou nt i ng r evea ls tha t a lmost the 60% of t he wel l-s ha ped or i ga mis c ont a ined mor e t ha n 3 out of 5 p oss ib l e p os it i ons oc cupi ed, a nd a lmost 80% c ons i der i ng mor e t ha n 2 occ upi ed p osit i ons. I n a ll, mor e t ha n 93% of the DNA arrays contained almost 1 ? -t hr omb in a tt a ched to the u nmodif i ed T BAs. 10. 2. Statistic studies to validate the ? -t hr ombi n bindi ng models Compa r ison of a ver a ges of t he t wo t yp es of bindi ng (r ight line a nd left line) wa s per f or me d using Student?s t distribution. Right line corresponded to the TBA2 and TBA1 staple strands, t he la t er met hyla t ed a nd subs equ ent l y r epa ir ed b y hAGT , a nd t he left one c ons ist ed of T BA1 and TBA2 staple strands, both unmodified. Binding of ? -t hr omb i n wa s supposed to ha ppe n si mi la r ly in b ot h lines a ft er hAGT demet hyla t i on. Compa r ison of a ver a ges r esult ed in a n insi gnif ica nt dif f er enc e b et ween them (p < 0. 5), whi c h c onfir med t ha t bindi ng wa s oc cur r ing equa l l y, wit hout a ny pr ef er enc e f or a ny line. 139 10. References Ma in text r ef er enc es: [4] c) N. V. Voi gt , T . T orr ing, A. Rot a r u, M. F. Ja cobs en, J. B. Ra vnsba ek, R. Subr a ma ni , W. Ma mdou h, J. Kjems, A. Mokhir , F. Bes enba c her , K. V. G ot helf, Nat Nanotechnol 2010, 5, 200. [1 8] M. Stolz, R. Got t a r di, R. Ra it er i, S. Miot , I. Ma r t in, R. I mer , U. Sta ufer , A. Ra duca nu, M. Duggeli n, W. Ba schong, A. U. Da ni els, N. F. Fr ieder ic h, A. Asz odi, U. Aeb i, Nat Nanotechnol 2009 , 4, 186. SI r ef er enc es : [1] F. M. Ruiz, R. Gil-R edondo, A. M or r ea l e, A. R. Ort iz, C. Fa br ega , J. Br a vo, J Chem Inf Model 2008 , 48, 844. [2] P. W. Rot hemu nd, Nature 2006, 440 , 297. [3] I. Smir nov, R. H. Sha fer , Biochemistry 2000, 39 , 1462. [4] M. Tra jkovski, P. Sket , J. Pla vec, Org Biomol Chem 2009, 7 , 4677. [5] M. T int or e, A. Avi no, F. M. Ruiz, R. Er it ja , C. Fa br ega , J Nucleic Acids 2010, 2010. [6] J. J. Li, X. Fa ng, W. Ta n, Biochem Biophys Res Commun 2002, 292 , 31. 140 Appendix 3 DNA nanoarchitectures: steps towards biological applications. 141 142 DNA nanoarchitectures: steps towards biological applications. Maria Tintor?, 1 Ramon Eritja 1 and Carme F?brega 1 * ChemBioChem (2014) Volume 15, Issue 10, pages 1374 ?1390 . DOI: 10.1002/cbic.201402014 . Impact factor: 3.06 1 IQAC-CSIC, CIBER-BBN Networking, Centre on Bioengineering Biomaterials and Nanomedicine. Cluster Building, Baldiri i Reixach 10, E-08028 Barcelona 143 144 DOI: 10.1002/cbic.201402014 DNA Nanoarchitectures: Steps towards Biological Applications Maria Tintor, Ramon Eritja, and Carmen Fbrega*[a] Dedicated to the memory of Dr. Francisco Snchez Baeza  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1374 CHEMBIOCHEM REVIEWS 145 Introduction Last year saw the sixtieth anniversary of the DNA double-helix model of Watson and Crick.[1] The advances in the field since then have been prodigious and far beyond expectations in areas as diverse as medicine, basic biology, genetics, forensics, and archeology, and culminated in the sequencing of the human genome.[2] Deoxyribonucleic acid is a self-assembling biopolymer that forms double helices directed by canonical Watson?Crick base pairing and is stabilized by hydrogen bonds, p?p stacking, and hydrophobic interactions. The B-form of these double-helical molecules have well-defined structures, which are repeated along the strands: the helical turn measures ~3.4 nm, the di- ameter is ~2.0 nm, and the twist angle between base pairs in solution is ~34.38.[1] The remarkable specificity of the molecular recognition between complementary nucleotides has made DNA an attractive molecule for scientists and engineers inter- ested in micro- and nanofabrication. Its predictability, rigidity, and precise structural control, as well as the creation of algo- rithms for de novo design of new self-assembled structures,[3] make it a useful building material to develop different kinds of nanotechnological platforms. Compared to other self-assem- bling molecules, DNA nanostructures offer programmable in- teractions and surface features for the precise positioning of other nanoparticles and biomolecules.[4] The field of DNA nanotechnology was pioneered by Ned Seeman, who set the basis for the use of DNA as a scaffold for nanoscale building.[5] Seeman?s original goal was the creation of regular 3D lattices of DNA that could be used as scaffolding for the rapid, orderly binding of biological macromolecules, in order to speed the formation of suitable crystals for 3D pro- tein?structure elucidation in X-ray diffraction studies.[5] This gave rise to the tile-based assembly method, which has been used to synthesize two-dimensional periodic lattices[6] and three-dimensional architectures (e.g. , a cube in solution,[7] and a solid-phase truncated octahedron).[8] In both cases a strategy that relies on repeated enzymatic treatment and purification was used. Another important breakthrough in the field of structural DNA nanotechnology has been the development of DNA ?ori- gami? by Paul Rothemund,[9] where a long scaffold strand is folded with the help of hundreds of short ?staples? to create the desired 2D shape. Since then, various 2D and 3D DNA motifs have been designed, and extensive studies are currently ongoing to apply these nanostructures to a large range of bio- medical, computational, and molecular motor purposes. In this review, we outline the evolution of DNA nanotechnol- ogy from simple to sophisticated, complex systems, with the aim of providing insights into the applications of the DNA- based nanostructures for the study of biological systems. 1. Basic Considerations in DNA Nano- technology The design and construction of DNA-based nanoarchitectures involves several major considerations. The design of the con- struct and its potential applications should be identified, and concerns, such as flexibility or rigidity of the scaffold, and static or dynamic devices, should be defined. It must be con- sidered whether symmetric or non-symmetric patterns of the array are required, and whether the growth is to be limited or infinite. The periodicity and growth dimensions of the scaffold are also important. All these requirements of the scaffold archi- tecture determine the DNA motif, as well as the building blocks design, which can comprise junctions, loops, crossovers, and single-strand binding sites. These motifs can be chemically modified to enable the attachment of non-nucleic-acids, such as proteins and nanoparticles. In addition, particular properties, such as length, specific hybridization of complementary strands, minimization of undesired hybridization, different binding sites for proteins, and restriction sites for characteriza- tion, need to be studied to achieve the final nanostructure with the best yield. Seeman developed the first program, SEQUIN, to facilitate the design of oligonucleotides for the preparation of arrays.[10] Since then, several automated software packages have been described.[11] An overview of the concepts and algorithmic ap- proaches of some programs was presented by Brenneman and Codon.[12] Finally, the designed DNA oligonucleotides have to be as- sembled in vitro, and it can be as easy as following a typical self-assembly process by simple mixing components. However, sometimes more complex protocols are needed, such as the DNA?s remarkable molecular recognition properties, flexibility, and structural features make it one of the most promising scaf- folds to design a variety of nanostructures. During recent de- cades, two major methods have been developed for the con- struction of DNA nanomaterials in a programmable way; both generate nanostructures in one, two, and three dimensions. The tile-based assembly process is a useful tool to construct large and simple structures ; the DNA origami method is suita- ble for the production of smaller, more sophisticated and well- defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed pat- terns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins, and be used as platform for genomic and drug delivery applications. In this review we examine recent pro- gresses towards the potential use of DNA nanostructures in molecular and cellular biology. [a] M. Tintor, Dr. R. Eritja, Dr. C. Fbrega Biomaterials and Nanomedicine IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering c/Jordi Girona 18?26. 08034 Barcelona (Spain) E-mail : carme.fabrega@iqac.csic.es  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1375 CHEMBIOCHEM REVIEWS www.chembiochem.org 146 assembly of single building blocks, different kinds of purifica- tion, structural integrity characterization by biochemical meth- ods like electrophoresis,[13] digestion and/or fragmentation analysis,[14] and visualization techniques such as atomic force microscopy (AFM),[6] transmission electron microscopy (TEM), and cryo-electron microscopy.[15] Major DNA motifs In the early 1980s, Seeman conducted pioneering work on the construction of artificial nucleic acid architectures by using branched DNA junctions containing three or four arms of double-stranded DNA helices with sticky ends (Figure 1A).[5] Variants with five and six arms were less stable.[16] Sticky ends at the junction arms enabled self-assembly into 2D lattices (by enzymatic or chemical coupling).[5] Three-dimensional ?dendrit- ic? structures labeled with different fluorescent dyes[17] have been generated with these junctions motifs, but they are con- sidered inappropriate for the assembly of regular architectures because of their high flexibility.[18] A study by Malo and co- workers demonstrated that the presence of a DNA-binding protein in the process of assembling a four-armed junction resulted in a grid of squares rather than hexagon?triangle latti- ces (Kagome-lattices).[19a] One-dimension ?railroad track? and two-dimensional lattices have been assembled with four arm junctions despite the high flexibility of these building blocks.[19b] The generation of ?44 tile? motifs (containing four-armed junctions with T4 loops at their arms) facilitated assembly as linear ribbons and two-dimensional arrays. He et al. assembled larger two-dimensional arrays from this motif by applying two new concepts (Figure 1B).[20] First, the motif was designed to be symmetric, and second, it followed a ?corrugation strategy?, consisting of two adjacent building blocks facing opposite di- rections in such a way that the small curvatures of the individ- ual motifs are canceled (by each other) instead of accumulat- ing throughout the structure. This system has been used to template streptavidin (STV) into periodic protein arrays, by in- Maria Tintor received her B.Sc. in Pharmacy at the University of Barcelo- na in 2009. She then obtained a Master in Molecular Biotechnology at the Uni- versity of Barcelona in 2011. She is cur- rently working on her Ph.D. thesis in the laboratory of Dr. R Eritja under the supervision of Dr. Fbrega at the IQAC- CSIC Institute, where she focuses on the design and biological evaluation of inhibitors of alkyl-transferases as po- tential enhancers in cancer therapy, by using DNA nanotechnology tools. Ramon Eritja received his Ph.D. in Chemistry from the University of Bar- celona in 1983. In 1990, after two post- doctoral positions, he became a group leader at CSIC, and in 1994 he trans- ferred to EMBL as a group leader. He returned in 1999 as a Research Profes- sor at IQAC-CSIC leading the Nucleic Acid Chemistry Group. In 2012 he was appointed director of the IQAC-CSIC. His research focuses on oligonucleo- tide and peptide synthesis for biomed- ical and nanotechnological applica- tions. Carmen Fbrega completed her Ph.D. on the incorporation of mutated bases in oligonucleotides at the University of Barcelona in 1997. After postdoctoral studies on aminoacyl tRNA synthetase and mRNA capping enzymes, she was appointed Tenure-Track Scientist at CNIO in 2003, where she researched inhibition of DNA repair enzymes. Since 2009 she has been a Senior Sci- entist in Dr. Eritja?s group developing new biosensing methods for the study of DNA repair mechanisms and its ap- plication to cancer. Figure 1. Major motifs in DNA nanotechnology. Arrows indicate the 3?-ends of DNA strands. A) Branched DNA junctions containing three or four arms/ strands. Double-helix arms form between strand pairs. B) A 44 DNA tile of nine unique strands. C) A DNA triangle contains three DNA duplexes, shown as rods (left) or as thin lines for single DNA strands (?st? right). D) A DX mole- cule results from double exchange between double helices. A TX molecule results from two successive double reciprocal exchanges. Both molecules contain exchanges between strands of opposite polarity. A PX molecule is formed when two double helices exchange strands at every point where helices come into contact. A JX2 molecule differs from PX in that it lacks the two central crossovers. The exchanges in PX and JX2 are between strands of the same polarity.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1376 CHEMBIOCHEM REVIEWS www.chembiochem.org 147 serting a biotin group into the T4 loops at the tile center.[21] A step toward versatile, size-controlled, and programmable DNA- based nanoarrays was reported with cross-shaped tiles, by using a stepwise hierarchical assembly technique.[22] DNA triangles are promising candidates for the fabrication of ?tensegrity? structures. These are constructs of rigid rods connected by short ?tendons? (here, branch points of DNA junction motifs) that are connected by short tensegrity seg- ments (Figure 1C).[23] As mentioned above, DNA junctions are rather flexible, and assembly does not often yield a regular structure. Seeman and co-workers solved this problem by cre- ating the most fundamental motif in DNA nanotechnology: the double crossover (DX) motif (Figure 1D). This consists of two double-stranded helices that interchange single strands at two crossover points.[24] DX motifs with sticky ends allow the constructions of 2D arrays.[6] Rinker demonstrated that it is possible to replace DNA strands in a DX motif with locked nu- cleic acid (LNA) strands in the formation of 2D arrays.[25] After the success of the DX motif, triple-crossover (TX) tiles were de- signed; these consist of three helices lying in a plane, connect- ed at crossover points (Figure 1D),[26] as found in linear lattices, 2D arrays,[26] and DNA tubes.[27] Various other tiles (other than square and rectangular) have been prototyped. At least three versions of triangular tiles have been described: in one the plane is tiled entirely with triangles,[23] and in the other two hexagonal patterns are formed.[28] Three-point[29] and six-point star[30] motifs have also been self-assembled into two dimen- sional arrays. Another important class of DNA motif is the paranemic crossover (PX) motif,[31] which is composed of two parallel heli- ces with crossovers at every possible site (Figure 1D). This motif can be topoisomerased to form the JX2 motif, where the relative positions of the two ends of the motif are rotated 1808 relative to PX (Figure 1D).[32] Extensive information about DNA major motifs, self-assembled DNA nanostructures and mechan- ical devices is found in several reviews.[33] 1.2. Polyhedra One of the major goals in DNA nanotechnology is the con- struction of three-dimensional DNA structures. However, 3D constructs have proved to be more difficult to build than 2D constructs.[34] The first 3D structures to be assembled were a cube[7] and a truncated octahedron[35] (Figure 2A and B), but these were obtained with low yield and were not suitable for biological applications. Seeman successfully engineered crys- tals with altered lattice dimensions in 2004,[36] thus pursuing his initial idea to generate 3D crystals for X-ray crystallographic study of ?guest? molecules, such as proteins.[37] A remarkable achievement was the design and synthesis of an octahedron with DX-like edges and PX motifs :[38] a 1.7-kb, ssDNA molecule amplified by polymerase and folded with five 40-mer synthetic oligodeoxynucleotides (Figure 2D).[15] Later, a chiral tetrahedral unit with short double helix edges was constructed in higher yield (Figure 2C).[39] By this approach, it was found that adding programmable linkers confers structural stability and resistance to deformation. The robustness of the structure was studied by AFM.[40] One of the edges of the tetrahedron was examined for mechanical movement by exchanging the DNA strands, thereby producing a reconfiguration of the DNA tetrahedral shape in a precise and reversible manner.[41] Covalently closed octahedral DNA cages were reported to be resistant to thermal and chemical denaturation, and could thus represent a step to- wards to the use of these devices as delivery systems.[42] More Figure 2. A) Cube formed from twelve equal-length double-helical edges ar- ranged around eight vertices. B) Double-helical representation of an ideal truncated octahedron. Reprinted with permission from ref. [35] . Copyright 1994, American Chemistry Society. C) DNA tetrahedron. Reprinted with per- mission from ref. [40] . Copyright 2005, American Association for the Ad- vancement of Science. D) 3D model of an octahedron. Secondary structures consist of five DX and seven PX motifs; hybridization of PX motifs for 3D for- mation. Reprinted with permission from ref. [15] . Copyright 2004, Nature Publishing Group. E) Polyhedral structures: tetrahedron, dodecahedron and buckyball, assembled from three-point star building blocks. Adapted with permission from ref. [45] . Copyright 2008, Nature publishing Group.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1377 CHEMBIOCHEM REVIEWS www.chembiochem.org 148 complex and modified polyhedra were also created in a single step.[43] Sleiman reported a new method to increase the range of 3D structures to include a prism, a cube, pentameric and hexame- ric prisms, a heteroprism, and a biprism.[44] Mao took advant- age of the ?sequence symmetry?[20] to generate three-point star motifs or tiles. By controlling the flexibility and the con- centration of the tiles and adapting the length of central single-stranded loop, his group synthesized a tetrahedron, a dodecahedron, and a buckyball from a minimal set of building blocks (Figure 2E).[45] By the same strategy, a five point start motif was designed and self-assembled into icosahedra.[46] Strategies to restrict polyhedral faces to those with an even number of vertices were used to assemble DNA cubes.[47] Triso- ligonucleotides with an organic vertex (C3h linker) were em- ployed to assemble a DNA dodecahedron, thus demonstrating the usability of this construct for multimodular machines by the introduction of extension arms that allow overhanging with other sequence.[48] Chirality is an essential feature in nature.[49] Enzymes and cell receptors have the ability to distinguish different substrates stereoisomers, thus leading to highly efficient and stereoselec- tive reactions and binding. Understanding and control of chir- ality is crucial for drug design,[50] encapsulation,[51] and other processes.[52] The chirality of DNA duplexes is indisputable; however, its extension to building stereoisomeric pure DNA nanostructures has been a challenge. Mitchell et al. described the self-assembly of chiral nanotubes,[53] and subsequently, He et al. reported a well-defined chiral octahedron.[54] The assem- bly of both structures was relatively straightforward, and their chiral features might enable the use of this type of structures for all the applications mentioned above. Another important tool in 3D DNA nanotechnology is ?ten- segrity?: the balance between components that are in pure compression and those in pure tension for stability. The Shih group reported nanoscale prestressed 3D tensegrity structures in which rigid bundles of DNA resist compressive forces exert- ed by single-stranded DNA segments that act as tension-bear- ing cables.[55] These tensile structural elements could be used to study molecular forces, cellular mechanotransduction and other fundamental processes. 1.3. DNA origami The implementation of DNA computing by Winfree?s group in 2004[3] formed the basis for the development of what would be one of the most important breakthroughs in the field: DNA origami, as reported by Rothemund in 2006.[9] This involves the folding of a long, circular, single-stranded DNA scaffold into ar- bitrary 2D shapes, directed by a collection of various (hundreds of) shorter single-stranded oligonucleotides that are comple- mentary to different regions of the scaffold (Figure 3A). The specific matching of the scaffold and ?staple? strands leads to well-shaped nanostructures, with high yield and reproducibility (Figure 3B). In addition, it avoids stoichiometric dependence, thus eliminating the need for purification and exact determina- tion of the concentration of the oligonucleotides, and thereby reducing the time and effort required for its assembly. One of the most attractive features of this technique is the addressa- bility of the surface, thus permitting the attachment of differ- ent biomolecules or nanoscale objects by the modification of specific staple strands at desired positions.[4] The origami approach was further developed for the con- struction of 3D nanostructures. Gothelf?s group assembled a box with a controllable lid from a combination of six origami sheets;[56] at the same time, Kuyuza and Komiyana designed another box-shaped origami by selective closing of a pre- formed motif.[57] A scaffold DNA origami of a closed DNA tetra- hedron was designed by Ke et al. ; this resembled the icosahe- dral structure of many viral particles.[58] Shih and co-workers in- troduced a strategy to generate 3D nanostructures by honey- combing (or square packing) scaffolded DNA[59] (Figure 3D). This group also described the insertion of twists and curves into 3D nanoconstructs, through addition or deletion of base pairs at specific positions within the helical layers.[60] The Yan group developed a method based on the organization of con- centric DNA rings of different radii to build DNA shapes with complex curvatures (Figure 3E).[61] They also reported the design of quasi-2D origami tiles with a Mobius topology, which implies a certain degree of twist and curvature, as well as flexi- bility and strength.[62] These novel 3D structures further dem- onstrate the robustness and potential of the origami technique in different kinds of application. An important limitation of this technique is that it results in a relatively small area, limited by the length of the standard scaffold. In many cases, the origami surface might not be suffi- cient for the precise positioning of functional molecules or for other applications. For this reason, other efforts in the field are focused on assembling larger structures, including approaches such as algorithmic assembly from origami seeds,[63] origami oligomerization[64] and polymerization,[65] the use of eight-helix bundles as staples,[66] long single-stranded PCR amplification products,[67] and double-stranded viral genomes[68] as scaffolds. An example of this oligomerization is given in Figure 3C.[64b] A more detailed description of these methodologies can be found in a review by T?rring et al.[69] A novel approach by Sugiyama?s group was to design RNA- templated DNA origami structures that were folded to form seven-helix, bundled rectangular shapes. These nanoconstructs were composed of an RNA transcript (as scaffold) with DNA staple strands, and have been exploited for specific applica- tions such as catalysis and RNA interference.[70] Recently, DNA origami technology is becoming more acces- sible to researchers from different fields because of the devel- opment of several computational tools for the design of di- verse structures, for example SARSE-DNA,[71] caDNAno,[72] and CanDo.[73] These software tools facilitate the sketching and pat- terning of the nanoconstructs and broaden the scope in terms of time, cost and need for researcher experience. Today, a few hours and basic computation fluency is sufficient to design ori- gami tiles for a wide variety of applications.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1378 CHEMBIOCHEM REVIEWS www.chembiochem.org 149 2. Biochemical Applications The advent of nanotechnology and the expansion of computa- tional algorithms has led to an explosion in the number of DNA scaffolds that can be created, and for a wide variety of applications, including nanomaterial assembly, biosensors,[41] molecular computation,[33d,74] biomolecular actuation, drug de- livery, and nanodevices.[33d] 2.1. Tiles and Polyhedra One great challenge in engineering functional materials and devices has been how to integrate and assemble them into hi- erarchical arrays with minimal defects. However, innovative methods by Mirkin[75] and Alivisatos[76] demonstrated that gold particles and surfaces are generally easily modified with oligo- nucleotides, and assembled into different packing densities and arrangements for a variety of applications. The design and generation of more-complex DNA tile motifs have been used for the assembly of lattices, particularity for allowing the incor- poration of components that protrude from the plane,[26] such as gold nanoparticles[77] of different size[78] (Figure 4A), and combinations of streptavidin and gold nanoparticles.[79] All these developments open the door to extensive work for the creation of nanoelectronic, nanophotonic, and optoelectronic devices, which are beyond the scope of this review. Mirkin[80] and Gang[81] demonstrated that DNA can be used to control the crystallization of nanoparticle?oligonucleotide conjugates; different DNA sequences guide the assembly of the same type of inorganic nanoparticles into different crystal- line states, thereby controlling distance and packing dynamics. Nanostructures for the sensitive detection of biomolecules have been reported by Niemeyer and co-workers by using DNA?streptavidin conjugates. Such DNA?protein conjugates could be versatile molecular tools in immunoassays, nanoscale biosensor elements, microstructural biochips, and supramolec- ular devices.[82] The streptavidin?biotin interaction has been the most explored method in the creation of molecular net- works.[21,22, 83] Efficient assembly into 2D DNA arrays of other ligands such as DNA aptamers[84] with target proteins such as thrombin or antibodies has been accomplished (Figure 4B).[85] In a similar approach, Krishnan and co-workers designed the I- switch, a DNA nanosensor that maps spatial and temporal pH changes inside living cells by FRET.[86] Organic and inorganic linkage connecting multiple DNA strands has been extended to include dendrimers,[84a, 87] macrocycles,[88] multibranched Figure 3. A) Formation of DNA origami. B) Example DNA origami nanostructures and their AFM images described by Paul Rothemund. Reprinted with permis- sion from ref. [9] . Copyright 2006, Nature Publishing Group. C) Example strategy for enlargement of DNA origami dimensions, by the union of ?jigsaw? pieces. Adapted with permission from ref. [64b]. Copyright 2011, American Chemistry Society. D) Strategy to generate 3D nanostructures by honeycomb or square packing of the scaffolded DNA. Reprinted with permission from ref. [59a] . Copyright 2009, Nature Publishing Group. E) Method to build DNA shapes with complex curvatures, based on the organization of concentric DNA rings of different radii. Adapted with permission from ref. [61]. Copyright 2011, American Association for the Advancement of Science.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1379 CHEMBIOCHEM REVIEWS www.chembiochem.org 150 structures,[89] and linear arrays containing functional mole- cules.[90] Many biochemical systems in biology require more than just one catalytic reaction by a single enzyme; for instance, with multienzyme reaction complexes,[91] the substrate is passed di- rectly from one catalytic site to the next. A benefit of special arrangements of enzymes is concerted action of several com- plementary enzymes to cooperatively perform a complex task. Artificial co-localization of enzymes (to improve reaction flux) has been attempted by a variety of approaches, such as with solid matrices, by chemical connections, by recombinant engi- neering, or by using protein?protein interaction domains.[92] In recent decades, there have been several attempts to apply DNA scaffolding for the creation of artificial multi- enzyme systems. Niemeyer and co-workers[93] demonstrated for the first time that DNA-directed assembly of two proteins could be the initial step for the efficient generation of artificial multienzyme complexes (Figure 4C). Later, they also reported the covalently linkage of enzymes like glucose oxidase (GOX) or horseradish peroxidase (HRP) to DNA to generate supra- molecular complexes.[94] In both cases, improved performance was observed when the proximity of the cooperative enzymes was constrained.[93,94] The first example of enzyme systems ar- ranged on a larger supramolecular DNA scaffold was demon- strated by Wilner et al.[95] Again with the GOX/HRP system, they reported a significant increase in overall activity when the two enzymes were held in close proximity by a hexagon-type DNA scaffold. In addition, they immobilized glucose dehydro- genase and its tethered cofactor NAD+ on the scaffold, and observed different activities for different tether lengths and distances. More recently, the same multienzyme system was in- corporated into a switchable DNA scaffold to dynamically con- trol a diffusion distance of intermediate products.[85c] In this way, the system reversibly regulated the reaction in situ.[96] An- other impressive achievement was the control of spatial organ- ization of a multienzymatic complex by using RNA molecules that are sequence-programmed to be isothermally assembled into predefined discrete, 1D and 2D structures in vivo. The re- action was optimized as a function of scaffold architecture.[97] Further studies could be useful for the development of novel catalysts for enzymatic processes or to perform multistep Figure 4. A) DNA scaffold deposited on mica combined with DNA-encoded nanocomponents of two sizes, and AFM and TEM images. Adapted with permis- sion from ref. [78a] . Copyright 2005, American Chemical Society. B) Aptamer-directed self-assembly of thrombin protein (green spheres) on a 2D DNA tile gen- erates a linear protein arrays. AFM images prior to and after thrombin binding. Adapted with permission from ref. [84a] . Copyright 2005, Wiley. C) DNA-direct- ed organization. Biotinylated recombinant enzymes are coupled to covalent DNA-streptavidin conjugates. D) Dynamic structural changes upon hybridization of a specific complementary strand in a prism structure. E) Tetrahedral delivery system for siRNA conjugated to cancer-targeting ligands. AFM image shows tetrahedron nanoparticles on mica. AFM image show the three upper edges of the tetrahedral. Adapted with permission from ref. [99c] . Copyright 2012 Nature Publishing Group. F) Tetrahedron containing a molecule of cytochrome c. Adapted with permission from ref. [100a]. Copyright 2006, Wiley.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1380 CHEMBIOCHEM REVIEWS www.chembiochem.org 151 chemical transformations of cheap precursors into drugs and fine chemicals. DNA nanostructures are also promising candidates for drug delivery, as they possess a combination of features. Firstly, they have a great flexibility in size and shape, thus offering a variety of parameters for optimization in order to improve cellular in- ternalization.[84b,98] Sleiman has reported a new method for the construction of the first dynamic 3D DNA capsules whose size is able to switch between three different lengths. These 3D switchable capsules can be used for drug delivery and dynamic DNA structures (Figure 4D).[44] In addition, DNA can be func- tionalized with different molecules as cargos, by covalent mod- ification,[98a] self-assembly of DNA polyhedral nanoparticles with a well-defined size which can deliver small molecules, siRNAs and antisense oligodeoxynucleotides into cells and si- lence target genes in tumors,[99] and encapsulation (Figure 4E and F).[100] Recently, Wollman et al. created a DNA molecular motor that performs a wide range of tasks, including self-or- ganization, capture, and concentration of cargoes transport signals for release and track or follow disassembly.[101] More- over, these structures are stable under physiological condi- tions[102] and, as natural material in living creatures, possess excellent biocompatibility.[98a] 2.2. DNA origami Since Rothemund?s original report,[9] a large number of re- search groups have adopted the DNA origami technique in several novel applications, from all type of chemical or enzy- matic single-molecule reactions to applications in photon- ics,[103] nanorobotics,[104] and nanomechanics.[105] Here we pro- vide a brief list of these applications. Gold nanoparticles have been selectively positioned and patterned on origami structur- es.[103b,106] Yan and co-workers connected gold nanoparticles to DNA origami nanotubes, a development which represents progress towards the goal of combining origami self-assembly and lithographic methods to design patterns on surfaces.[107] Precise DNA sequences with sugar moieties covalently at- tached were used for the site-specific immobilization of fluo- rescent silver nanoclusters at predefined positions on the nanoscaffold;[108] the resulting high density array of emissive nanoclusters could have potential applications in the nano- fabrication of semiconductor nanostructures. A methodology combining gold and silver nanoparticles using a predetermined pattern was developed by Pal et al.[103c] A general method for arranging single-walled carbon nanotubes (SWNT) in two di- mensions by using DNA origami was reported by the Winfree group; it has potential use in the synthesis of multi-SWNT logic gates or memory circuits.[109] As a final example, Stein et al.[110] achieved a combination of multistep energy transfer in a photonic wire-like origami structure using fluorophores that undergo an energy-transfer cascade. This work demon- strates the wide and multidisciplinary potential of DNA origami nanotechnology for the above-mentioned applications. Given that the field is so extensive, here we focus on the biological and biochemical applications of DNA origami and their pros and cons for specific uses; we do not review the more me- chanical and computational approaches, which have been cov- ered in many excellent reviews.[4,111] 2.2.1. Material organization: There is almost no limitation in attaching functional biomolecules to DNA origami, as chemi- cally modified sequences can be incorporated to staple strands, or specifically modified DNA can be readily attached by hybridization to staples strands.[112] Several research groups have demonstrated the possibility of displaying nucleic acids on the origami surface: when mRNA probes are attached to DNA origami structures, they can be used for the detection of gene expression at the single-mole- cule level.[113] These findings open the door for using the origa- mi technique for a great variety of diagnostic applications. By using the same basic concept, the complete sequence of a syn- thetic oligonucleotide can also be tracked down over an origa- mi surface by using complementary probes, with broad appli- cations for the recognition of unknown DNA sequences.[114] In addition, a huge number of proteins have been pro- grammed over DNA nanoconstructs. Niemeyer and co-workers decorated DNA origami with three types of proteins by using coupling systems orthogonal to the biotin?streptavidin interac- tion (Figure 5A).[115] This study raised the question over prefer- ence for face-down or face-up orientation of the origami struc- tures when deposited over mica; this was found to depend principally on the method used for the binding of the proteins to the origami tile. A possible application of the origami technique is the study and control of the binding distances between DNA and the protein partners. For example, Rinker et al. studied the multiva- lent interactions of a-thrombin and its two a-thrombin bind- ing aptamers (TBA), and the distance dependence for simulta- neous binding (Figure 5B).[116] By taking advantage of the spa- tial accuracy of this platform, it was found that a-thrombin binds the dual system with a tenfold better recognition than with a single aptamer, and it was shown for the first time that 5.3 nm between the two aptamers is optimal for bivalent bind- ing. This group used a similar strategy to study the distance- dependent kinetic processes associated with the GOX/HRP enzyme pair. The distance between enzymes was systematical- ly increased from 10 to 65 nm, thereby revealing strongly enhanced activities for these enzymes when closely spaced, whereas the activity dropped dramatically when as little as 20 nm apart. They also observed that strong activity enhance- ment was not simply achieved by reducing the inter-enzyme distance but also by restricting the diffusion of intermediates to the 2D surface connecting the enzymes.[117] These results further demonstrate the high resolution and spatial accuracy of the technique, and the possibility of using it for the visuali- zation and characterization of single-molecule events. A new method was reported to direct protein nanopattern- ing over origami nanoconstructs, by using nitrilotriacetic acid and histidine-tag metal-linked interactions. This report de- scribes a new way of selectively binding a staple strand to the target protein?highly relevant in protein immobilization on DNA origami.[118] Although it provided a novel way of attaching proteins to the DNA origami surface, this method needs to be extended to other tags, as the histidine tag is not well tolerat-  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1381 CHEMBIOCHEM REVIEWS www.chembiochem.org 152 ed by all proteins, and can prevent folding and/or activity. The chemical functionalization of the target molecules could be also be a drawback, so will need further development to extend this method to a broader range of proteins. Streptavidin nanoarrays were created by using periodical nanometer-scale wells embedded in DNA origami in which single streptavidin molecules could be held (Figure 5C).[119] This represents an innovation because all in previous attach- ments nanomaterials were anchored to DNA origami at the surface of the array. In this design, gold nanoparticles were nanopatterned, by selectively capturing a single nanoparticle in each well of the punched DNA origami. Subsequently, they combined alternating organic (streptavidin) and inorganic (gold nanoparticle) materials in a defined pattern.[120] Recently, zinc-finger proteins (ZFP) were assembled on an origami scaffold (Figure 5D).[121] Orthogonal targeting of the specific locations in the structures was demonstrated by using two adaptors, and Escherichia coli lysate containing the adap- tor-fused proteins successfully afforded the expected protein? DNA assembly. The diversity of target DNA sequences and the semi-programmable design of ZFPs offers orthogonal adaptors, thus enabling the placement of multiple engineered proteins at different locations on DNA-origami structures. Nature uses multiple enzymes in close proximity to efficiently carry out chemical reactions and signal transduction. Such assemblies of multiple proteins could be realized in vitro by using DNA ori- gami structures that have predefined binding sites and various kinds of ZFP adaptor-fused proteins. 2.2.2. Single-molecule reaction: Single-molecule studies are crucial to understand the trajectories of molecules as they un- dergo reactions. For this reason, considerable effort has been made in this area, and the application of the origami tech- nique has given the opportunity to solve many spatial and iso- lation problems because of its nanometric resolution and ad- dressability. In addition, any functionality that can be conjugat- ed with DNA can be placed on the origami surface, thereby serving as a molecular recognition probe. Some of the applica- tions described in the above could be also considered single- molecule reactions, for example RNA hybridization assays or the distance-dependent thrombin-aptamers binding. One of the first reports to show the possibility of detecting single molecule reactions in real time at positions of an origa- Figure 5. Examples of precise material organization on DNA origami surfaces. A) Orthogonal decoration of DNA origami, raising the controversy of face-up or face-down disposition of the nanostructures over mica surfaces for visualization. Reprinted with permission from ref. [115] . Copyright 2010, Wiley. B) Distance- dependence study of multivalent interactions of a-thrombin with two binding aptamers (TBA) on an origami platform. Adapted with permission from ref. [116] . Copyright 2008 Nature Publishing Group. C) Construction of a streptavidin nanoarray on a DNA origami, by size-selective capture of one streptavi- din tetramer in each well. Adapted with permission from ref. [119] . Copyright 2009, Wiley. D) Assembly of zinc-finger proteins (ZFP) on DNA origami struc- tures, to enable placement of multiple engineered proteins at different locations. Reprinted with permission from ref. [121a] . Copyright 2012, Wiley.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1382 CHEMBIOCHEM REVIEWS www.chembiochem.org 153 mi platform was conducted by Voigt et al.[122] They performed three successive cleavage and bond-forming reactions with high yield and chemoselectivity, and they demonstrated the feasibility of post-assembly chemical modification of DNA nanostructures, as well as their potential use as locally address- able solid supports. Furthermore, they gave a different per- spective to the face-up/face-down discussion by finding that in their case almost all of the constructs had the chemical modifi- cations pointing towards the solution, thus keeping the ques- tion raised by Niemeyer and co-workers[115] open to debate. Several methods exist for the detection of single-nucleotide polymorphism (SNPs),[123] as these differences are the major basis for phenotypic individuality and genetic variation. The ability of DNA nanotechnology to detect SNP unimolecularly is straightforward, as probes for the polymorphism can be placed over the origami sequence with full control of the loca- tion of each probe. Seeman and co-workers employed the ef- fectiveness of kinetic methods based on branch migration[124] to detect single molecule polymorphisms by AFM over an ori- gami surface (Figure 6A).[113b] However, these methodologies are still far from feasible as diagnostics, as they require AFM, which is neither quick to use nor affordable for many labs. In addition, DNA microarrays are extensively used for the same purpose, with good efficiency and simpler manipulation. The application of the DNA origami platform for detecting SNPs needs to be simplified and cost-reduced to be implemented as a diagnostic system. An interesting application of DNA nanotechnology involves extension of the study of kinetics and conformational changes to different types of molecules. The kinetics of the formation Figure 6. Study of different single-molecule reactions by the DNA origami approach. A) Detection of single nucleotide polymorphisms ?SNPs?; by AFM over an origami surface. Adapted with permission from ref. [113b]. Copyright 2011, American Chemistry Society. B) Detection of the formation of G-quadruplexes in G-rich DNA sequences on a frame-like DNA origami. Adapted with permission from ref. [126]. Copyright 2010 American Chemistry Society. C) Methyl trans- fer reaction of EcoRI methyltransferase and analysis of the structural effect of this methylation by using a frame-like DNA origami. Adapted with permission from ref. [130]. Copyright 2010, American Chemistry Society. D) Nanosensor to analyze DNA repair activity of hAGT by a-thrombin interaction with methylated DNA aptamers, substrates of the protein. Reprinted with permission from ref. [133]. Copyright 2013, Wiley.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1383 CHEMBIOCHEM REVIEWS www.chembiochem.org 154 of a duplex DNA structure can be monitored on an origami tile?studying in real time the binding and unbinding events and estimating association/dissociation rates. As well as static analysis, dynamic processes in the sub-second range can be in- vestigated on an origami platform in real time, and the results are comparable with those obtained by traditional methods to detect ensemble events.[125] In addition, conformational changes of biomolecules can also be analyzed with this plat- form. A novel method to detect the formation of G-quadru- plexes in G-rich DNA sequences was developed by Sugiyama?s group.[126] G-quadruplexes are four-stranded structures formed by the square arrangement of a tetrad of guanines, stabilized by the formation of Hoogsteen bonds.[127] They are promising anticancer agents, as they inhibit telomerase.[128] A DNA origa- mi frame structure was designed: two sets of connection sites were introduced for the hybridization of duplex DNA. Each duplex contained a single strand rich in guanines in the upper and lower region in the presence of K+ it changed its confor- mation to a G-quadruplex. This event was visualized by AFM in real time (Figure 6B). The secondary DNA binding site of human topoisomerase I was identified by using an origami construct designed by Knudsen and co-workers.[129] A bait DNA protrusion was at- tached to the rectangular DNA nanoarray, by a 21-nucleotide extension of one of the staple strands. The secondary interac- tion of topoisomerase was discovered by adding either TopoIB?DNA complex (T1 mode) or by stepwise addition of the TopoIB and cleavage complex (T2 mode), and by monitoring the process by AFM. These results led to the conclusion that topoisomerase can bind positively and negatively supercoiled DNA. However, this should be confirmed by other methods, as this was the first time that a second binding site for topoiso- merase was identified. The study of the mechanism of enzymatic reactions in real time is crucial for understanding their biological functions, and great advances in DNA nanotechnology could help. Numerous publications have described the detection of enzymatic activity by DNA origami, and most of the studied enzymes have bio- medical implications. Regulation of DNA methylation by using different tensions of double strands was studied and moni- tored by AFM by Sugiyama and co-workers.[130] They intro- duced two different double-helical tensions (tense and relaxed) into an origami frame, to control the methyl transfer reaction of EcoRI methyltransferase and examine the structural effect of this methylation (Figure 6C).[130] A further development was a versatile nanochip for direct analysis of DNA base-excision repair by 8-oxoguanine glycosylase and T4 pyrimidine dimer glycosylase.[131] The exact positioning and displacement of the enzymes in the reaction were monitored and analyzed. By using the same origami model, Suzuki et al.[132] developed a method for the visualization of Cre-mediated site-specific re- combination events. Controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold the process was regulated, thus influencing the synapsis and the outcome of the reaction. These systems could be extended to the direct observation of various enzymatic phenomena in designed nanoscale spaces, even though currently the methodology is only useful for DNA-related mechanisms. Another study of DNA methylation was conducted in our lab:[133] the use of the DNA origami as a nanosensor to analyze the DNA repair activity of human O6-methylguanine alkyltrans- ferase (hAGT) by a-thrombin interaction with methylated DNA aptamers, substrates of the hAGT. This DNA repair enzyme is a cancer prognosis indicator. Inhibition of hAGT can enhance the efficiency of chemotherapeutic treatment. Previously, we demonstrated that introduction of a methyl group into any of the guanine tetrads of TBA destabilizes the G-quadruplex formed by this G-rich sequence, thus preventing thrombin from recognizing/binding to it.[134] The introduction of several methylated TBAs at specific positions of the origami allowed us to observe the loss of thrombin binding. When the O6- methylguanine of the modified TBA was repaired by hAGT, a- thrombin was able to bind to the aptamers, as they could form the quadruplex structure required for the interaction (Fig- ure 6D). This detection of the DNA repair activity of hAGT in an origami could be further developed to design hAGT activity assays for the identification of potential inhibitors as chemo- therapy enhancers. Accurate quantitative determination of microRNA was per- formed on a DNA origami motif. Zhu et al. developed a method for detecting single-strand displacement based on the interaction of streptavidin and quantum dots, measured by AFM in a linear and direct way.[135] This methodology has ad- vantages over fluorescence signal detection: it is simple, time- and material-saving, potentially multitarget in one unique motif, and accurate enough for certain impure samples, as the signals are directly counted by AFM. All these studies assumed that immobilization of biomole- cules on a 2D platform does not affect their activity. By using fluorescence-based assays, Tinnefeld demonstrated that DNA origami does not influence the function of biomolecules (by comparison with data obtained in solution), and confirmed that DNA origami is a biocompatible surface.[136] 2.2.3. Carriers for delivery: In previous sections, we mostly re- viewed applications of 2D DNA origami, as one of the most rel- evant features is precise positioning control on the planar sur- face. In contrast, many origami platforms developed as carriers for drug delivery have employed 3D nanostructures. Many reports have described preliminary results pointing to the use of DNA origami as nanocarriers for cellular delivery. An important consideration is stability of these nanostructures under physiological conditions (maintenance of folding): DNA double helical domains and the connecting cross-overs should remain intact for the correct performance. Dietz?s group re- ported that DNA origami objects built with a average staple length and cross-over density can be safely incubated at 37 8C (typical for cell-culture application).[73] They also proved that these nanoconstructs are stable in cell-culture medium (DMEM), and in Tris buffer with BSA or salted dextran (crowd- ing agents), and at low pH. Furthermore, they tested stability in the presence of different nucleases, and found that their scaffolds were stable against T7 exonuclease, E. coli exonu- clease, Lambda nuclease, and Micrococcus MseI restriction exo-  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1384 CHEMBIOCHEM REVIEWS www.chembiochem.org 155 nuclease. However, they were degraded by DNAseI and T7 en- donuclease, although they were more stable to DNAseI than double stranded DNA (degraded after 1 hour instead of 5 min at 37 8C). In addition, Mei et al. demonstrated that DNA origami are stable in cell lysate; this allows them to be easily separated from lysate mixtures, unlike natural single- and double-strand- ed DNA.[137] These findings illustrate the potential of DNA ori- gami in cellular applications, and overcome some of the main problems that other delivery systems suffer. Visualization of the intracellular location and stability of DNA origami with a label-free fluorescent probe was performed suc- cessfully by Shen et al.[138] They reported a strategy to study the distribution and stability of DNA origami nanostructures in living cellular systems, by using carbazole-based biscyanine as a probe. After 12 h incubation, most DNA origami nanostruc- tures were localized in lysosome. After 60 h, most were disas- sembled or unfolded. Their results provide important informa- tion for the development of DNA origami as a biocompatible drug delivery vehicle, with the potential to accomplish con- trolled release. Kjem, Gothelf and co-workers designed an origami box with a controllable lid; opening and closing was demonstrated by FRET experiments (Figure 7A).[56] Programmable access to the interior of the box could yield several interesting applications, for example the controlled release of nanocargos. A further de- velopment of this is the logic-gated DNA nanopill that was de- signed by Douglas et al. for the selective delivery of molecular payloads to the cell.[139] These nanopills are capable of induc- ing a great variety of responses in cell behavior. The biological- ly active payloads can be bound indirectly by antibody frag- ments, thus enabling applications in which the nanorobot is able to stimulate signals for activation and inhibition tasks before target delivery (Figure 7B). Another cutting-edge cellular application of DNA origami was the design of DNA-based channel that can introduce pores into the cell?s lipid membrane (Figure 7C). Single-mole- Figure 7. 3D origami structures (and AFM or TEM images) for cellular delivery. A) DNA box with a controllable lid (potential drug carrier). Reprinted with per- mission from ref. [56] . Copyright 2009 Nature Publishing Group. B) Logic-gated DNA nanopill for selective delivery of molecular payloads. Reprinted with per- mission from ref. [139]. Copyright 2012, American Association for the Advancement of Science. C) DNA-origami based channel to punch pores into the cell membrane, with ability to discriminate single DNA molecules. Reprinted with permission from ref. [141]. Copyright 2012, American Association for the Ad- vancement of Science. D) DNA origami as a carrier for circumvention of doxorubicin resistance. Adapted with permission from ref. [142]. Copyright 2012 American Chemistry Society. E) Incorporation of CpG oligonucleotides into a DNA origami for the induction of strong immune responses. Adapted with per- mission from ref. [143]. Copyright 2011, American Chemistry Society.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1385 CHEMBIOCHEM REVIEWS www.chembiochem.org 156 cule translocation experiments showed that the synthetic DNA channels can be used to discriminate single DNA molecules.[140] DNA origami has also been used as a carrier to circumvent drug resistance.[141] Doxorubicin (a well-known anticancer drug) was attached to a DNA origami by intercalation, thereby ach- ieving high efficiency of drug loading when the nanostructures were administered. As a result, the cellular internalization of doxorubicin was increased, thus contributing significantly to enhance the cell-killing effect in doxorubicin-resistant MCF-7 cells (Figure 7D). In addition, H?gberg developed two DNA ori- gami structures with precise degrees of global twist to deliver doxorubicin in three different breast cancer cell lines.[142] They were able to regulate encapsulation efficiency and the drug release kinetics by tuning the nanostructure. With the aid of these DNA tiles, the cytotoxicity of doxorubicin was increased as the intracellular elimination rate was decreased, as com- pared to free drug. These results suggest that DNA origami has immense potential as an efficient and biocompatible drug delivery vehicle, although how exactly the origami nanostruc- tures deliver the anticancer drugs remains unclear. As mentioned above, many functional nucleic acids (includ- ing aptamers, antisense oligonucleotides, short interference RNA, and microRNA) have high diagnostic and therapeutic po- tential. As the carrier and the cargo are essentially the same type of molecule, the loading process can be simplified: nucle- ic acid hybridization or integration of the required sequence into the carrier. However, it is necessary to take architectural issues into account when designing the nanostructure, to avoid undesired interactions (between the cargo and the carri- er) that could reduce the efficacy of the delivery process. The incorporation of cytosine-phosphate-guanosine (CpG) oligonu- cleotides to a DNA origami tube represents a good examina- tion of this. Schller et al.[143] tested the immune responses induced by hollow 30-helix DNA origami tubes covered with up to 62 CpG sequences in freshly isolated spleen cells (Fig- ure 7E). The DNA constructs showed no detectable toxicity (in contrast to lipofectamine) and did not affect the viability of the splenocytes, but triggered a strong immune response, characterized by cytokine production and immune cell activa- tion. Given that DNA oligonucleotides can be easily modified to incorporate a wide variety of biomolecules, this approach could be extended to combinations with viral moieties to gen- erate vaccines and enhancers designed with nanometer preci- sion and high biocompatibility. 2.2.4. DNA nanomachines and nanodevices: In addition to the static DNA origami structures, continuous advances in DNA nanotechnology have made possible the construction dynamic nanosystems that combine walkers, cargo, tracks and drive mechanisms to achieve complex motion on 2D or 3D surfaces. These DNA nanomachines and nanodevices are promising for biological applications. Several nanomachines have been reported, such as a system that integrates a spider-like molecular walker with ability to move along a 2D substrate track assembled on a DNA origami array.[104b] DNA transporters that moved along a designed track were constructed on a DNA origami device, and the multistep motion of the motor strand was traced by AFM and TEM.[104a,144] Reck-Peterson and co-workers built a programma- ble synthetic cargo by using 3D DNA origami to which differ- ent DNA oligonucleotide-linked motors were attached, thereby allowing control of motor type, number, spacing, and orienta- tion.[145] This cargo system was useful to determine and charac- terize the motile behavior of microtubule-based motor ensem- bles, depending on polarity of orientation. The behavior was found to be similar to that observed in vivo. Further develop- ment of these nanoconstructs might result in bidirectional powerful transport nanodevices to investigate the motile prop- erties of motors and the mechanisms of motor regulation. A DNA origami nanomachine with the potential for in vivo biosensing and intelligent delivery of biological activators was produced by Firrao?s group.[146] This 3D DNA nanodevice per- formed its function in response to an external signal : in the presence of a small amount (picomols) of its target DNA, the robot moved a flap, thereby exposing its cargo DNA and self- assembly of the latter into a stable DNAzyme. A versatile sensing system for the detection of a variety of chemical and biological targets at molecular resolution was designed by Kuyuza et al.[147] Their sensing methodology was based on a nanomechanical device formed by two DNA origa- mi levers connected by a fulcrum, and was used to visually detect (by AFM) the biomolecule and its shape. All of the described mechanical detection mechanisms are suitable for targets of interest and can be used orthogonally with different- ly shaped origami devices in the same mixture on a single plat- form. A development of previous work concerning material organ- ization and single-molecule reactions[94,95,117] was conducted by Fan and co-workers.[148] They designed DNA nanostructures with unprecedented properties as nanoscale bioreactors, to study enzyme activities and cascades in highly organized and crowded cell-mimicking environments. These artificial systems were developed in a reliable and single-step strategy with high speed and cooperativity. A different type of biological application was addressed by Lin et al.[149] They designed sub-micrometre nanorods that act as fluorescent barcodes and produce up to 216 barcodes that can be decoded unambiguously by using epifluorescence or total internal reflection fluorescence microscopy. The potential applications of this novel approach are of great variety: they could be used to improve tagging techniques, to develop a set of versatile imaging tools for single-molecule biological reactions and for biomedical diagnostics. They could also be modified with diverse biomolecules, such as monoclonal anti- bodies for immunophenotyping applications, or they could be used for in situ labeling of multiple cell types. By following the same approach, Yin and co-workers developed a multiplexed 3D cellular super-resolution technique for imaging, by using DNA origami to obtain multiplexed images for studying com- plex biomolecular systems in cell cultures.[150] Upon refinement of their features and performance, these dynamic nanodevices could be further developed for many dif- ferent biological and biomedical applications because of their predictable biophysical and biomechanical behavior and inter- action with the cellular environment.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1386 CHEMBIOCHEM REVIEWS www.chembiochem.org 157 Outlook and Prospects The biological applications reviewed here are just a starting point to show the potential of DNA nanotechnology for bio- medical applications. Since the establishment of structural DNA nanotechnology as a field of research, scientists from dif- ferent disciplines have been working together to develop nu- merous innovations over more than three decades. It has ad- vanced from static scaffolds to dynamic functional devices that include other synthetic and natural materials. It has been ap- plied for a variety of purposes from nanoelectronics, nanopho- tonics, and nanorobotics to more biological implementations, which could evolve into diagnostic and therapeutic tools. However, some current difficulties must be addressed for the application of nanotechnology for biomedical purposes. Yield and scale-up of complex constructs remain a challenge; apparently, both are inversely related to the complexity and density of the desired structures. Deeper investigation into the kinetics and thermodynamics of annealing and assembly might come up with solutions to increase the stability and yield of the nanodevices and thus enable better prospects for in vivo application. Methods to expand the dimensions of the constructs should be addressed, as well as to increase the rate of encapsulation of biomolecules and inorganic material for delivery, and to broaden the diagnostic and therapeutic possi- bilities. The performance of single-molecule analytical tech- niques requires improvement of spatial resolution and time- scale, and additional methodologies will be required for a better understanding of unimolecular processes, and the appli- cation to diagnostics. A number of previously unfulfilled goals have been ach- ieved, thus leading to successful progress of DNA nanotechnol- ogy. On this basis, we believe that further development of these techniques holds great potential for biological applica- tions, hopefully with highly programmable and controllable ca- pabilities in the very near future. Acknowledgements The Communities FUNMOL, ?Fondo de Investigaciones Sanitarias? (grant PI06/1250) and by ?Ministerio Ciencia e Innovacin? (grant CTQ-2010-20541-C03-03) are acknowledged for financial support. C.F is grateful to Generalitat de Catalunya and Instituto de Salud Carlos III for a SNS Miguel Servet contract. M.T. was supported by a pre-doctoral fellowships from MINECO. Keywords: biosensors ? DNA nanotechnology ? DNA origami ? DNA structures ? DNA tiles ? nanostructures [1] J. D. Watson, F. H. Crick, Nature 1953, 171, 737?738. [2] a) F. Sanger, G. M. Air, B. G. Barrell, N. L. Brown, A. R. Coulson, C. A. Fiddes, C. A. Hutchison, P. M. 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Firrao, Small 2014 ; DOI: 10.1002/smll.201400245.  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1389 CHEMBIOCHEM REVIEWS www.chembiochem.org 160 [147] A. Kuzuya, Y. Sakai, T. Yamazaki, Y. Xu, M. Komiyama, Nat. Commun. 2011, 2, 449. [148] Y. Fu, D. Zeng, J. Chao, Y. Jin, Z. Zhang, H. Liu, D. Li, H. Ma, Q. Huang, K. V. Gothelf, C. Fan, J. Am. Chem. Soc. 2013, 135, 696?702. [149] C. Lin, R. Jungmann, A. M. Leifer, C. Li, D. Levner, G. M. Church, W. M. Shih, P. Yin, Nat. Chem. 2012, 4, 832?839. [150] R. Jungmann, M. S. Avendano, J. B. Woehrstein, M. Dai, W. M. Shih, P. Yin, Nat. Methods 2014, 11, 313?318. Received: February 6, 2014 Published online on June 20, 2014  2014 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2014, 15, 1374 ? 1390 1390 CHEMBIOCHEM REVIEWS www.chembiochem.org 161 162 Chapter 4 A fluorescence biosensor for hAGT activity. 163 164 A fluorescence biosensor for hAGT activity. Maria Tintor?,1 Santiago Grijalvo,1 Ramon Eritja1 and Carme F?brega1 In preparation. 1 IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine. c/ Jordi Girona 18-26. 08034 Barcelona (Spain). 165 O6-alkylguanine-DNA alkyltransferase (hAGT) activity provides resistance to cancer chemotherapeutic agents and its inhibition enhances chemotherapy. In chapter 4, we present the development of a novel fluorescence assay based on the fluorescence transfer from a duplex oligonucleotide containing a fluorophore-quencher pair to the active site of hAGT, for the detection of its DNA repair activity. For this purpose, we designed a double-stranded DNA sequence containing a fluorophore-quencher pair, where the fluorophore was attached to a synthetic 06-benzylguanine. This precursor was synthetized through a novel route which comprises the use of the Mitsunobu reaction for the introduction of the benzyl group into the O6 position of the guanine. We performed HPLC studies of the repair of this benzylguanine by hAGT, to ensure that this bulky substituent can enter the active site of the protein. The repair activity of hAGT was detected by fluorescence transfer, producing an increase in intensity upon repair of the alkylguanine. This assay can be used for the evaluation of potential inhibitors of hAGT in a straightforward manner and avoiding radioactivity. The compounds described in chapter 1 as possible candidates to inhibid hAGT activity were tested by this methodology and the preliminary results obtained are included as Appendix 4. 166 A fluorescence biosensor for hAGT activity. M . Tintor?, a , b S.Gr ijalvo, a , b R.Eritja *a,b and C.F?brega *a, b a Inst it ut e for Advance d Chemi st ry of Catal oni a (IQAC), Span ish Nat io nal Research Counci l (CSI C), C/ Jordi Giro na 18 -26, 080 34 Barc el ona , Spai n; Tfn : +34 93 40061 0 0 . Fax: +34 93 204 590 4 e-mai l: cgcnq b@ci d. csic. es ; recgma@cid. csic. es . b Netwo rkin g Cent er on Bioe ngi nee rin g, Bioma t eri al s and Nano medi cin e (CI BER - BBN). K e y w or d s : Biose nsor, fluore scence , DNA repa ir, O 6 -alkylgua ni ne -DN A alkylt ransf erase, inhi bitio n -act ivi t y Abstr a c t: O 6 -al kylgu ani ne -DN A alkylt ransf erase (hAG T ) act ivity provi des resi st ance to cance r chemo t hera peu t ic age nt s and its inhi bi t io n enh ance s chemo t he rap y. We here in prese nt the deve lo pmen t of a nove l fluoresce nce assay ba sed on a duple x oligo n u c l e o t i d e conta i ni n g a fluor o p h o r e -que n cher pai r for the det ect io n of the DNA repa ir act ivi t y of hAGT . This assay can be used for the eval uat io n of potent ial inhi b i t ors of hAG T in a strai ght f orward mann er and avoi ding radi oact ivit y. 167 1 . Introd ucti on Alk ylati ng age nts are chemothe rap eut ic antic anc er drugs that produc e their cytoto xic effe ct by ge ner ati ng add uc ts at multi pl e sites in DNA. [ 1] The most rel evant add u ct in terms of mutage nes is and carc in ogen es is is the alkyla tion of the O 6 positi on of guani n e s . [ 2] In partic ul ar, the alkyla tin g age nt 1,3 -b i s - ( 2 -ch lo roe thyl) -1 -ni tros our ea (BCN U) initi al ly atta ck s at the O 6 guani n e posi t i o n , caus i n g its cross -li nk with the opp os ite cytos in e, blockin g DNA re pl ic ati on and produc in g G 2 /M arre st . [ 3] In addition to the well -known side effe ct s and limitati ons of chemothe rap eut ic age nts , thes e subs tan ces also show acqui red tumour res is tan ce probl ems. The DNA -rep ai r prote in human O 6 - al kylg uan in e DNA alkyltr ans fera se (hAGT or MGMT) is res pon si bl e for removing al kyl add uc ts from the O 6 positi on of gua ni nes , there by bloc king the cytoto xic effe ct s of the alkyla ting age nts and maki ng a cruc ia l contri but io n to the res istanc e mech ani sm. [ 4] It is well estab li sh ed that tumoral cells show gr eat er expres si on of this protei n, which expla in s their low sensi tivity to chemothera peu tic drugs in a large numbe r of canc ers . [ 5] On the contra ry, the promoter methylati on of hAGT has been asso ci ated with patients? lo nge r survival. [ 6] Therefore , pha rmaco lo gi ca l inhi bi tio n of hAGT has the poten t i a l to enhan c e the cytot o xi c i t y of a diver s e rang e of antic a n c e r agent s . [ 7] hAGT is a DNA -b i n d i n g prote i n that contai n s a highly conse rv e d inter n a l cyste in e, which act s as th e acce pt or sit e for alkyl grou ps. It beh aves like a suicid al non-enzym e, inactivat ing itself since the S-alkylcyste in e formed is not rege nera t ed. [8 ] For this reaso n, intense resea rch effort has been devo t ed to the i denti f icatio n of smal l molecul es capa bl e of inhibi t in g hAGT act ivity and signi f icant ly enh anci ng the cytoto xic effect of BCNU in prostat e, breast , colon and lung tumou r cells. [7 ] Given the pot ent ia l relevance of hAG T as a prog nost ic marker of cance r and as a thera peu t ic targe t , severa l met hod s are avai la b le to chara ct eri ze its act ivi t y. Moreo ver, they are als o abl e to eval uat e the capa cit y of smal l molec ul es for inhi bi t in g hAG T . Most of these met hod s invol ve radi oact ivi t y assays, whil e others are base d on mult ip le -st ep enzyma t ic react io ns. [9 ] However, the first met hod s requ ire the use of rigorou s saf et y proce dure s and the others are discont in uou s and time -consu min g due to the nece ssit y of multip le steps. Recent ly, two met hod s base d on the conf ormat io nal ch ang e of an intramol ecul ar G -qua drup le x were deve lo ped by our group . [1 0 ] In this paper we descri be the developme nt of a one -step FRET assay which improves the effici ency of our previous met hod s beca use it increases t he repa ir rat e of hAG T by using do ubl e -st rand ed DNA, the nat ural subst rat e of the prot ein. In 168 add it io n, the det ect io n of a fluorescence incre ase that is prop ort io nal to the repair rat e of hAG T represent s a low -cost , strai ght f orw ard and rapi d met hod . For this purp ose, we desi gne d a doub le -st rand ed DNA sequ ence cont ai ni ng a fluorop hore -que nche r pai r (Sche me 1) . The fluorop hore wa s post -synt het ica lly and coval ent ly attache d to a modi f ied O 6 -ben zylgu ani ne ( F dG ) and the que nche r was introdu ced in a very cl ose po sit io n of the compl emen t ary strand of the dup le x ( Q d T - compl emen t ary or Q d T -missma t ch ), blockin g the fluorescence. The fluorop hore was transf erred toge t her with the benzyl grou p to hAG T ?s active sit e when the prot ei n repa ire d the DNA , res t ori ng the guani n e . The remova l of the fluoroph ore bro ugh t it apa rt from the que nc her, prod uci ng a sig ni f ica nt increase in fl uore scence whic h allow e d to measure the repa ir reactio n of hAGT . Sc heme 1. A. C he mi c al str uc tur e of the O 6 - b en zyl g ua ni n e with the flu o rop h ore co v al en tl y att ac he d to the al kyl grou p. B . C he mi cal str uc tur e of the qu en che r gro up (Dab cyl ) , cov al en tl y att ac he d to a thymi di ne . C . Sche ma ti c re pre sen ta tion of the flu ore s ce nce assa y. 1 ) . An ne al in g of the two stra nd s con ta in in g a fluo r op ho re and a que nch er gro u p, with the co rre spo nd en t exti nc tion of fluo r es cen c e. 2 ) . hAG T rep ai r ac tiv ity over the benz yl gu an in e , drag gi n g the fluo r es cei n grou p an d sig ni fic an t l y incr ea si n g fluo re sce nce . 2 . Res ults and Disc u s s io n T he A d G oli gon ucl eot ide was pr epa red usi ng the app ropri at e modi f ie d gua nosi ne pho spho ra mid it e ( 7 ) in the aut omated DNA synthe sizer using stand ard prot ocol s [1 1 ] . Derivative 7 was prepared from ?-d eoxyguano s in e and the protec ted 4 - triflu oro ac etyl - amino meth yl ben zyl alcohol ( 1 ) (Sch eme 2A). Its inco rpo ration at the O 6 positi on of the 169 g u a n i n e was perfo r me d thro u g h a Mitsu n o b u reac t i o n . The gua ni ne amino funct ion was protec ted with dimethylamino meth yl id en protectin g group. Final ly, the protec tio n and funct io nal ization of the two hydro xyl groups were carri ed out und er stan dard cond iti ons . Scheme 2. A) Chemical synthes is of the O 6 - benzyl- ?-deoxyguanosine phosphoramidite. a) TEA, EtTFA in DCM, 0?C to r.t. o/n b) Ac 2 O, DMAP, TEA in DMF, r.t. 4h; c) Compound 1, PPh 3 , DEAD in Dioxane, r.t. o/n: d) MeOH :NH 3 (1:1), 55?C 4h; e) (CH 3 O) 2 C H - N(CH 3 ) 2 in DMF; f) DMT-Cl, DIEA in Pyr, r.t. o/n; g) DIEA, CNEOP(Cl)N(iPr) 2 in DCM, r.t. The ben zylgua ni ne -co ntai ni ng oligonu cl eot id e and its compl ementa r y stran ds with a quenc her group (dabc yl, Q) eithe r at the complement ary pos iti on or at one nuc le oti de shift were synth es iz ed, puri fie d by HPLC and ch ar ac teri se d by MALDI -T OF (see Supp l ementa ry Dat a ). Subs equ ent ly, the fl uor es ce in label (F) was atta ch ed to the O 6 - ben zylg uan os in e amino group thro ugh a fluore scei n isothiocyana te (FIT C) react io n. [12] The DNA dup le xes , one comp le mentary and the othe r conta ining a mismatc h due to the pos iti oni ng of the T -quench er fos fora midi te pairin g with the modif ie d F G , were pre par ed from the flu oro pho re -que nc her pai r of olig onu cl eoti des und er proper anne a l i n g cond i t i o n s (Tab l e 1 ). We explo red to differ ent pos iti on s of the que nc her group in the complement a ry seque nc es to ens ure the maxi mal que nc hi ng of flu ore sc ence and to mini mize steri c effe ct s and comple ment ary disc rep anc ie s. In the first 170 d u p l e x , the plac ement of the que nc her in the complem ent ary bas e of the modi f i e d gua nos in e provok es a mismatc h in the seque nce (G:T) inst ead of a (G:C ) pai r due to the fac t that there is no commerci al ly avail abl e cytid in e covalent ly modif ie d with the dab cyl group and the use of a thymid in e was requi red . The secon d dup lex conta in s the que nc her grou p at one nuc le oti de shift with res pec t to the ben zylgua ni ne in the complement ary seque nce avoid in g the introd uc tion of a mismatch and expect in g that the flu ore sc ence extinc tio n woul d remain effi ci ent . Name Sequ enc e Olig o - F dG: C omple men tar y - Q T ?- ATC TTC TC F G ATT CA - 3? 3?- TAG AAG AGC Q T AA GT - ? O ligo - F dG:M is match - Q T ?- ATC TTC TC F G ATT CA - 3? 3?- TAG AAC AG Q T TAA GT - ? Table 1. Sequences of the two different duple xes used in this work . Fi rs t, to see the possi ble effe ct of this two bulky grou p s in the dup le x formati on , their therma l stabi li ty was studi ed at 260 and 48 5 nm and compared to eac h other (see Tab le S2 at Supp le mentary Data, SD ). In gen era l , we observed des tabi li za tio n of the dup le x at 260 nm in the prese nc e of the bul ky subst itu ent in the O 6 position of the central guan i n e , compar ed to the contro ls . Howe ver, both dup le xes are stable at 30? C. CD spec tra of the two dup le xes were rec ord ed, showin g that both struc ture s formed a duple x at 25? C (See Figur e S1, SD) . In order to be abl e to use these two dup le xes in a fluore sc en ce -que nc h i n g assa y, we studi ed how the stabi li ty of the dup le xes affe ct s the emis si on of flu ore sc enc e by moni tori ng the emitte d flu ore sc ence at exci tati on wavelen gth of 485 nm (See Figur e S2, SD). Signi fic ant differ enc e s in the melti ng tempera ture were obs erved due to the rel eas e effe ct of the que nc her and a great incr eas e in the flu ore sc ent signal was detec ted . In the case of the mismat ch ed seque nc e, this incr ease in flu ore sc enc e is produc ed at 26 ?C while in the ful ly complement ary dup le x, the que nc her effe ct persist ed until 53 ?C, allo wing us to use it at the enzymat ic tempe ratu re range for hAGT activity. Effi ci ent reac tio n with hAGT re qui res the F G modif ic ati on to correc tly acco mmoda te in the active site. Thi s bri ngs the CH 2 whic h is atta ch ed at the O 6 positi on in clos er proximity to the th yol ate d ion Cys1 45, maki ng the react io n pos si bl e. For thi s reaso n, we performed a study on the hAG T dea lk ylation activity over the F - ben zyl modif ie d dou bl e -st ran ded subs trat e s . The reac tio n was fol lo wed by HPLC. Thi s stud y 171 was performed in the abs enc e and prese nc e of the que nc her in the complem ent ary stran d. Figure 1. HPLC profile of the repair reaction of the F dG double stranded sequence in the absence of the quencher group in the complementary stra nd . The HPLC analysis was perfor med 37 ?C. For thi s purpos e, the ful l -le ngt h hAGT was over -expres se d and purifi ed as previous ly des cr ib ed. [ 10a, 13] Increa sing conc ent ratio ns of the protei n were inc uba ted wit h the dou bl e -strande d F dG seque nc e for 90 min at 30? C and anal y z ed usin g HPLC. Figur e 1 shows one of the HPLC profil e s of the reaction?s final products of hA*T at 3?&. The app ear anc e of a peak with a shorte r rete ntion time corres pon ds to the rep aired seque nc e, formed by the removal of the F -benz yl group. Our res ul ts confi rmed that hAGT has the capac ity to acco mmoda te bul ky groups , inclu di ng a fluoroph ore , in the active site with out upse ttin g its rep air activity. We then tes ted the effe ct ivene ss o f our propos ed flu ores ce nc e method to evalu ate the repai r activity of hAGT. We performed the hA GT react io n usin g a 10 nM conc ent rati on of the fluores ce ntl y -dab cyl label led dup le x for 20 min, usin g differen t hAGT conc ent rati ons . We determine d 10 nM to be th e mini mu m amoun t of dou bl e - st ran ded DNA requi re d to achi eve a detec tab le and rel ia bl e differ enc e in inten si ty compared to the backgroun d. As expec ted , the flu oro phore -que nc her pai r gave low bac kg rou nd flu ore sc ence, bec aus e of the que nc hi ng effe ct produc ed by the proximit y of these two grou ps in the bas e paired seque nce. The presence of hAGT produc ed a remark abl e incr eas e in flu ore sc ence inten si ty caus ed by the tran sf er of the F -ben zyl group to its active site, thro wing it apa rt fro m the que nc her . The incr ea se rate in flu ore sc ence inten si ty correla ted direc tly with the amoun t of hAGT in the react io n mixture (Figur e 2). Moreover, the inac tive mutant hAG T -C145 S did not exhib it any dec rea se in flu ore sc ence due to its inabi li ty to repair the alkyla ted DNA (se e SD ). 172 Figure 2 . hAGT activity assay . A) Inc rease of fluoresce nce with respect to the backgro u nd fluorescence of the olig onucle otide during 20 minutes. B) Total increase of fluorescence after 20 minutes of reac tion with different concentrations of hAGT. In view of all these obs ervatio ns , we corrob ora ted that our initia l hypoth esi s and des ig n of the new FRET metho dol ogy is cons istent. 3. Concl usi ons In summary , we have des cr ib ed a novel route for the synth es is of a O 6 -ben zyl -? - deo xygua n o s in e precu rso r whi ch inc orp ora ted to an oli gonu cl eot id e seque nc e, can bec ome a fluore sc ent ly label le d subst rate for meas uri ng hAGT repai r activity. Anne al in g with a comple mentary qu enc her strand produc es a dupl ex with low bas al fluore sc enc e. Using thi s label le d oligon uc le o tide, we ha ve devel ope d a new assa y for quant i f yi n g the DNA repai r prot e i n hAGT acti v i t y and ident i fyi n g pote n t ia l inhi bi t o r s as chemothe rap y enh anc ers . The novel ty of thi s method compared to exis tin g one s is the singl e -st ep and real -time meas ure ment of th e react io n, repres ent ing a rapid and strai ght forwa rd method that reduc es cost , time and effo rt. In contra st with our previo us devel ope d method s to qua nti fy hAGT activit y, [ 10] this syst em uses a doubl e -st ran ded olig o nuc le otide, whic h is the natura l subs trat e for the hAGT activity, imp roving effi ci enc y and rel ia bi li ty due to its eas y qua nt ifi ca tio n of the flu ore scenc e incr ease upo n activity. We bel ie ve that thi s new method will fac il ita te the searc h for new and more po ten t inhi bi tors whic h enha nc e the effec t of che mothera peu tic dru gs. Thi s method ol ogy could be impl emented for the study of hAGT dea lk ylati on in cell cultu re and in ani mal model s, thro ugh the trans fec tio n of flu ore scent olig o n u c l e ot id e s and visua l i z a ti o n of the incr eas e in fluores ce nc e usin g confocal micro sc opy. However, the thermal stabil ity of the olig onucl eoti des should be improved to ens ure their stab il ity at physi ol ogi cal tempera t ure . Work in thi s directio n is curren tly und erway. 173 A c k now led ge m e nts T he Commun it ie s MULT I F UN (cont ract NMP4 -L A -2 0 1 1 -3 , ?Fondo de InYestigaciones6anitarias? grant 3I1 and by ?Ministerio&iencia e InnoYaciyn? (gran t s CTQ -2 0 1 0 -2 0 5 4 1 -C 0 3 - 0 3 and CTQ2014 -525 8 8 -R ) are acknow le dge d for financi al supp ort . M.T. was supp ort ed by a pre -do ct oral fellowship (FPI) from MINEC O . C.F is grat ef ul to TV3 Marat o 201 2 for a resea rch cont ract . Ex pe r i m e n ta l data : Deri vative 7 was pr epa red from 5?,3 ? -di a ce yl -? -deo xygua nos in e and 4- amino meth yl ben zyl alcohol (1.2 eq) , pre vious ly protec ted with a triflu oroace tyl group v i a a Mits uno bu react io n in the prese nc e of dieth yl azodi ca rbo xyla te (3 eq.) an d triphe nylph os phi ne (3 eq.) . The n, the gua ni ne amino funct io n was protec ted with the dimethylami nometh ylide n protec tin g group. Final ly, the pro tec ti on and funct io nal ization of the two hydrox yl gr oup s for the DNA synt hes is were carrie d out und er stand ard condi tio ns . See SD for furth er info rmatio n. The olig onucl eot id e sequen ce s were synth es ized usin g standa rd protoc ol s and purifi ed by revers e d -pha se HPL C . Th e inco rporatio n of the O 6 -( N -4 - ((hydrox yme thyl)b enz yl) -tri flu oro ac etylmid e) -? -d eoxyguano s in eph os pho ramidi te ( A dG) in the seque nc e was carrie d out manua ll y wi th a coupl ing time of 15 min, obtai ni ng a yield of 75% . The lengt h and homog ene ity of the oli gonucl eot id e were verifi ed by MALDI-T OF (T abl e 1, SD . The ? -ATCTTCT C A GATTC A - 3? oligonucleotide was left to rea ct pos t -synth eti ca ll y with flu ore scei n isothiocyana te (FIT C) throu g h its free amin o group . The coupl in g effici enc y was determine d by HPLC anal ys is . See SD for furt her detai ls . Melti ng curv es of all the pos si bl e pairs of complement ary olig onu cl eot ides were meas ure d by monit ori ng the abs or ban ce hyperc hro mici ty at 260 nm, he ati ng the sample over the ran ge 10 -80 ?C . 4 ?M of th e dup le xs in 10 mM sod iu m pho sp hat e pH 7.0 and 100 mM NaCl. The CD spec tra of all the pos si ble pai rs of compleme nta ry olig o n u c l e ot id e s were regi s t e r e d at 25?C over a range of 220 ?320 nm usin g the same conc ent rati ons and buffer as for the melti ng tem per atu re exper iment s . See SD for more deta i l e d infor ma t i o n . HPLC proof of conc ept of the deal kyla tio n of A d G was perform ed by inc uba tin g the dup le xes with FL hAG T 90 minut es at 30? C and ana lyzi ng by HPLC on a Nucl eos il ana lytic al column at 37?C with a gradi ent of 10 ?40% acetonitri le in 20 m in. See SD for more detai le d info rmatio n. 174 The flu ore scenc e assay was perfo rmed incu bat in g incr easin g conc entratio ns of hAGT (50, 100 and 200 nM ) with the flu oro pho re -que nc her dup le x subs trat e (10 nM ). Fluor e sc ence was mea su red during 20 min at exci tati on and emis si on wavelen gth s of 485 and 535 nm, res pec tivel y. Average s over thre e re adi ngs were tak en for eac h condi tio n teste d. Each experiment was performed in tri pl ica tes . All the experi mental pro ce dur es are furth er deta iledi n the Suppl ementa ry Data . 175 Re feren ces : [1] M. R. Middleton, G. P. Margis on, Lance t Oncol 2003 , 4 , 37- 44 . [2] a) A. Sabharwa l, M. R. Middleton, Curr Opin Phar maco l 2006 , 6 , 355 - 363; b) A. E. Pegg, B. Singer, Ca ncer Inves t 1 9 8 4 , 2 , 18; c) R. Saffhill, G. P. Marg ison, P. J. O'Connor, Biochim Biophys Acta 1985 , 8 2 3 , 111-145; d) B. Singer , Cance r Res 1986 , 4 6 , 4879- 4885 ; e) K. A. Jaeck le, H. J. Eyre , J. J. Townsend, S. Schulman, H. M. Knudson, M. Belanich, D. B. Yarosh, S. I. Bearman, D. J. Giroux, S. C. Schold, J Clin Onco l 1 9 9 8 , 1 6 , 3310 - 3315; f) M. Belanich , M. Pastor, T. Randall, D. Guerra, J. Kibitel, L. Alas, B. Li, M. Citron , P. Wasser man, A. White, H. Eyre, K. Jaeck le, S. Schulman, D. Rector, M. Prados, S. Coons, W. Shapiro , D. Yarosh , Canc er Res 1996 , 5 6 , 783- 7 8 8 ; g) R. S. Foote, S. Mitra , B. C. 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Herman, N Engl J Med 2000 , 343 , 1350- 1354; b) M. E. Hegi, A. C. Dise rens, T. Gorlia, M. F. Hamou, N. de Tribo let, M. Weller, J. M. Kros , J. A. Hainfellner , W. Maso n, L. Marian i, J. E. Bromberg, P. Hau, R. O. Mirimanoff, J. G. Cairncross, R. C. Janz er, R. Stupp, N Engl J Med 2005 , 3 5 2 , 997 - 1003; c) R. Tuominen, R. Jewell, J. J. van den Oor d, P. Wolter , U. Stiern er, C. Lindho lm, C. Hertzman Johansson, D. Linden, H. Johansson, M. Frostvik Stolt, C. Walk er, H. Snow den, J. Newton- Bishop, J. Hansson, S. Egyhaz i Brage, Int J Cancer 2014 ; d) X. Li, F. Hu, Y. Wang, X. Yao, Z. Zhang , F. Wan g, G. Sun, B. B. Cui, X. Dong, Y. Zhao, Biomed Res Int 2 0 1 4 , 2014 , 236361; e) J. M. Brandwe in, J. Kass is, B. Lebe r, D. Hogge, K. Howson -Jan, M. D. Minden, A. Galarne au, J. F. Pouliot, Br J Haematol 2 0 1 4 , 1 6 7 , 664- 670. [7] A. E. Pegg, K. Swenn, M. Y. Chae, M. E. Dolan, R. C. Moschel, Biochem. Pharmacol. 1 9 9 5 , 5 0 , 1141- 1148. [8] a) K. S. Srivenug opal, X. H. Yuan, H. S. Fried man, F. Ali - Os man, Bioc hemistry 1996 , 3 5 , 1328 - 1334; b) M. Xu-Welliver, A. E. Pe gg, Ca rcinogenesis 2002 , 2 3 , 823 - 8 3 0 . 176 [9] a) B. D. Wilso n, M. Strauss, B. J. Stickells, E. G. Hoal - van Helden , P. van Helden, Ca rcinogenesis 1994 , 1 5 , 2143- 2148; b) M. E. Dolan, D. Scicchitano, A. E. Pegg, Ca ncer Res 1988 , 4 8 , 1184- 1188; c) A. M. Moser, M. Patel, H. Yoo, F. M. Balis, M. E. Hawkins , Anal Bioch em 2 0 0 0 , 281 , 216- 222; d) R. S. Wu, S. Hurst -Ca lderone, K. W. Kohn, Ca ncer Res 1987 , 4 7 , 6229 - 6235. [10] a) M. Tintore, A. Avino, F. M. Ruiz, R. Eritja, C. Fabrega, J Nucleic Acids 2 0 1 0 , 2 0 1 0 ; b) M. Tintore, I. Gallego, B. Manning, R. Eritja, C. Fabre ga, Angew Chem Int Ed Engl 2013 , 5 2 , 7747- 7750. [11] C. B. Reese, Org Biomol Chem 2 0 0 5 , 3 , 3851- 3868. [12] S. Perez -Re ntero, N. Kielland, M. Terraz as, R. Lavilla, R. Eritja, Bioconjug Chem 2 0 1 0 , 2 1 , 1622- 1628. [13] F. M. Ruiz , R. Gil -Re dondo, A. Morreale, A. R. Ortiz , C. Fabrega, J. Bravo, J Chem Inf Model 2008 , 4 8 , 844 - 8 5 4 . 177 178 S upple me nta ry Da ta A fluorescence biosensor for hAG T ac ti vi t y M Tin t o r? , a ,b S Grija lvo , a,b R Erit ja *a,b and C F?b reg a *a,b Ta ble of conte nts: 1. Ma t e ria ls an d me th od s Pa ge 1 2. Ch e mica l synt he sis of th e 6 - be n zyl - ?- d eo xygua no sin e pho sph o ra mid it e pre cu rso r Pa ge 2 3. Synt he sis an d pu rif ica t io n of oligo nu cleo t ide s Pa ge 5 4. Ove re xp re ssio n of hA G T Pa ge 6 5. Th e rma l stab ilit y stu d ie s Pa ge 7 6. Circu la r Dich ro ism Pa ge 8 7 . HPL C ana lysis of hA G T de a lkyla t ion Pa ge 7 8 . Flu o rescen ce assa y Pa g e 7 Tab le S1 : Seq ue n ce of olig on u cle o t id e s an d its cha ra cte riza t ion by MA L DI - TOF Pa ge 8 Tab le S2 . Me lt in g te mp e ra t u re s of th e du p lexe s Pa ge 9 Fig u re S1. CD sp e ct ra of th e two fluo ro ph o re - q uen che r du p le xe s Pa ge 9 Fig u re S2. Me lt in g te mp e rat u re c u rve s of th e two flu o r op ho re - qu en che r dup le xes at 48 5n m Pa ge 1 1 Fig u re S3. HPL C prof ile s of the co n t ro l seq ue nce s Pa ge 1 1 Fig u re s S 4 an d S 5 : Con t ro l exp e rimen t s of the f luo re scen ce assa y Pa ge 1 2 1 H - N MR, 13 C - N MR 19 F - N MR an d 31 P - NMR sp e ct ra Pa ge 1 3 179 1 . Ma te r ia l and me thods 1.1 Abbre via t ions: A c: ace t yl, Ac 2 O: ace t ic anh yd rid e , ACN: aceto n it rile , AcO Et : eth yl ace t at e , anh : anh yd rou s, Ar: aro ma t ic, BCNU: bis -ch lo roe th ylnit ro so u re a , Bz: be n zyl, Bzl: ben zo yl, CP G : con t ro lled po re gla ss, Da bcyl: dimet h yla mino -a zo b e n z o ic acid , DCM: dich lo ro me th an e , DEA D : d ie th yl azod ica rbo xyla te , DIE A : N, N -d iisop rop yle th yla mine , DMA P : N, N -d ime t h yla min op yrid ine , DMF: N, N -d ime t h ylf o rma mide , dmf : dime t hyla mino me th ylid en e, DMT: ,?-d ime th o xyt rit yl, DMT-C l: ,?-d ime th o xyt rit yl ch lo ride , DMS O : dime t hylsu lf o x ide , DTT: dith io th re it o l, EtTFA : eth yl trif lu o ro a ce ta t e, F: f lu o re sce in, F G : f lu o re sce in -b en zylgu an osin e, FITC: Flu o re sce in isot h io cyan at e , FRE T: f lu o re scen ce re so na nce en e rg y tran sf e r, hA G T: hu man O 6 -a lkylgu an ine -DNA alkylt ra n sf e rase , LCAA : lon g cha in amin o alkyl, MA L DI: ma t rix -a ssist e d la se r diso rp t io n/ io n iza t ion , Me O H: me th an o l, PPh 3 : t riph en ylp ho sp h ine , Pyr: pyrid ine , Q T: (N- 4 ' -ca rbo xy -4 -(d imet h yla min o ) -a zo be nzen e ) -a minoh e xyl -3 -a cryla mid o] - 2 ' -de oxyu rid in e , RP -HP L C: re ve rse pha se hi g h pre ssu re liqu id ch ro ma to g ra ph y, TCA: trich lo roa ce t ic acid , TEA: triet h yla min e , TEA A : trie th yla mmon iu m ace ta te , THA P : trih yd ro xya cet op he no ne mo no h yd ra te , THF: te t ra h yd rof u ran e , TL C: th in -la ye r ch ro ma to g ra ph y, TOF: time of f ligh t, UV : ult ra vio let . 1 . 2 Che mic a ls Re a ge nt s fo r olig on u cle o t id e synt he sis in clu d in g pho sph o ra mid it e mo no me rs of dA Bzl , dC Bz l , dG dm f and T, the ?-de b lo cking so lu t io n (3 % TCA in DCM ), act ivat o r so lu t io n (0 .4 M 1H-t e t ra zo le in ACN), CA P A so l u t io n (A c 2 O /P yr/ THF), oxid izing so lu t ion ( 0 .0 2 M io d ine in THF/ P yr / wat e r (7: 2 :1 ), su pp o rt s an d LCAA -CP G we re pu rcha sed f ro m App lied Bio syste ms (PE B io syst e ms Hisp an ia S.A . , Spa in ) and Lin k Te chn o lo g ie s Ltd. (S cot la nd ) an d used as rece ived . Sep had e x G -2 5 (NAP - 1 0 ) co lu mn s we re pu rcha sed f ro m GE He a ltc a re, US A . Th e 96-we ll Op t ica l re act ion plat e s we re pu rcha sed to Nu n c, US A. HiTra p TM FF co lu mn we re pu rch a se d at GE He a lth ca re . FITC (iso me r I) an d th e re st of ch e mica ls we re pu rcha sed f ro m Ald rich , Sig ma , or Flu ka (S ig ma -A ld rich Qu imica S.A. , Spa in ), and u se d wit ho ut fu rt he r pu rif ica t io n . An hyd rou s so lven t s an d de u te ra te d so lve nt s (CDCl 3 , CD 3 O D and DMS O - d 6 ) we re ob ta ine d f ro m re pu ta b le so u rce s an d used as re ce ive d . Th in -la ye r ch ro ma t og rap h y (TL C) wa s ca rrie d out on alu min iu m -ba cke d Silica -G e l 60 F 254 plat e s . Co lu mn ch ro ma to g ra ph y wa s pe rf o rme d using Silica Ge l (6 0 ?, 230 x 400 me sh ). 1 . 3 Instrume nta t ion O lig on u cleo t id e se qu e n ce s we re syn t he sized on an App lied Bio syste ms 34 00 DNA Syn th e size r (A pp lie d Bio syste ms, US A ). RP -HP LC pu rif ica t io n s we re pe rf o rme d on a W ate rs ch ro ma t og rap h y syste m using Nucle o sil se mi -p re pa ra t ive 120 C 18 (25 0x4 mm) co lu mn s. An a lyt ica l RP - +3/& was performed using a ;Bridge 26T &1 . ?m co lu mn and a Nu cle o sil An a lyt ica l co lu mn 120 C18 (2 50 x4 mm). Olig on u cle ot id e s we re qua n t if ie d by UV ab so rp t ion at 260 nm wit h a Ja sco V6 50 spe ct ro ph o to me te r . Ma ss sp e ct ra we re re co rd ed on a MAL DI P e rse pt ive Voyag e r DE TM RP time - o f -f ligh t (TOF) 180 sp e ct ro me te r (A pp lie d Bio syste ms, USA ) equ ip ped wit h nit rog en la se r at 33 7 nm using a 3n s pu lse . Th e ma t rix used co n ta ine d 2,4 ,6-t rih yd ro xya ce top he no ne (THA P, 10 mg / mL in ACN/ wa te r 1:1 ) and ammo n iu m cit ra te (50 mg / mL in wa te r). NMR sp e ct ra we re me a su red on Va rian Me rcu ry -4 0 0 . Che mica l sh if t s are given in pa rts pe r millio n (p p m); J valu e s are given in he rt zs (Hz). A ll sp ect ra we re in te rn a lly ref e ren ced to th e app ro p riat e re sidu a l und eu te ra te d so lven t . UV ana lyse s we re pe rf o rmed using a Ja sco V -6 50 in st ru me nt equ ipp ed wit h a th e rmo re gu la te d ce ll ho lde r. Se t te mp e ra t u re was co n t ro lle d wit h an 890 90 A Ag ile nt Pe lt ie r de v ice and +ellma Tuartz cuYettes were used 1 ?/ Yolume . CD sp e ct ra we re re co rde d on a CD on a JAS CO spect ro po la rime te r J -8 10 . Flu o ro me t ric me a su re men t s we re pe rf o rmed on a sp ect rof lu oro me t e r Ja sco FP 62 00 at 25? C an d wit h a Mu lt i-De t e ct ion Micro p la t e Rea de r Spe ct raMa x M5 ( Mo le cu la r de vice s Bio No va cie n t if ica , Sun n yva le, USA ). 2 . Che mic a l synthe s is of the 6 -be nzyl- 2? - de ox igua nosine phosphora mid ite pre c ursor 2. 1. Pre para tion of N- (4 -(h ydrox yme thy l)be nzy l ) -tri fluoroa c e ta mide (1 ) 4 -a min o me th ylb en zyla lco ho l (50 0 mg, 2.8 9 mmol) wa s disso lved in 3 ml DCM. TEA (3 . 0 eq, 1.16 9 ? l, 8.4 mmo l) wa s add ed drop wise an d kep t un de r argon atmo sp he re . Af t e rwa rd s, 400 ?l (1 .2 eq, 3.3 6 mmo l) of EtTFA we re adde d dro p wise and the mixt u re wa s stirre d in arg on atmo sp he re at ro o m te mpe ra tu re fo r 4 hr. Th e re a ct io n wa s fo llo wed up by TL C (DCM: Me O H 5%). Th e re a ct io n mixtu re disso lved in DCM an d wa s wa she d wit h brin e and the orga n ic ph a se dried wit h Mg S O 4 . Th e org an ic ph a se wa s f ilte re d and evapo ra te d . Th e crud e of the re a ct io n wa s pu rif ied by ch ro ma to g rap h y (silica ge l), elu te d with a grad ie nt of 0 -5% Me O H in DCM. Yie ld ( 1 , 500 mg, 76 %). TL C R f =0 . 36 (DCM: Me O H 95 :5 ), MS (E I 70 eV ) m/z ca lcd fo r C 10 H 14 F 3 N 2 O 2 (M + + NH 4 + ) 251 . 10 02 fo un d 25 1. 10 02 . 1 H-N MR (4 0 0 MHz, C DCl 3 ) ? [pp m] 4.52 (d , 2H J=5 .8 Hz, Bz -CH 2 NH), 4.7 0 (s, 2H, Bz - CH 2 O ), 6.5 (s, 1H, OH), 7.2 5 (d , 2H J = 8 Hz, Bz-H ar om ), 7.37 (d , 2H J = 8 Hz, Bz-H ar om ). 13 C N MR (1 0 0 MHz, CD Cl 3 ) ??? [pp m] 43. 64 ( B z -CH 2 NH), 64 . 7 9 ( B z -CH 2 O ), 115 .7 0 (q , J = 287 . 9 Hz, CF 3 ), 127 .5 5 ( 2 C ar om ), 12 8 .1 8 (2 C ar om ), 13 5. 16 (C q- ar om ), 14 1. 0 (C q- ar om ), 15 7 . 09 (q, J = 15 7. 19 Hz, CO CF 3 ). 19 F NMR (3 7 6 MHz, CDCl 3 ? >ppm@ -7 5 .8 4 (CO C F 3 ). 2 . 2. Preparation of 3?,5? -O -dia ce tyl- 2? -de ox yguanosine (2 ) ?-De o xyg ua no sine (1 g, 3.78 mmo ls, 1 eq. ) wa s drie d by co e va po ra t io n wit h anh yd rou s DMF and disso lve d in 20 ml of an hyd ro u s DMF. Th en , 90 mg of DMA P (0 . 74 mmo ls, 0.2 eq . ) we re disso lve d in 2 ml of an h yd ro u s DMF and ad de d dro p wise . Ne xt , 2.6 mL of TEA (1 8 .5 mmo ls, 5.0 eq . ) an d 1.0 5 ml of Ac 2 O (1 1. 1 mmo ls, 3.0 eq. ) 181 we re add ed and th e mixt u re disso lved co mp let e ly. The so lu t io n wa s lef t to rea ct fo r 4 hou rs at r.t . Af te rwa rd s co ld DCM wa s add ed to pre cip it at e the de sire d co mp ou nd . Th e mixt u re wa s va cuu m -f ilt e red and the de sired co mp ou nd dried . Yie ld ( 2 , 1.1 g, 91 . 6%). MS (E I 70 eV ) m/z ca lcd fo r C 14 H 18 N 5 O 6 (M + + H + ) 3 52 . 12 fo un d 35 2. 12 1 H-N MR (4 0 0 MHz, Me O D ? [pp m] 1.93 (s, 3H, CH 3 O ), 2.01 (s, 3H, C H 3 O ), 2.4 7 (m, 1 +, ? , .1 m, 1+, ?? , . m, 1 +, ? , . m, +, ?, ?? , .3 m, 1+, 3? , .1 m, 1 +, 1? , .6 (s, 1H, 8). 2 . 3. Pre pa ra tion of O 6 - ( N -4 -( (hydrox y me thy l) be nzyl) -tr if luoroa c e tylmide )- 3?,5? -O - dia c e tyl- 2? -de ox ygua nosine (3 ) 3?,?-O -d ia ce ty l-d eo xygu an o sin e ( 2 , 620 mg , 1.7 9 mmo l) wa s drie d 3 time s by evap o rat ion of DMF. PP h 3 (1.4 2 g; 5.4 mmo l, 3 eq ) an d th e pro te cte d ph eno l ( 1 , 50 0 mg , 2.15 mmo l, 1,2 eq ) wa s ad de d an d lef t un de r va cuu m fo r 2 h. The n , th e mixt u re wa s disso lve d in an h yd ro us dio xa ne (1 0 .0 ml) an d ma in ta ine d un de r an arg on atmo sp he re . Fina lly, DEA D (8 60 ? l; 5.4 mmo l 3 eq ) wa s ad de d dro p wise. Th e re act ion mixt u re wa s stirre d fo r 4 hou rs at ro o m te mpe ratu re and the pro g re ss of the rea ct io n wa s mon it o red by TL C (DCM: Me O H 9:1 ). Th e ora ng e re a ct ion wa s con cen t rat ed in va cuu m to ob t a in a viscou s oil. Th e oil wa s pu rif ie d by f la sh ch ro ma to g ra ph y using a gra d ie n t of Me O H in DCM to yie ld a wh it e pro du ct (75 0 mg , 75 %). TL C R f =0 . 42 (DC M: Me O H 9:1 ), MS (E I 70 eV ) m/z ca lcd f o r C 24 H 26 F 3 N 6 O 7 (M + + H + ) 56 7 . 18 fo un d 567 . 18 1 [M + ] . 1 H-N MR (4 0 0 MHz, CDCl 3 ? >ppm@ .8 (s, 3H, CH 3 CO ), 2.12 (s, 3H, CH 3 CO ), 2.52 m, 1+, ? , .8 m, 1+, ?? , .33 m, +, ?,?? , .5 2 m, 1+, ? , 4 .9 0 (s, 2 H, B z- CH 2 1+ , .3 m, 1+, 3? , . (s, 2H, B z-CH 2 O ), 6.28 (dd , 1H, J  +z, 1? , . d, 2 H , J = 8 Hz, Bz -H ar om ), 7.49 (d, 2H, J = 8 Hz, Bz -H ar om ), 7.74 (s, 1 H, 8). 13 C N MR (1 0 0 MHz, CDCl 3 ? >ppm@ . CH 3 CO ), 20 .9 3 ( CH 3 CO ), 36. 69 (C- ? , 4 3 .6 0 (B z-CH 2 NH), 63 .7 9 (C - ? , .1 Bz-CH 2 O ), 74 .6 1 (C- 3? , .3 &- 1? , . &- ? , 11. T, J = 28 7 Hz, CF 3 ), 116 .1 1 (C-5 ), 128 . 06 (2 C ar om ), 128 . 77 (2 C ar om ), 135 . 74 (C -8 ), 136 . 49 (C q- ar om ), 13 7 .5 6 (C q- ar om ), 153 . 53 (C -4 ), 156 . 63 -15 7 .74 (q , J = 37 Hz COCF 3 ), 159 .1 5 (C-2 ), 16 0 .8 7 (C-6 ), 17 0. 27 (CO ), 170 .6 1 (CO ). 19 F NMR (3 7 6 MHz, CDCl 3 ? >ppm@ -7 5 .7 6 (CO C F 3 ). 2 . 4. Pre para tion of the de prote c te d O 6 - (N -4 -((hydrox y me thy l)be nzy l) -tr if luoro - a ce tyl mide )- 2? -de o x y gua nosine (4 ) Co mp ou nd ( 3 ) wa s disso lved in 3 ml of Me O H an d tran sf e rre d to a po lyp ro p yle ne co n ica l tub e (5 0 ml) with a stirrin g ba r. Ammo n ium hyd ro xid e (2 0%) 3 mL wa s add ed and the mixtu re was stirre d du rin g 2 hou rs. The re a ct io n wa s fo llo wed up by TL C. The so lven t wa s eva po ra ted and the cru de wa s pu rif ie d by f la sh ch ro ma t og rap h y (DC M: Me O H 95 : 5 to 90 : 10 ), yie ld ing th e exp e ct ed co mp ou nd 5 (11 2 mg , 46 %). TL C 182 R f =0 . 18 (DCM: Me O H 95 : 5 ), MS (E I 70 eV ) m/z ca lcd f o r C 20 H 22 F 3 N 6 O 5 (M + + H + ) 483 . 15 98 fo un d 48 3. 15 99 [M + ] . 1 H- 1M5  M+z, Me2' ? >ppm@ .31 m, 1+, ? , . m, 1+, ?? , 3.3 dd J = 12 Hz, J = 3, 1H, ? , 3.3 dd J = 12 Hz, J 3, 1+ ?? , .3 m, 1+, ? , . 43 (s, 2H, Bz - CH 2 NH 2 , . m, 1+, 3? , . s, +, B z -CH 2 O ), 6.28 (d d , 1H J = 8 Hz, J  +z, 1? , 7 . 27 (d, 2H J = 8 Hz, Bz -H ar om ), 7.45 (d , 2H J = 8 Hz, Bz -H ar om ), 8.0 1 (s, 1H, 8). 13 C N MR (1 0 0 MHz, Me O D) ? [pp m] 39. 73 (C - ? , . B z -CH 2 NH), 62 .3 2 (C- ? , 6 7 .2 5 ( B z -CH 2 O ), 71. 73 (C- 3? , .3 &- 1? , . &- ? , 11. &-5 ), 116 . 0 4 9 (q J = 286 . 7 Hz, CF3 ), 127 . 4 (2 C ar om ), 128 .3 1 (2 C ar om ), 135 . 91 (C-8 ), 13 7. 01 (C q- ar om ), 138 . 65 (C q- ar om ), 15 2 .8 7 (C-4 ), 15 7. 54 (q , J = 37 Hz COCF 3 ), 159 . 9 (C-2 ), 16 0. 66 (C -6 ). 19 F-N MR (3 7 6 MHz, Me O D): ? >ppm@ -77 .2 0 (CF 3 ) 2 . 5. Pre para tion of the N 2 - (N?,N? -dime thy la mino) me thy le n) -O 6 - ( N -4 - ((hydrox y me thyl ) -be nzy l)-tr if luoroa c e tyla mide ) - 2? -de ox ygua nosine (5 ) Th e pro du ct ( 4 , 0. 23 mmo l ) wa s drie d by eva po ra t ion wit h an hyd rou s DMF an d disso lved in 2 ml of DMF und e r arg on . Dmf pro t ect in g grou p 1 ?/ (4 eq , 0.9 3 mmo l) wa s add ed and the mixt u re wa s stirred du ring 4 hou rs. Th e rea ct ion wa s f o llo wed by TL C. Th e so lven t wa s eva po ra te d an d cru de wa s pu rif ied by f la sh ch ro ma to g rap h y ove r silica ge l yie ld in g th e exp e ct ed co mpo und 5 (112 mg , 46 %). TL C R f =0. 28 (DC M: Me O H 90 : 10 ), MS (E I 70 eV ) m/z fo r C 23 H 27 F 3 N 7 O 5 (M + + H + ) 538 . 20 2 fo un d 538 . 20 17 [M + ] . 1 H- 1M5  M+z, Me2' ? >ppm@ .3 m, 1+, ? , . m, 1+, ?? , 3. s, 3+, N(CH 3 ) 2 ), 3.14 (s, 3H, N(C H 3 ) 2 ), 3.7 3 (d d J = 12 Hz, J = 3, 1H, ? , 3, dd J = 12 Hz, J 3, 1+ ?? , .1 m, 1+, ? , .3 s, +, B z -CH 2 1+ , . m, 1+, 3? , . s, +, B z - CH 2 O ), 6.41 (d d , 1H J = 8 Hz, J  +z, 1? , . d, + J = 8 Hz, Bz -H ar om ), 7.4 8 (d , 2H J = 8 Hz, Bz -H ar om ), 8.24 (s, 1H, 8), 8.6 5 (s, 1H, CH=N). 13 C N MR (1 0 0 MHz, Me O D) ? >ppm@ 3 4 .5 8 (C - ? , ., .3 &, 1 CH 3 ) 2 ), 43 . 1 4 ( B z -CH 2 NH 2 ), 62 . 5 4 (C - ? , . B z -CH 2 O ), 71 .8 8 (C- 3? , .3 &- 1? , .3 &- ? , 1 16 . 77 (q J = 286 . 7 Hz, CF3 ), 117 .6 0 (C -5 ), 128. 0 1 (2 C ar om ), 12 8. 56 (2 C ar om ), 136 .7 7 (C -8 ), 137 .5 7 (C q- ar om ), 140 .6 2 (C q- ar om ), 153 . 42 (C-4 ), 15 8 .9 4 (q , J = 37 Hz COCF 3 ), 159 . 6 (C-2 ), 16 0. 41 (C-6 ), 162 . 60 (CH = N ). 19 F-N MR (3 7 6 MHz, Me O D): ? >ppm@ -7 7 .1 6 (CF 3 ). 2 . 6. Pr eparation of 5? -O - (4,4? -dime tox ytrit y l ) - [ N 2 - (N,N? -dime thy la mino)-O 6 - ( N -4 - ((hydrox y me thyl )be nzyl ) -tr if lu oroa c e tylmide )- 2? -de ox ygua nosine (6 ) A so lu t ion of (90 mg , 167 ? mo ls) of prod uct (5 ) was drie d th re e time s wit h an h yd rou s pyrid ine (3 ? 5 mL ) and f ina lly disso lved in an h. pyr (4 ml) an d pla ce d und e r arg on . Th en , 125 mg (1 .4 eq , 0.3 2 mmo ls) of DMT - Cl and 14 mg (0 .5 eq , 0. 11 5 mmo ls) of DMA P we re add ed . Th e re a ct io n wa s stirred und e r arg on ove r nig h t at ro o m 183 t e mpe ra tu re an d prot e ct ed f ro m th e ligh t . The pro du ct f o rmat io n was fo llo we d up by TL C. The n , th e re a ct io n was que nche d wit h 1 ml of Me O H and the so lve nt wa s evap o rat ed . The cru de prod uct wa s pu rif ied by fla sh ch ro ma to g rap hy ove r silica ge l (DC M 10 0 %, DCM: Me O H 1 -10 % + 2% Et 3 N ) to give 117 mg (8 3 %) of co mpo un d 6 . TL C R f =0. 68 (DCM: Me O H 95: 5 ). 1 H-N MR (4 0 0 MHz, CDCl 3 ? >ppm@ 2 . 49 m, 1+, ? , .5 m, 1+, ??), 2.92 (s, 6H, N(CH 3 ) 2 ), 3, 41 m, +, ? ?? , 3.6 6 (s, 6H, DMT -O -CH 3 ), 4 . 1 m, 1+, ? , . s, +, B z -CH 2 1+ , . m, 1+, 3? , . s, +, B z -CH 2 O ), 6.3 3 (t, 1H J  +z, 1? , . d, 2 H J = 8 Hz, DMT-H ar om ), 6.68 (d , 2H J = 8 Hz, DMT -H ar om ), 7. 0 6 - 7. 34 (m, 13 H DMT - H ar om , B z -H ar om ), 7.8 7 (s, 1H, 8), 9.3 2 (s, 1H, N=C H ). 19 F-N MR (3 7 6 MHz, CDCl 3 ): ? >ppm@ - 75 .5 6 (CF 3 ). 2 . 7. Pr eparation of 5? -O - (4,4? -dime tox ytrit y l ) - [ N 2 - (N,N? -dime thy la mino)-O 6 - ( N -4 - ((hydrox y me thyl )be nzyl ) -tr if luoroa c e tylmide ) - 2? -de ox ygua nosine - 3? -O -(N, N- di i sopropy l-2 -c ya noe thyl pho sphora mid ite (7 ) A so lu t io n of prod uct (6 ) (117 mg , 1 3 6 ?mo ls) wa s disso lve d in anh yd ro u s CH 2 Cl 2 und e r arg on and at ?&. Then, 'I3(A  ?/, 5 4 0 ? mo l, 4 eq ) an d 2 -cyan oe th yl diisopropylphosphoramidochloridite  ?/,  ?mo l) we re add ed . Af te r 15 min th e re a ct ion wa s allo we d to rea ch r.t an d stirred fo r 1 h o u r . Th e rea ct io n wa s qu en che d wit h brine , ext ra ct ed wit h CH 2 Cl 2 , drie d (Mg S O 4 ) and con ce n t ra t ed . Th e re sidu e wa s used dire ct ly with ou t f u rth e r pu rif ica t io n fo r oligo nu cle ot ide syn th e sis . TLC R f =0 .6 8 (h e xan e/ E tO A c 20 : 70 + + 2% Et 3 N ). 1 H-N MR (4 0 0 MHz, CDC 3 ? >ppm@ 1 .2 5 (s, 6H, NCH(CH 3 ) 2 ), 1.2 6 (s, 6H, NCH(CH 3 ) 2 ), 2. 50 m, 1+, ? , . m, 3 +, ??, CH 2 CN ), 2.92 (s, 6H, N(C H 3 ) 2 ), 3.24 m, +, ? ??), 3. 41 (m, 4H, CH 2 CH 2 O P, NCH( CH 3 ) 2 ), 3 .66 (s, 6H, DMT -O -CH 3 , .1 m, 1+, ? , .2 (s, 2H, Bz -CH 2 NH), 4.58 m, 1+, 3? , .4 (s, 2H, Bz -CH 2 O ), 6.33 (t, 1H J  +z, 1? , 6 . 65 (d d , 4 H J = 7 Hz, DMT-H ar om ), 7.0 6 -7. 34 (m, 13 H DMT -H ar om , B z -H ar om ), 7.8 7 (s, 1H, 8), 9.32 (s, 1H, CH = N ). 13 & 1M5 1 M+z, Me2' ? >ppm@ 2 0 .4 5 (CH 2 CN), 24 .4 7, 24 .5 2 , 24 . 54 , 24. 59 (4 C, P- N CH(CH 3 ) 2 ), 3 9 . 28 (C- ? , 4 3. 18 (2 C, N( CH 3 ) 2 ) , 4 3 .3 1 (2 C, C(CH 3 ) 2 ), 43 . 55 (B z- CH 2 NH 2 ), 55 . 16 (2 C, DMT -O CH 3 ), 57 . 94 (CH 2 CH 2 O P ), 63 .4 8 (C- ? , .31 (Bz-CH 2 O ), 74 .3 8 (C- 3? , 4 . 5 9 (C - 1? , 5 . 8 0 (C - ? , 86.4 3 (DMT- C q ), 11 3 .1 1 (4 C, DMT-C 3 ), 11 7. 46 (CF3 ), 117 .6 9 (CN), 119 . 15 (C -5 ), 127 . 28 (2 C, C ar om ), 12 8. 05 (2 C, C ar om ), 128 . 18 , 128 . 72 , 12 9 .9 3, 129 .9 5 (9 C, DMT -C 3- ar om ), 13 5 .05 (2 C, DMT - C q- ar om ), 13 5 .5 6 (B z -C q- ar om ), 13 5 . 9 7 (C -8 ), 14 0 .1 2 ( B z -C qAr om ), 14 0 . 7 0 (DMT- C qAr om ), 15 1 .4 5 (2 C, DMT -C qAr om ), 152 . 50 (C -4 ), 158 . 4 7 (2 C, COCF 3 , C-2 ), 160 . 57 (C -6 ), 16 2 .5 6 (N=CH). 19 F-N MR (3 7 6 MHz, CDCl 3 : ? >ppm@ -75 .5 6 (CF 3 ). 31 P -N MR (1 6 1 MHz, CDCl 3 ): ? >ppm@ 14 8 .8 3. 184 3 . Synthe sis and purific a tion of oligonuc le ot id e s 3.1. Oligonuc le otide synthe s i s Th e oligo nu cleo t ide seq ue n ce s we re syn th e size d at 1 ?mo l sca le usin g th e sta nd a rd pro to co ls (Tab le 1). Th e in co rp o rat io n of th e O 6 - (N -4 -((h yd ro xyme th yl)b en zyl) - t rif luo roa cet ylmid e ) - ?-de o xyg ua no sine ph osph o ramid it e ( A d G ) in th e seq ue n ce wa s ca rrie d ou t man ua lly wit h a co up ling time of 15 mi n , ob ta in in g a yie ld of 75 % . Th e in co rpo ra t io n of th e Q T-n u cle ob a se s to th e co mp le men ta ry se qu en ce s wa s au to ma t ica lly pe rf o rme d on the syn th esize r. In all th e syn t he se s we used dime t hylf o rma mid in o - p ro te ct ed gu an ine pho sp ho ra mid ite ( 7 ) . Th e syn th e se s we re co mp let ed using the DMT - O FF pro t o co l , exce pt fo r the seq ue n ce con ta in ing A d G , wh ich wa s syn t he sized in DMT - O N mo d e to he lp pu rif icat io n . 3 . 2 . Oligonuc le otide s de prote c tion and purific a tion . Af t e r olig on u cle o t id e syn th e sis, th e so lid supp o rts we re tra nsf e rr ed to scre w -ca p via ls and in cub at ed wit h a so lu t io n of con cen t ra t ed aq ue ou s ammon ia ove rn ig h t at roo m te mpe ra tu re , an d ad d it io na l 15 min in cub a t io n at 55 ? C we re ne ed ed f o r the se qu en ce co n ta in in g Bz G . Th e so lut io n s we re the n f ilt e red usin g ste rile co t to n a nd tra n sf e rred in to a 2 mL ep pe nd o rf tu be . The so lu t ion s we re eva po ra te d usin g a nit rog en syste m to re mo ve the ammon iu m. The re su lt ing prod u cts we re de sa lt ed by Seph ad e x G -25 using wa t e r as elu en t . The se qu en ce con ta in ing Bz G wa s pu rif ie d by reve rse d -p ha s e HP L C using the DMT - O N pro to co l in a Nu cle o sil 12 0 10 -C18 10 ?m (250 x8 mm) co lu mn wit h a f lo w ra te of 3 mL / min and an in crea sing grad ie nt of ACN (15 % to 80 %) ove r 0.1 M aq ue ou s TE A A pH 6.5 , du rin g 20 minu te s. The re te nt ion time for th e olig on u cleo t id e was 10. 23 min . Th e pu re f ra ct ion s we re co mb ine d and evap o ra t ed to dryn e ss. The ob ta ine d re sidu e s we re de t rit yla te d by add in g 1 mL of 80 % acet ic acid so lu t io n fo r 30 min at ro o m te mpe ra tu re . The dep ro te ct ed olig onu cle ot ide s we re de sa lt ed and fu rt he r pu rif ied b y a se con d ro un d of ch ro mat og ra ph y, using th e DMT -O FF pro to co l: 20 min ut e s of lin ea r gra d ie n t f ro m 5% to 50 % ACN ove r 0.1 M aqu eou s TEA A pH 6.5 . Th e yie ld and pu rit y ob ta ine d fo r the pro du ct s wa s aro und 85 % in all ca se s. The len gt h an d ho mo ge ne it y of th e o ligo nu cleo t ide we re ve rif ie d by MA L DI -TOF (ta b le 1) . The DNA -st ra nd con cen t rat io n wa s de te rmin ed by ab so rba nce mea su re men t s (26 0 nm) and its ext in ct io n co ef f icie nt s . Olig on u cleo t id e sa mp le s we re ke p t at 4 ?C un t il fu rt he r use . 3 . 3 . Pre para tion of the flu ore sc e nt F G oligonuc le otide . The ?-A TCTTCTC Bz G A TTCA - 3? olig on u cle ot id e wa s lef t to re a ct wit h f luo re sce in iso t h io cyan at e (FITC) th ro u g h its f ree amino gro u p as fo llo ws. 5 O.D. of th e olig on u cle o t id e we re dissolYed in  ?/ of an aTueous solution of . M 1a+&2 3 (p H 9) and 1 eT of a solution of FIT& solYed in  ?/ 'MF were added and left to react at rt for h. Then, 1 ?/ of an aTueous solution of . M 1a+&2 3 (pH 9) and an 185 a dd it io na l 10 equ iv of FITC in 1 ?/ of 'MF were added and the mixture was left to re a ct ove rn igh t at rt. The mixtu re s we re co n cen t ra te d to dryn e ss an d the re sid ue re su spe nde d in 1 mL of wa t e r. Th e so lu t ion w a s pu rif ied by Se ph ad e x G -2 5 an d ana lyzed by RP -HP L C using an XB ridg e OS T C1 8 2.5 ? m co lu mn . The co up lin g eff icien cy wa s de te rmin ed by HP L C ana lysis ( 8 6 %). Th e pu rif ie d oligo me rs we re ana lyzed by MS (MA L DI- TOF) , se e tab le 1. DNA du p le xe s we re ob ta ine d by ann ea lin g eq u imo la r co n ce nt ra t io n s of th e con t ro l, Bz G and F G se qu en ces wit h the co mp le me nt a ry oligon u cleo t id e stran d s at 72 ?C f o r 5 min and th en allo w in g the m to slo wly coo l do wn to ro om te mpe ra tu re . 4 . hAG T ex pre ssion and purific a tion I n vit ro assa ys we re ca rrie d ou t usin g re co mb inan t hAG T , ove re xp re sse d an d pu rif ied as pre vi ou sly de scrib ed . 13 Brief ly, hA G T pro t e in wa s exp re sse d in th e E. co li stra in Ro se tt a , ind u ce d by ad d ing 1 mM IPTG an d lef t to exp re ss fo r 4 h at 30 ?C. Th e pe lle t wa s disrup t ed by so n ica t ion an d ce nt rif u ge d. The su p e rn at an t wa s f ilte red , loa de d int o a HiTra p TM FF co lu mn an d elut ed with an Imid a zo le grad ie nt (20 -50 0 mM) in th e fo llo wing buf f e r of 35 0 mM Na Cl, 20 mM Tris pH 8, and 1 mM BME . Fina lly, th e prot e in wa s loa de d in t o a Sup e rd e x 75 16 /6 0 be in g the buf f e r 200 mM Na Cl, 20 mM Tris pH 8.0, 10 mM DTT an d 0.1 mM EDTA . The pro te in wa s co n ce nt ra te d to 2 mg / ml in th is buf f e r and kep t at -2 0 ?C in th e pre sen ce of 40 % glyce ro l. Th e sa me pro to co l wa s used f o r the pu rif ica t ion of th e ina ct ive mu ta n t hA G T -C1 45 S exp ressed in the E . co li stra in BL2 1 . Th is mu ta n t wa s used as neg a t ive con t ro l in man y expe rime n t s. 5 . T he rma l sta bili ty studie s. Me lt in g cu rve s of all the po ssib le pa ir s of comp le men t a ry oligo nu cleo t ide s we re me a su re d by mo n it o ring th e ab so rb an ce hyp e rch ro micit y at 26 0 nm. UV/ V is ab so rp t io n sp e ct ra we re re co rde d at 1?C/ min in t e rva ls, with a 1 -min equ ilib ra t io n time at ea ch te mpe ra tu re ; the sa mp le wa s hea te d ove r th e ra ng e 10 -80 ?C. Th e buf f e r so lu t io n s used we re 10 mM so d iu m pho spha t e pH 7.0 an d 100 mM Na Cl. Sa mp le con cen t rat ion was around  ?M. (ach sample was allowed to eTuilibrate at the initial temperature wit h ou t an y ext e rn a l con t ro l of te mp e ra t u re f o r 5 min bef o re the me lt in g expe rime n t beg an . Th e me lt in g te mp e ra t u re s (Tm) are th e ave ra ge va lue of at lea st on e pa i r of T m expe rime n t s. Th e da ta we re an a lyze d by th e den a tu ra t io n cu rve pro ce ssing prog ra m, Me ltW in v. 3.0 . Me lt ing te mpe ra tu re s (Tm) we re de te rmin ed by co mp ut e r f it t ing of the f irst de riva t ive of ab so rb an ce with respe ct to 1/T. Th e rma l sta b ilit y expe rime n t s re co rd in g the emit t ed f luo re sce n ce at excita t ion wa ve len gt h of 485 nm we re also pe rf o rmed fo r t h e do ub le stran d s con ta in ing a f lu o rop ho r or a f luo rop ho r-q ue n ch e r pa ir . The se exp e rimen t s we re re a lize d in the sa me co nd it ion s, co n ce nt ra t io n , ra ng e of te mp e ra t u re an d buf f e r de scrib e d ab ove . 186 6 . Circ ula r Dic hroism Th e CD spe ct ra of all the po ssib le pa irs of co mp le me n ta ry olig on u cle o t id e s we re re g ist e re d at 25? C ove r a ra nge of 22 0 ?32 0 nm, wit h a scan n ing spe ed of 50 nm?min ?1 , a re spo n se time of 4s, dat a pit ch o f 0.5 nm, and a ban d wid t h of 1n m. Th e du p le xe s concentration was  ?M in a buffer solutions of 1 mM sodium phosphate p+ . and 1 00 mM Na Cl. Th e sa mp le s we re ann ea led bef o re re co rd ing of sp e ct ra . 7 . HP LC ana lys is of h AG T dea lk yla tion . In ord e r to me a su r e th e dea lkyla t io n of F G, 14 2 pmo l an d 350 pmo l of th e dup le xe s we re in cuba t ed wit h 40 pmo l of fu ll-le ng th -h AGT to a f in a l vo lu me of 40 0 ?l in a re a ct ion buf f e r (20 0 mM Na Cl, 50 mM Tris pH 8.0, 1 mM DTT, 5 mM EDTA ). Th e re a ct ion wa s in cub at ed fo r 90 min at 37 ?C and sto pp ed by hea t ing the sa mp les at 72 ?C f o r 5 min . The rea ct ion prod u ct s we re ana lyzed by HPL C on a Nu cle o sil ana lyt ica l co lu mn at 37 ? C. The HP L C f lo w ra t e was 1 ml/ min , and a grad ien t of 10 ?40 % ace to n it rile in 20 min wa s use d. 8 . Fluore sce nce a ssa y for hAG T ac tivit y . Th e f lu o re scen ce assa y wa s pe rf o rme d with fu ll-le n g th hA G T an d the hA G T -C1 45 S (in a ct ive mu ta nt ) wa s use d as a ne ga t ive co n t ro l. Th e rea ct ion wa s acco mp lishe d in a total Yolume of  ?l in each well, incubating increasing concentrati on s of hA G T (5 0 , 100 and 200 nM) in re a ct ion buf f e r (2 00 mM Na Cl, 50 mM Tris pH 8.0 , 1 mM DTT, 5 mM ('TA,  mM .&l . The assay was initiated by the addition of  ?l of fluoro ph o re - q ue n ch e r dup le x su b st ra te (10 nM, 0.5 pmo l ) and th is so lu t ion wa s the n pla c ed in a micro p la te re ad e r syste m. Fluo re sce n ce wa s me a su re d eve ry minu te f o r 20 min at excit at io n an d emission wa ve len g th s of 485 and 535 nm, re spe ct ive ly. Ave rage s ove r th ree rea d ing s we re ta ke n fo r ea ch co nd it io n teste d . Each expe rime nt wa s pe rf o rmed in trip lica te s. 187 Ta ble S1 : Se que nc e of oligonuc le otide s and it s cha rac te riza tion by MALD I -T O F Na me Se qu en ce Ma ss fo un d (ca lcu lat ed ) Olig o - Bz G ?- A TC TTC TC Bz G A TT CA - 3? 431 0 .9 (e xp ect ed 430 6 ,9 4 ) Olig o - F G ?- A TC TTC TC F G A TT CA - 3? 47 0 2 . 2 (e xp ect ed 4 69 5 .3 1 ) Co n t ro l ?- A TC TTC TCG A TT CA - 3? 419 0 .0 (e xp ect ed 418 7 .7 7 ) Co mp le men ta ry ?- TGA ATC GA G AA G AT - 3? 4 33 3 .8 (e xp ect ed 433 2 .7 7 ) Co mp le men ta ry - Q T ?- TGA A Q T C GA G AAG AT - 3? 474 1 .2 (e xp ect ed 474 0 .3 8 ) Misma t ch - T ?- TGA ATT GA G AA G AT - 3? 4 34 8 .2 (e xp ect ed 434 7 .7 7 ) Misma t ch - Q T ?- TGA AT Q T GAG AAG AT - 3? 4 75 7 .0 (e xp ect ed 475 5 .3 9 ) Se qu en ce s of the dif f e ren t olig on u cle ot id e used in th e de ve lop me nt of the hA G T f lu o re scen ce assa y. Bz G rep re sen t s O 6 -be nzyl-2 ?-d eo xy gu an ine . F G corre sp on d s to th e O 6 - Flu o re sce in -be n zy l- ?-de o xygu an in e, wh e re f lu o re sce in is a f lu o rop ho re gro up . Q T re p re se n t s a mo d if ie d T wit h the que n ch e r gro up (Da b c yl). Ma ss sp e ct ro me t ry ana lysis (MA L DI -TOF) an d exp e ct ed ma ss. 188 Ta ble S2 . Me lting te mpe ra ture s of the duplex es. ?-A TCTTC TC XA TTCA - 3? 3 ?-T A G AA G A ZW TAAG T - 3? Name XA ZW Tm (2 6 0 nm) Tm (4 8 5 nm) Co n t ro l: Co mp le me nt a ry G A CT 54 .6 N d Co n t ro l: Misma t ch - T G A TT 45 .0 N d Olig o - Bz G : Co mp le me nt a ry Bz G A CT 33 .3 N d Olig o - Bz G : Misma t ch - T Bz G A TT 36 .4 N d Olig o - Bz G : Misma t ch - Q T Bz G A Q TT 40 .2 N d Olig o - Bz G : C Q T Bz G A C Q T 41 .3 N d Olig o - F G: Co mp le men t a ry F G A CT 33 .0 N d Olig o - F G: C Q T F G A C Q T 30 .3 28 .0 /5 4 .1 Olig o - F G: Misma t ch - T F G A TT 27 .9 N d Olig o - F G: Misma t ch - Q T F G A Q TT 34 .3 26 .1 nd : no t de te rmin ed . 189 Figure S1: CD spectra of the different duplexes. A) CD spectra of complementary duplexes. B) CD of the mismatch duplexes. All de experiments were performed in 10 mM sodium phosphate pH 7.0 and 100 mM NaCl with a 2 uM of duplexes. Figure S2: Curves of the melting temperatures of the two fluorescent-quencher duplexes at 485nm. 190 Figure S3 : HP LC profi le s o f the contro l se que nce s. A) u n mo d if ied oligo nu cleo t ide se qu en ce wit h its co mp le me n ta ry strand . B) f luo re sce in -be nzyl-mo d if ie d olig on u cle o t id e seq ue n ce with its co mp le men ta ry stra nd . Th e HPL C wa s pe rf o rmed at 37 ?C. 191 Figure S 4 : Control ex pe rime nts of the f luo re sc e nc e assa y . Th e ba ckg ro un d f lu o re scen ce of the do ub le stran de d se qu en ce is ob se rve d in the ab sen ce of hA G T and in the presen ce of an ina ct ive mu ta nt . An in crease of f luo re scen ce is ob se rved in the pre sen ce of th e sa me con cen t rat io n of act ive hAGT (1 50 nM). Figure S5 . K ine t ic da ta of f igu re 2 (ma in text ). Th e emission of f luo re sce n ce co rre spo nd ing to 3 co n ce nt ra t ion s of hAG T (1 0 , 20 an d 30 nM) is me a su re d fo r 20 min u t es. 192 1 H- NMR , 13 C- NMR 19 F- NMR and 31 P - NMR spe c tra 193 194 2 195 3 196 4 197 5 198 6 199 7 200 201 202 Appendix 4 In vitro assay to evaluate potential inhibitors of hAGT by a new fluorescence method: preliminary results 203 204 In vitro assay to evaluate potential inhibitors of hAGT by a new fluorescence method In chapter 4 we describe the development of a novel methodology to measure hAGT activity through the fluorescence transfer from a labelled alkyl-guanine to the active site of hAGT. The repair reaction produces the removal of fluorescein, which is withdrawn with the alkyl group and brought apart from the quencher group, which remains in the duplex sequence. Using this methodology, we have performed a first screening of the compounds described in chapter 1. The results obtained are preliminary, but represent a proof of concept of the usability of the assay for the evaluation of potential inhibitors of hAGT. The assay was performed using the mismatch duplex described in chapter 4. hAGT was incubated with each compound at 30?C during 30 minutes before adding the fluorescent duplex. Immediately, emission of fluorescence was monitored at 25?C during 20 minutes. Figure 1 represents the kinetic data obtained for compounds 1 to 9. Figure 1. Kinetic data of the inhibitory activity of compounds 1 to 9. This results suggest that compounds 1, 2, 4 and 8 may inhibit hAGT activity in vitro, while compounds 3, 6, 7 9 do not seem to affect hAGT activity. Compound 5 seems to slightly inhibit hAGT during the first minutes of the assay but at minute 20 seems to reach the same fluorescence as hAGT. Inhibition at 20 minutes is represented in figure 2. Figure 2. Inhibition of hAGT by compounds 1 to 9 at 20 min, calculated from the increase of fluorescence with respect to time 0. The basal increase of fluorescence of the duplex was subtracted from all the measurements. 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 % Flu or es ce nc e Inhibition at Time = 20 m in 205 Compound 8 seems to be the most promising inhibitor in vitro, judging by the results obtained in these experiments. This is in agreement with the results described in chapter 1, where compound 8 was found to form a complex with hAGT by ESI-MS and was observed to be non-toxic per se and a good enhancer of carmustine activity in cellular experiments. However, these experiments are preliminary and should be confirmed by the repetition of the assays at 37?C. 206 Experimental details: The fluorescence assay was performed using previously overexpressed and purified full-length hAGT (chapter 1)? he reaction ?as accomplished in a total volume o? 5? ?l in each well, incubating 150 nM of hAGT with 750 nM of each compound (hAGT:compound 1:5) in reaction buffer (200 mM NaCl, 50 mM Tris pH 8.0, 1 mM DTT, 5 mM EDTA, 20 mM KCl). The compounds were dissolved in 1 mL of DMSO to prepare a stock concentration of 100 ?M. Enough volume of each compound was added and left to incubate with hAGT during 30 minutes prior to the repair reaction with DNA. The assay was initiated by the addition of 5 ?l o? ?luorophore-quencher duplex substrate (15 nM) and this solution was then placed in 96-well plates with black bottom (Nunc). Fluorometric measurements were performed on a spectrofluorometer Jasco FP6200 at 25?C and with a Multi-Detection Microplate Reader SpectraMax M5 (Molecular devices BioNova cientifica, Sunnyvale, USA). Fluorescence was measured every minute for 20 min at excitation and emission wavelengths of 485 and 535 nm, respectively. Averages over three readings were taken for each condition tested. Each experiment was performed in triplicates. 207 208 Chapter 5 Molecular biosensing using gold-coated superparamagnetic nanoparticles functionalized with DNA aptamers. 209 210 Molecular biosensing using gold-coated superparamagnetic nanoparticles functionalized with DNA aptamers. Maria Tintor?,1 Stefania Mazzini,2 Laura Polito,3 Marcello Marelli,3 Alfonso Latorre,4 ?lvaro Somoza,4 Anna Avi??,1 Carme F?brega,1 and Ramon Eritja1*. ChemBioChem, submitted 1 IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine. c/ Jordi Girona 18-26. 08034 Barcelona (Spain). 2 Department of Food, Environmental and Nutritional Sciences (DEFENS), Division of Chemistry and Molecular Biology, University of Milan, Via Celoria 2, 20133 Milan, Italy. 3 Institute of Molecular Science and Technologies, ISTM-CNR, Via G. Fantoli 16/15, 20138 Milan, Italy. 4 IMDEA Nanociencia & Nanobiotecnolog?a (IMDEA-Nanociencia), Unidad Asociada al Centro Nacional de Biotecnolog?a (CSIC) Madrid, Spain 211 Gold and iron-based magnetic nanoparticles seem to be one of the most promising nanoparticles for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core provides the benefits from both nanoparticles, adding the magnetic properties to the robust chemistry provided by the thiol functionalization of the gold coating. In this chapter, we describe the use of gold-coated magnetic nanoparticles for molecular biosensing. Binding of ?-thrombin to two aptamers conjugated to these nanoparticles causes aggregation, given that the aptamers bind cooperatively to opposite sites of the protein, forming a molecular network. This phenomenon can be observed by UV, DLS and MRI. These techniques discriminate even a single methylation in one of the aptamers, which prevents aggregation due to the inability o? ?-thrombin to recognize it when it is not correctly folded. A parallel study with gold and ferromagnetic nanoparticles is also detailed, concluding that the gold coating of Fe3O4 nanoparticle does not affect the performance of the iron-based nanoparticles and that they are suitable for the development of more complex biosensors. These results prove the high detection potency of gold coated superparamagnetic nanoparticles for biomedical applications. This work is the result of a doctoral short stay in the University of Milan and in the CNR. The long-term objective of this detection method is to implement it as a detection method for hAGT repair activity, as it is able to detect a single methylation in guanines, the substrate for hAGT repair activity. 212 FULL PAPER Mo l ecul ar bi osensin g usi ng gold -coated superparam agneti c nanoparti cl es functi onal iz ed with DN A aptam ers. Ma ria Tin t o r? , [a] Stef an ia Ma zzin i, [b] Lau ra Po lit o, [c ] Marce llo Ma re lli , [c ] Alfo nso Lat o rre , [d ] ?lva ro So mo za , [d ] Anna Avi?? , [a] Carme F?b re ga , [a] a nd Ra mo n Erit ja * [a] Ab str act: A u - and ir on- b as ed m agn etic n an op art ic l es (NPs ) ar e pr omis i ng NPs f or b i om edic al ap pl ic at i ons d u e t o t h eir un iq u e pr op er ti es . T h e c omb in ati on of a g old c oati ng over a m agn etic c or e pr ovi d es th e b en ef i ts f r om ad di ng t h e m agn et ic pr op ert i es t o t h e r ob us t c h em is tr y pr o vi d ed b y th e t hi ol f u nc ti on ali z ati on of g ol d. H er e, th e us e of A u-c oat ed m agn et ic NPs f or m ol ec u l ar b i os ens i ng is described for the first time. Binding of ? - thr om bi n t o t wo ap t am ers c onj ug at ed t o th es e N Ps c aus es ag gr eg ati on , a p h en om en on t h at c an b e obs er ved b y UV, D LS and MR I. T h es e t ec h ni qu es dis c ri min at e a s in gl e m et h yl at i on in on e of t h e apt am ers , pr e ven ti ng aggregation due to the inability of ? -th r omb in t o r ec og ni z e i t. A p ar al l el s tu d y wi th g ol d an d f err om ag n etic NPs is d et ail ed, c onc lu di ng t h at t h e Au c oati ng of F e3O 4 N P d oes n ot af f ec t th eir p erf or m anc e an d t h at t h ey ar e s u it abl e f or t h e d ev el op m ent of c omp l ex bi os ens ors . T h es e r es ults pr ove th e h ig h d et ec t i on p ot enc y of A u-c oat ed S PI O N s f or bi om ed ic al app l ic at i ons . Introduc tion I n r ec ent y ear s , a gr eat v ar i et y of c h em ic al m et h ods h as b e e n d ev el op ed t o s ynt h es i z e fu nc t i on al i z ed n an op ar t ic l es f or b i om ed ic al ap p l ic at i ons s uc h as dr u g d el i v er y, c anc er th er ap y , d i ag n os t ic s , tis s u e en g in e er i n g and m ol ec u l ar b i ol o g y, an d th e s t r uc t ur e- f u nc t i on r el at i ons hi p of th es e fu nc t i on al i z e d n an op ar t ic l es h as b een ext ens i v el y ex am i n ed. [1 ] In p ar t ic u l ar , th e c ont r ol l ed as s em b l y of g ol d n an op ar t ic l es (Au NPs ) h as b een a su bj ec t of gr eat int er es t ov er th e p as t d ec ad e du e t o th e p ot ent i al ap p l ic at i ons of t h es e p ar t ic l es in n an ob i ot ec h n ol og y. [2 ] Th ei r un i qu e p h ys ic al pr op er t i es , [3 ] par t ic u l ar ly th ei r loc al i z ed s ur f ac e p l as m on r es on anc e ( LS P R) an d t h eir ef f ic i en t int er ac t i on w it h m ol ec u l es w it h a fr ee t h i o l gr ou p m ak e Au NPs at t r ac t i v e b u i ld i n g b l oc k s f or n an os c al e el ec t r on ic and ph ot on ic d ev ic es . [4 ] Sinc e t h e f ir s t DNA s ens or w as d es i g n ed b y M ir k in an d c o- w or k er s , [5 ] t h e d ev el op m en t of Au N Ps - b as ed c ol or i m et r ic bi os ens or s h as b e en inc r eas i n g l y ap p l i ed f or th e d et ec t i on of a lar g e v ar i e t y of t ar g et s , inc lu d i n g nuc l eic ac ids , pr ot ei ns , s ac c h ar i d es , s m al l m ol ec u l es , m et a l ions , an d ev en c el ls . T h is t ec h n iq u e t ak es ad v ant ag e of t h e c ol or c h an g e t h at ar is es fr om t h e int er p ar t ic l e pl as m on c oup l i n g dur i ng A uN P ag gr eg at i on (r ed - t o - p ur p l e or b l u e) or r ed is p er s i o n of an Au N P ag gr eg at e ( p ur p l e- t o - r ed) . [2 a , 2 b, 2g ] It is q uic k l y b ec om i n g an imp or t ant al t er n at i v e t o c onv en t i on al d et ec t i o n t ec h ni q u es , as f l u or es c enc e- b as ed as s ays , and h ol ds gr e at p ot ent i al in c l in ic al d i ag n os t ic s , dr u g dis c ov er y an d en v ir on m ent al c ont am i n ant an al ys is , am on g ot h er s . In c ont r as t , s u p er p ar am ag n et ic ir on o xi d e n an op ar t ic l es (SP IO Ns ) p os s es s d if f er ent int er es t in g f eat ur es f or n an om ed ic i n e. S P IO Ns ar e w el l k n ow n as in n ov at iv e ag en t s in d i ag n os t ic s , du e t o th ei r ad v ant ag es as M ag n et ic R es on an c e Im ag i n g (M R I) c ont r as t ag en t s . [6 ] In c om p ar is on w it h t h e tr ad it i on al g ad ol i n i um - b as ed c ont r as t ag en t s , SP I ONs pr od uc e low er t oxi c it y, s t r ong er en h anc em ent of pr ot on r el ax at i on an d h av e a low er d et ec t i on lim it . [7 ] Fur t h er m or e, S P IO Ns h a v e s ev er al ot h er app l ic at i ons in b i om ed ic i n e, es p ec i al l y f or d el i v er y pur p os es , d u e t o t h eir r ed uc ed s i z e, th e ab i l it y t o b e tr ans p or t e d in bi ol og ic al s ys t ems [8 ] an d th e p ot ent i al us e f or th er ap y b y m ag n et ic h e at i n g. [9 ] G ol d an d ir on- b as ed m ag n et ic n an op ar t ic l es (A u SP I ONs ) h av e a pr om i n ent p ot ent i al in b i om ed ic al ap pl ic at i on s d u e t o th eir u n i qu e pr op er t i es . Th e c om bi n at i on of a g ol d c o at i n g ov er th e m ag n et ic c or e pr ov i d es t h e b en ef it s fr om b ot h n an op ar t ic l es , ad d i ng t h e m ag n et ic pr op er t i es t o t h e r ob us t c h e m is t r y pr ov id ed by t h e t h i ol f u nc t i on al i z at i on of th e g ol d c oat in g. F or th is r eas on, th er e is an inc r eas i n g int er es t on th e s y nt h es is and ap p l ic at i ons of th is ty p e of g ol d - c oat ed n an op ar t ic l es . [8 c , 8 d, 10 ] In th is w or k , w e d es c r ib e f or th e f ir s t tim e t h e u s e of g ol d c oat ed m ag n et ic n an op ar t ic l es as m ol ec u l ar b i os ens or s , thr oug h t h eir f u nc t i on al i z at i on w it h D NA apt am er s th at ar e recognized by the protein ? - t hr omb i n. F or t h is p ur p os e, w e conMugated the ?- t hr om b in b in d i ng apt am er s 1 an d 2 (T BA 1 an d TB A 2) , and a m et hy l at ed v er s i on of T BA 1 (O 6 - M eG- T BA 1) (T ab l e 1) t o g ol d- c oat ed ir on- oxi d e n an op ar t ic l es , t o ir on - o xi d e n an op ar t ic l es an d g ol d n an op ar t ic l es , in or d er t o as s es s th e ad v ant ag es of e ac h ty p e of N Ps . T h e TB A 1 an d T BA 2 s eq u enc es ar e k n ow n t o bi n d c oop er at i v el y t o t w o s p ec if ic an d almost opposite epitopes of ? - thrombin, forming a ?molecular sandwich? complex.[1 1 ] TB A 1 (pr im ar i ly f i br i n og en - r ec og n it i on [a] M. T i ntor ?, Dr . A. A vi ??, Dr . C. F ?br ega, and Pr of. R Er i tj a. Depar tm ent of Chem i c al and Bi om ol ec ul ar Nanotec hnol og y IQ AC - C S IC, C IB ER - B BN Networ ki ng Cen tr e on Bi oe ngi neer i ng, Bi om ater i als and Nanom edi c i ne C/ J or di G ir ona 18 - 26. 08 034 Bar c el ona, Spai n. E - m ai l : : ram on.er i tj a@i qac .cs ic .es [b] Dr . S. Ma zzi ni , Depar tm ent of Food, En vi r onm ental a nd Nutr i ti onal Sc i enc es ( DEF ENS) , Di vi s i on of Chem i s tr y an d Mol ec ul ar Bi ol og y, Uni ver s i ty o f Mi l an, Vi a Cel or i a 2, 20133 Mi l an, It al y. [ c ] Dr . L. Pol i to, Dr . M. Mar el l i Depar tm ent Ins ti tute of Mol ec ul ar Sc i enc e and T ec hnol ogi es IST M - C N R , Vi a G . Fantol i 16/15, 20 138 Mi l an, Ital y [d] Dr . A Lator r e, Dr . A Som oz a IMD EA Nanoc i enc i a & Nano bi otec nol o g? a (IMD EA - Nanoc i enc i a) , As oc i ada al Centr o Nac i onal de Bi otec nol og? a (CS IC) C/ Far ada y 9, 280 49 Ma dr i d, Spai n 213 FULL PAPER ex os it e b i n di n g) [1 2 ] is a 1 5m er nuc l eot i d e c omp os ed of tw o G - t et r ads th at ar e c on n ec t ed b y thr ee ed g e - w is e l o ops , f or mi n g a w el l- c h ar ac t er i z ed int r am ol ec u l ar c h ai r - l ik e, ant i p ar al l e l qu ad r u p l ex. In c ont r as t , TB A 2 (2 9m er nuc l eot i d e, h ep ar i n - b in d i ng e xos it e) f or ms a c om b i n ed qu ad r u p l ex/ du p l e x s t r uc t ur e. [1 1 ] T ab le 1. 2ligonucleotide seTuences of the three ? - thr om bi n bi ndi ng aptam ers . Nam e Seque nc e T BA1 ?- H S - T 15 G GT T G GT GT GGT T GG - 3? T BA2 ?- H S - T 5 AGT CC GT G GT AGG G CAG GT T G G GGT G ACT - 3? O 6 - MeG - T BA1 ?- H S - T 15 G GT T G Me GT GT GGT T GG - 3? Th e m i xt ur e of T BA 1 an d T B A2 c onj ug at ed n an op ar t ic l es s h ou ld f or m a tr i d im ens i on al n et w or k in presence of ? - t hr omb i n, [8 e ] as repr es en t ed in Sc h em e 1. T h is int er ac t i on c an b e d et ec t ed in a st r ai g ht f or w ar d m an n er us i n g thr ee t yp es of t ec h ni q u es : UV, D yn am ic L i ght Sc at t er i n g (D L S) an d M R I. Au S PI O Ns an d Au N Ps h av e a m axi m u m of abs or b anc e du e t o th eir s ur f ac e p l as m on r es on an c e at 5 2 0 n m th at s h if t s to h ig h er w av el en gt hs wh en ag gr eg at i on oc c ur s an d t h is c h an g e is eas i l y d et ec t ed b y UV- s p ec t r os c op y. A ggr eg at i on of th e thr ee ty p es of n an op ar t ic l es c an b e d et ec t ed b y D L S, m eas ur i n g th e m ai n hy dr od y n am ic d i am et er (H D) of t h e n an op ar t ic l es r es u lt i n g in a hu g e inc r eas e when ?- t hr omb i n is ad d ed t o th e m i xt ur e of n an op ar t ic l es c ar r y i n g th e T B As . F in al l y, S P IO Ns an d Au63I21s allow the detection of the complex between ? - t hr omb i n and th e n an op ar t ic l es by m e ans of M R I, b ec aus e t h e y ar e c ont r as t ag ent s f or im ag e en h an c em ent . Sch em e 1. Repr es entati on of the T BA- c onj ugate d nano par ti c l es and the tridimensional networN formation in presence of ? - thr om bi n for the unm odi fi ed T BAs . T BA1 and T BA2 ar e repr es ented fol ded i n t hei r c hai r l i ke s tr uc tur e i n dar k bl ue a nd i n gr een res pec ti ve ly ? - thr om bi n i s repr es ented i n vi ol et. Fur t h er m or e, w e als o e xp l or ed t h e d is c r im i n at i on c ap ac it y of th e A uS P IO N n an op ar t ic l es t o d et ec t a sin g l e m et h y l at i on in t h e DN A apt am er , u p on d es t ab i l iz i n g th e qu adr u p l ex s t r uc t ur e of TB A 1 by th e inc or p or at i on of a m et h y l G in on e of it s t et r ads . In this c as e, t h e m i xt ur e d o es n ot f or m a tr i d im ens i on al n et w or k because ?- t hr omb i n is n ot ab l e t o r ec og n i z e th is m od if i e d v er s i on of th e apt am er . [1 3 ] M or e ov er , th is in ab i l it y t o f or m th e n et w or k al l ow ed us t o us e th is s et of n an op ar t ic l es as a d et ec t i on pr ob e f or a s ing l e m et hy l at i on. T his s ys t em c an b e fur t h er d ev el op ed f or th e d et ec t i on of t h e ac t i v it y of D NA r ep ai r pr ot ei ns w h ic h h av e alk y l at ed g u an i n es as s ubs t r at e. Re sult s and Disc uss ion Sy nt he s is of go l d s up er p ar a m ag ne t i c ir o n ox i d e na no p ar t i c l es ( AuS P I ON) Th e pr ep ar at i on of th e g ol d c oat ed n an op ar t ic l es w as p er f or m e d f ol l ow i n g a pr oc es s c ons is t i ng of t w o m ai n s t eps , t h e pr ec ip it at i on of th e f er r om ag n et ic s eeds f oll ow ed b y th ei r c oat in g w it h g ol d ac et at e. [1 0 ] Th e pr ec ip it at i on of f er r om ag n et ic s eeds w as obt ai n ed in g o od y i el d, an d t h e r es u lt i ng n an op ar t ic l e c or es w er e c h ar ac t er iz ed b y TE M. TE M im ag es ( F i gur e 1 A) s h ow h om og en eit y in s i z e an d s h ap e of th e f er r om ag n et ic c or e wh ic h is an in d is p ens ab l e c on d it i on t o ac h i ev e t h e f i n a l Au S PI O N. Th e c oat i ng of th e f er r om ag n et ic s eeds w it h g ol d ac et at e w as p er f or m ed s u bs eq u ent ly w it h h i g h y i el d of c oat in g. Th e p ur if i e d g ol d- c oat ed m ag n et ic n an op ar t ic l es w er e an al ys ed by T E M (Fi g ur e 1 B) , s h ow i ng an inc r eas e of ar ou nd 1. 6 nm in t h e c or e d i am et er , w h ic h c onf ir ms t h at t h e c ov er i ng w as obt ai n e d s uc c es s f ul l y. F ig u re 1 . T EM i m ages of the gol d -c oated m agneti c nanopar ti cl es , AuSP IO N. A. Im ages and p ar ti c l e s i ze di s tri buti on of the f er r om agneti c c or es . B. Im ages and par ti c l e s i ze di s tri buti o n of the gol d- c oated SP IO N. Co nj ug at i o n of the AuN P s, AuS P I ON s a nd S PI ON s w it h T B A1, T B A2 a nd O 6 - M eG- T B A1 TBA1 and TBA, that bind to opposite sites of ? - t hr om b in, an d O 6 - M eG- T BA 1 w er e c onj ug at ed s ep ar at el y an d s uc c es s f u l ly t o th e t hr ee ty p es of n an op ar t ic l es s el ec t ed in t his w or k . Th e c onj u g at i on of T BAs t o A uN Ps (c om m er c i al l y av ai l ab l e) an d Au S PI O Ns w as d on e b y inc ub at i on of N Ps w it h th i ol at ed - ol i g on uc l eot i d es f ol l ow ed b y s l ow s al t ag in g as d es c r ib ed . [1 4 ] Th e s u p er p ar am ag n et ic n an op ar t ic l es w er e pr ep ar ed as rep or t ed [1 5 ] an d c oat ed wit h d i m er c apt os uc c in ic ac i d (DM S A) . [1 6 ] Tw o t y p e of li nk er s (m al ei m i d e [1 7 ] and d is u lf i d e, [1 8 ] Fig ur e 2) w er e us ed t o fu nc t i on al i z e th es e n an op ar t ic l es w it h T BAs . In or d er t o int r od uc e th es e fu nc t i on al i t i es , th e c ar b o xy l at e gr ou ps 214 FULL PAPER of DMSA were activated using EDC/N HS and then reacted with the amino groups of the corresponding linkers bearing a maleimide or disulfide moiety. Subsequently, the resulting SP I ONs were reacted with the corresponding thiolated- oligonucleotides. The degree of functionalization of SP IO Ns with TBAs was independent of the type of linker and they were further used indistinguishably. Figure 2. Schematic representation of the functionalization of SP IO Ns nanoparticles with TBAs (in green) using the two type of linkers maleimide (left) and disulfide (right). The functionalization of all the nanoparticles was confirmed by the quantification of the decrease in the concentration of the oligonucleotide present in the solution, before and after conjugation (data not shown). All nanoparticles were stable after centrifugation and resuspension with aqueous buffers. UV s tudy of the complex formation between ? -thrombin and AuN Ps or AuSP I ONs f unctionalized with TBAs The behaviour of TBAs functionalized nanoparticles when incubated with ?-thrombin was monitored by means of U V- spectroscopy observing the red-shift broadness of the surface Plasmon band. [2a, 5] Binding interaction of the modified AuNPs-TBAs and AuSPIO Ns- TBAs with ?-thrombin was carried out in phosphate buffer with additional K + at 25? C. As expected, the UV spectrum for TBA1 and TBA2 anchored to AuNPs or AuSPIO Ns showed a maximum of absorption at 520 nm before incubation with ? - thrombin (Figure 3). This maximum was displaced to higher wavelengths in a continuous way when ? -thrombin was added, until reaching stabilization (3 0 nm). These results confirmed that ?-thrombin was interacting with both TBA1 and TBA2, creating a NP network formed by the binding of thrombin to both TBA1 and TBA2 sequences. In addition, the formation of this network of interactions between ?-thrombin and TBAs is not affected by the nature of the nanoparticles, as both AuNPs and AuSPIO Ns showed the same response. In contrast, the interaction between ? -thrombin and the mixture of O 6 -MeG-TBA1 and TBA2 anchored to the two types of nanoparticles (AuNPs or AuSPIO Ns) was evidently smaller. As it can be seen in Figure 3B and 3D, at equal concentrations of ?-thrombin the maximum of absorption was not changed in AuNPs (Figure 3B) or slightly displaced in Au SP IO Ns (Figure 3D). This small difference in the displacement rate observed in Au SP IONs (less than 10 nm), can be explained by the only interaction of AuNPs-TBA2 or AuSPIO Ns- TBA2 with ?-thrombin. These observations proved that ? -thrombin was not able to form the network with the same performance as in the case of the unmodified TBAs, and that its K D is much higher when one guanine in the central tetrad of the TBA sequence is modified with a methyl group in the O 6 position. This simple modification prevents the formation of its quadruplex structure, and for this reason, it cannot be recognized by ? -thrombin. Moreover, when we forced the concentration of ? -thrombin versus TBAs (molar ratio 4: 1) , we could observe a small displacement of the maximum in the UV spectrum of the mixture containing the methylG-TBA1 conjugated with both types of NPs. This effect could be due to the formation of small clusters, even if in a reduced way if compared to the non-methylated mixture of TBAs (1 0 nm for AuSPIO Ns and 15 nm for AuNPs). As the protein itself is able to recognize the sequence of methylated TBA and force it to fold in its quadruplex structure suitable for the binding [1 3a] , we presume that the methylated sequence is also recognized as a result of the excess of protein in the mixture. It is interesting to note that ?-thrombin attempts to fold it and this may result in some degree of binding. Figure 3. U V- Visible spectra of the complex formation between TBAs and ? - thrombin at a molar ratio of (1:1). The curves represent the mixture of NPs- TBAs in the absence (dashed lines) and in the presence (orange continuous lines) of the ? -thrombin. Blue represents unmodified TBAs and green, methylated TBAs A. and B. display spectra recorded for AuNPs. C. and D . display spectra recorded for AuSP IO Ns . UV seems a proper and simple way to measure the interaction between ?-thrombin and the TBAs nanoparticles, as the change can be visualized within a direct step and it is not cost nor time consuming. This result confirmed that macromolecular aggregation processes can be studied indistinguishably by these two types of nanoparticles, demonstrating that the gold coating maintains the chemical and optical properties of gold itself. 215 FULL PAPER DLS study of the complex formation between ? -thrombin and NPs-TBAs. The study of the complex formation between the three types of NPs- TBAs and ?-thrombin by dynamic light scattering was carried out in phosphate buffer with additional K + at 25? C. Figure 4 and Table 2 show the particle size distribution for the mixture of the different types of TBAs nanoparticles with and without ? - thrombin. Particle size analysis was performed after the addition of ?-thrombin into a mixture of TBA1 and TBA2 anchored to the three different types of nanoparticles (AuNPs, SP I ONs and AuSPIO Ns). In all three cases the average diameter of the nanoparticles immediately increased by 25, 21. 5 and 7 fold (see Table 2). Even if a small polydispersity is observed in the initial state of the mixture with AuSPIO N, we can clearly detect the formation of the ?-thrombin-TBAs network. No precipitation was observed at these complexes concentration during the measurements. Similar results were observed for the SP IO Ns independently of the linker used for the functionalization. Figure 4. Particle size distribution of NPs-TBAs at 25? C recorded by DLS. The mixtures of TBA1 and TBA2 NPs (left) and O 6 - MeG-TBA1 and TBA2 NPs (right) are displayed in dashed blue, while the same NP mixtures in the presence of ? -thrombin are represented in orange. In all cases, the molar ratio of ? -thrombin:TBAs was 0.5:1. From top to bottom: A. AuNP, B. S P IO Ns and C. AuSP IO N. In contrast, the interaction between equal concentrations of ? - thrombin with the mixture of each one of the three types of O 6 - MeG-TBA1 and TBA2 nanoparticles was inexistent or clearly smaller (see Table 2). These observations indicated that ? - thrombin is not able to form the network with the same efficiency when the TBA1 is substituted by methylated TBA1, and thus the particle size average remains similar. In these cases, the interaction between NPs- TBA2 and ?-thrombin was not identified. We presume that this may be due to the small variation in the size of the nanoparticles, which is too low to be observed by D L S. Table 2 : Size distribution (HD, nm ) by DLS of TBAs nanoparticles in the absence and in the presence of ? -thrombin. Nanoparticles type (- ) ? -thrombin HD (nm ) (+) ? -thrombin HD (nm ) AuNPs TBA1 & TBA2 38 ? 5 947 ? 28 3 O 6 -MeG-TBA1 & TBA2 38 ? 5 56 ? 7 SP IO Ns TBA1 & TBA2 36 ? 5 781 ? 18 5 O 6 -MeG-TBA1 & TBA2 39.5 ? 11 39.5 ? 11 AuSP IO Ns TBA1 & TBA2 91 ? 25 633 ? 22 5 O 6 -MeG-TBA1 & TBA2 91 ? 25 109 ? 34 Magnetic resonance imaging assay to compare the complex formation between ? -thrombin and NPs-TBAs The study of the complex formation between ? -thrombin and the ?-thrombin binding aptamers by magnetic resonance imaging was already been described by Yigit et al [8e] . In our work we went a step forward in the application of the MRI technique using gold-coated magnetic nanoparticles as contrast agents, studying also the effect produced in the MRI contrast when TBA1 is substituted by a methylated TBA1. A 1: 1 mixture of TBA1 or O 6 -MeG-TBA1 and TBA2 anchored to SP I ONs or AuSPIO Ns was prepared in phosphate- K + buffer at 25 ?C. Table 3 summarizes the formation of the molecular network due to ?-thrombin binding to the nanoparticles, monitored by the changes in brightness of the T2- weighted MR images of the solution. A 2. 4 ug Fe/m L concentration of the SP IONs resulted in a T2 of 50 ? 3 ms. After aggregation of the mixture by adding ?-thrombin, the magnetic relaxation properties changed, reducing the T2 relaxation time to 40 ? 3 ms. Even if the concentration of the AuSPIO Ns nanoparticles was required to be 100 0 times higher, due to the fact that the gold coating decreases the magnetic response, a similar reduction in the T2 was observed. Again, the T2 values for the mixture containing methylG- TBA1 did not decrease when ?- thrombin was added, indicating that the network could not be formed. These observations are in agreement with our previous results obtained by U V and DLS studies, and prove that gold- coated magnetic nanoparticles can be used as contrast agents for MRI detection of biomolecules. Table 3 : T2 relaxation times in the absence or presence of ? -thrombin. SP IO Ns were measured at 1 /1000 ( 2.4 ?g Fe/m L) and the AuSP IO Ns 1 /1 (2.4 m g Fe/m L). These results represent the average of at least three independent measurements. Nanoparticles type Concentration of ? -thrombin 0 5 nM SP IO Ns TBA1 & TBA2 50 ? 3 ms 40 ? 3 ms O 6 -MeG-TBA1 & TBA2 51 ? 4 ms 51 ? 5 ms AuSP IO Ns. TBA1 & TBA2 70 ? 1 ms 62 ? 1 ms O 6 -MeG-TBA1 & TBA2 63 ? 2 ms 62 ? 1 ms 216 FULL PAPER Conc lus ions W e h av e obt ai n ed in h i g h yi el d an d p ur it y g ol d - c oat ed m ag n et ic n an op ar t ic l es . Th ei r func t i on al i z at i on w it h ol i g on uc l e ot i d es is c om p ar ab l e w it h th e on e obt ai n ed f or th e g ol d n an op ar t ic l es . Our r es u lt s pr ov e t h e b i os en s i n g c ap ac it y of A uS P IO Ns f or t h e fir s t tim e, as th ey w er e us ef u l f or t h e d et ec t i on of a pr ot ei n b y thr ee d i v er s e t ec h n iq u es . Au S PI O Ns w er e s u it ab l e f or U V exp er i m ent s w it h th e s am e p er f or m anc e as Au N P, w it h ou t m aj or c h an g es in th e e xec ut i on n or in th e d et ec t i on. A uS P IO Ns s h ow ed s i m il ar b eh av i ou r in D LS m e as ur em ent s t o A u NPs an d SP I ONs , r es u lt i n g in v er y s im i l ar p l ot s of s iz e d is t r ib ut i on. F in al l y , Au S PI O N w er e us ef u l f or MR I e xp er im ent s , ev en if t h ey s h ow e d a sh i el d i n g of th e m ag n et ic r es on anc e d u e t o t h e g ol d c oat i n g an d r eq u ir ed h ig h er c onc ent r at i ons t o r eac h th e s am e s i gn al as SP I ONs . Fur t h er m or e, th is w or k pr ov es th at A uS P IO N c an dis c r im i n at e a n or m al T BA fr om a m et h yl at ed on e in t h e an al y s is of t h e complex formation between TBAs and ? - t hr om b i n b y b i op hys ic al m et h ods . As O 6 - m et h y lg u an i n e is th e s u bs t r at e f or DN A r ep ai r en z ym es , c r it ic al f or c h em ot h er ap y r es is t anc e, t h is m et h od c an als o b e us ed t o m on it or th e ac t iv it y of th e s e D N A rep ai r pr ot ei ns . D if f er ent d et ec t i on m et h ods f or t h e ev al u at i on of th e r ep ai r ac t i v it y of on e of th em, alk y l gu an i n e - D NA- t r ans f er as e (hA GT) [1 9 ] hav e b een pr ev i ous ly d es c r i b ed. [1 3 ] Th es e m et h od s ar e al s o b as ed on t h e c onf or m at i on al c h an g e of T BA u p o n m et hy l at i on of a s in g l e g u an i n e. It s r ep air r es t or es th e G - qu ad r u p l ex an d it s r ec og n it i on b y ?- t hr omb i n. W e en v is ag e t h at Au S PI O Ns ar e us ef u l f or th e d ev el op m ent of a n e w m et h od ol og y t o d et ec t hA GT ac t iv it y in a st r ai g ht f or w ar d m an n er . W or k in th is d ir ec t i on is c ur r ent ly on g oi n g in ou r lab or at or y. W e b el i ev e t h at t h e m u lt i d is c i p l in ar y d et ec t i o n c a p ac it y of A u SP I ONs c an b e ev ol v ed t o d es i g n m or e c om p l e x b i os ens or s f or b i om ed ic al ap p l ic at i ons as w el l as f or dr u g d el i v er y an d i n v i v o im ag i n g. E x perime nta l Se c tion Abb re vi at i ons : A uNP s: gol d n an op art i c l es, AuS P I ONs: g ol d -c oat ed sup er p ar am ag n et i c ir on oxi d e n an op art i c l es, DLS : dy n am i c light sc at t eri ng, dm f: dim et h yl f orm am i di n o g r oup, D MS A : dim erc apt osuc c i ni c ac i d, DTT : dit hi ot h r ei t ol , ED C: 1- et h yl - 3- (3 - di m et hyl am i n opr op yl )c ar b odi i m i d e, F e 3 O 4 : i r on oxi d e, hA GT: hum an O 6 - al k yl gu ani n e- DNA alk yl t r ans f er as e, HD: Hy dr ody n am i c diam et er, HP LC: high p erf orm anc e liqui d c hr om at ogr aph y, LS P R: loc al i z ed sur f ac e pl asm on r es on anc e, MRI : m agn et i c r es on anc e im agi ng, NHS : N - hyd r oxy suc c i ni m i d e, NP s: nan op art i c l es, O 6 - M eG: O 6 -m et h yl gu ani n e, O 6 -M eG-TB A 1: O 6 - methylguanine ? - t h r om bi n -bi n di ng apt am er, S P I ONs : sup er p ar am ag n et i c ir on oxi d e n an op art i c l es, T2: s pi n - spi n r el axat i on time, TBA: ? -t hrom bi n-bi ndi n g apt am er, TCE P ? HCl : t ri s (2 - c ar b oxy et hyl )p h osphi n e h ydr oc hl ori d e, TE A A c : t ri et hyl am m oni um ac et at e, TE M: t r an sm i ssi on el ec t r on m ic rosc opy, T MA O H: t et r am et h yl am m oni um hyd r oxi d e, Tri s: Tri s (h ydr oxym et h yl ) am i n om et h an e. Chem i ca ls : +uman ? -t hr om bi n w as pu rc h as ed fr om H eam at ol ogi c Tec h n ol ogi es I nc . All reag ent s an d dr y s ol v ent s w er e pu rc h as ed fr om Sigm a-A l d ri c h or Flu k a an d w er e u s ed w it hout furt h er p uri fi c at i on. Ot h er c hem i c al s fr om s p ec i fi c c om m erc i al s ou rc e w il l b e sp ec i fi ed as th ey ar e n am ed. St an d ard p h osp h or am i di t es w er e purc h as ed fr om c om m erc i al s ou rc es. S ol v ent s f or oli g on uc l eot i d e s ynt h esi s w er e p urc h as ed fr om Appl i ed Bi os y st em s (US A ). Gol d n an op art i c l es (10 nm ) st abi l i z ed in aqu eou s s ol ut i on of c it rat e w er e p urc h as ed fr om Sigm a. L ow bindi n g epp end or f t ub es w er e us ed in th e pr ep ar at i on of th e n an op art i c l es i n ord er t o av oi d ads orpt i on t o th e tub e. Mat ri x f or MA L DI -TOF exp eri m ent s were ?,?,?-T ri hy dr oxi ac et oph en on e m on oh yd r at e (T HA P , Aldri c h) an d Amm oni um c it rat e dib asi c (Fl u k a). Sol v ent s f or c hr om at ogr aphi c an al y si s w er e pr ep ar ed u si n g t ri et h yl am m oni um ac et at e (TE A A , Merc k ) an d ac et oni t ri l e ( Merc k ) as m obi l e ph as e. Ult r ap ur e w at e r ( Mi l l i p or e, US A ) w as us ed in all exp eri m ent s. Ins trum ent at i on : A n al yt i c al RP -HP LC w as p erf orm ed usi n g an XB ri dg e 26T &1 . ?m column and a 1ucleosil Analytic column 1 &1 (25 0 x4m m ). Oli gon uc l eot i d e s eq u enc es w er e d et ec t ed by UV ab s orpt i on at 260 nm on a J A S CO V -65 0 in st rum ent equi p p ed w it h a therm or eg ul at ed c el l h ol d er. Mas s s p ec t r a w er e r ec or d ed on a MA L DI Voy ag er DE TM RP tim e of- fl i ght (TOF ) sp ec t r om et er (A p pl i ed Bi os y st em s, US A ). UV m easu r em ent s w er e p er f orm ed on a sp ec t r ofl u or om et er J asc o V-650 at 25? C. Set tem p er at u r e w as c ont r ol l ed w it h an 8909 0A Agi l ent Pel t i er d evi c e and H el l m a q u artz cuYettes were used 1 and  ?/ v ol um e). Dy n am i c light sc at t eri ng st udi es w er e p erf orm ed on a Dyn am i c Light Sc at t eri ng (DLS ) s p ec t r om et er ( LS Inst rum ent s, 3 D c r os s c or r el at i on m ul t i pl e- sc at t eri n g) equi pp ed w it h a He -N e l as er (632. 8 nm ) w it h v ari ab l e int ensi t y. MRI : MRI st u di es w er e c arri ed out usi ng a st and ar d b or e B ru k er Av anc e AV 60 0 sp ec t r om et er (B ru k er Bi ospi n Gm bH, Rh ei nst et t en, G erm an y) eq ui pp ed w it h a 10 m m 1H m ic ro - i m agi ng pr ob e and a v ari abl e -t em p er at u r e c ont r ol uni t . Ac qui si t i on an d d at a pr oc es si ng w er e p er f orm ed w it h Par aV i si on v. 4. 0 (B ru k er Bi oS pi n MRI Gm bH, Et t l i ngen, G erm an y). TE M an al y si s w as p erf orm ed on a Tr an sm i ssi on El ec t r on Mic r osc op e (TE M ) LI B RA ? 200FE w it h an el ec t r on b eam s ourc e of 200 k eV , a r ess ol ut i on p ow er of 0. 24 nm and a m agni fi c at i on of 8 x - 1, 0 00, 0 00 x. Ol ig onu c le ot ides syn the si s : T h e t hr ee oli g on uc l eot i d e s equ enc es (T abl e 1) w er e sy nt h esi z ed on an AB I 3400 DNA Sy nt h esi z er (A p pl i ed Biosy st em s, US A ) u si ng t h e 20 0 - nm ol sc al e s ynt h esi s an d fol l ow i ng t h e st and ar d pr ot oc ol s. For the introduction of the thiol group at the ?- en d we used ?-t hi ol m odi fi er C 6 S -S ph os ph or am i di t e f r om Lin k Tec h n ol ogi es (S c ot l and, UK ). For st r an ds c ont ai ni ng O 6 - M eG, w e u s ed G d mf ph os ph or am i di t e. O 6 - M eG -TB A 1 w as d epr ot ec t ed in am m oni a s ol ut i on, ov er ni g ht at r oom t em per at u r e. Th e r esul t i ng pr oduc t s w er e d es al t ed b y S eph ad ex G - 25 ( NA P -1 0, Am er sh am Bi osc i enc es, US A ) and an al y z ed by r ev ers ed -ph as e HP LC. Th e l engt h an d h om og en ei t y of the oli g onuc l eot i d es w er e c h ec k ed by MA LDI -T OF. H O(C H 2 ) 6 -S -S - (C H 2 ) 6 -T 1 5 T BA1 [ M] = 96 17. 1 ( exp ec t ed 96 12. 6 ), H O( CH 2 ) 6 -S -S -( CH 2 ) 6 - T 15 -O 6 - Me G-T BA1 [M] =96 21. 4 ( exp ec t ed 96 26. 6 ), HO(C H 2 ) 6 -S -S - (C H 2 ) 6 -T 5 -T BA2 [M] =1 07 95. 5 ( exp ec t ed 1079 7. 9). Th e DNA -st r an d c onc ent r at i on w as d et erm i ned by abs orb anc e m eas ur em ent s at 26 0 nm . Oli g on uc l eot i d e s am pl es w er e k ept dr y at -20 ? C unt i l u s e. S ynthe si s of SP I ONs : D MS A c oat ed F e 3 O 4 n an op art i c l es w er e obt ai n ed in good yi el d t hr ou gh t h erm al dec om p osi t i on, f ol l ow i n g t h e pr ot oc ol r ep ort ed b y S al as et . al . [ 1 5a ] S ynthe si s of m al eim ide l ink er [1 7 ] : 2- (2 - a m i n oet h oxy ) et h an ol (2 m L, 18 mm ol ) w as pr ot ec t ed w it h (B OC) 2 O in TH F, f ol l ow ed by a st and ar d w or ku p. Th e r esul t i n g pr ot ec t ed c om p ou nd w as l eft t o r eac t w it h m al ei m i de ( 2. 1 g, 22. 5 m m ol ) in THF, an d a fr es hl y s ol ut i on of PP h 3 (3. 6 g, 13. 5 m m ol ) an d DI A D (3. 6 m L, 18 m m ol ) f ol l ow i ng t h e c ondi t i on s d esc ri b ed. [ 1 7 ] Th e p r od uc t w as p r ec i pi t at ed i n Et 2 O, fil t er ed and p uri fi ed by fl ash c hr om at ogr aph y (H ex an e/ A c OE t 5: 2 to 1: 1). Th en, it w as d ep r ot ec t ed w it h t ri fl u or oac et i c ac i d an d puri fi ed t o obt ai n a fin al yi el d of 89% . The r esul t i n g pr od uc t had th e s am e ph y si c al and sp ec t r osc opi c al pr op ert i es as th e on e d esc ri b ed by W eb er et . al . [ 2 0 ] S ynthe si s of thi op yr idin yl lin ker ( PD A* HC l). [1 8 ] : The t hi op yri di n yl link er w as obt ai n ed f ol l ow i ng r ep ort ed p r ot oc ol s. Bri efl y, t o a st i r r ed s ol ut i on o f al dri t hi ol (2 13 m g, 0. 96 mm ol ) in MeOH, 2 -m erc apt oet hyl am i n e 217 FULL PAPER hyd r oc hl ori d e ( 109 m g, 0. 96 mm ol ) w as ad d ed. Aft er st i rri ng 1h, th e s ol v ent w as ev ap or at ed an d th e r esi d u e w ash ed w it h c ol d Ac OE t t hr e e t im es. PDA *HCl w as obt ai n ed as a w hit e s ol i d in 51% yi el d. Thi s pr oduc t is th e s am e as th e on e d esc ri b ed in r ef er enc e 1 8 and it s p hy si c al an d sp ec t r osc opi c al c h ar ac t eri z at i on is th e s am e. Fu nct i ona l iz at i on of D MS A c oated Fe 3 O 4 nan op art ic le s w it h m ale im id e or PD A*H Cl li nker : T o 1 m L of SP I ON s at 2. 4 m g Fe/ m L, the m al ei m i d e or P DA *H Cl link er ( 50 m m ol / g F e), 1 equi v al ent of N aO H, 150 m m ol of ED C/ g Fe an d 75 mm ol of NHS / g F e w er e ad d ed. Thi s m ixt ur e w as st i r r ed at r oom t em p er at u r e du ri ng 1 6 h, and w as w as h ed by it er at i v e c ent ri fug at i on and r edi sp er si on i n m il l i Q w at er f or at l east 3 tim es. S ynthe si s of AuS PI ON s : Synthe si s of Fe 3 O 4 MNP a s seed s [1 0 a ] : Iron (I I I ) ac et yl ac et on at e ( 0. 35 5 g, 1 mm ol ) w as di s s ol v ed in ph enyl et h er (10 m L) w it h ol ei c ac i d (1 m L, 3 mm ol ) and ol eyl am i n e (1 m L, 2 mm ol ) in ar g on at m osph er e w it h vig or ou s st i r ri ng. 1, 2 -h ex ad ec an edi ol (1. 2 9 g, 5 mm ol ) w as add ed an d the s ol ut i on w as h eat ed und er r efl u x at 21 0 ?C for 2 h our s. Then, t h e m ixt ur e w as l eft t o c ool d ow n t o r oom t em p er at ur e. Th e fin al s ol ut i on w as us ed in th e fol l ow i ng st ep s w it hout fu rt h er p uri fi c at i on. Redu c t i on of Au -ac eta te (c oat ing ) [1 0 b ] : 2. 5 m L of th e F e 3 O 4 n an op art i c l es s ol ut i on p r evi ou sl y pr ep ar ed ( app r oxi m at el y 0. 1 66 m m ol Fe 3 O 4 ) w er e dil ut ed t o a fin al v ol um e of 7. 5 m L in phenyl et h er an d m ixed u p w it h Au(OOC CH 3 ) 3 (0. 5 5 m m ol , 0. 208 g), ol ei c ac i d ( 0. 37 5 mm ol , 0. 125 m L), ol eyl am i n e (1. 5 m m ol , 0. 75 m L) and 1, 2 - h exad ec an edi ol ( 3 mm ol , 0. 775 g), un d er arg on at m osp h er e w it h vig or ou s st i rri n g. T em per at u r e w as inc r eas ed f ol l ow i ng a r am p of 10? C/ m i n unt i l reac hi ng 1 90? C, and th e m i xt ur e w as r efl u xed f or 1. 5 h. Th en, AuS P I ONs w er e p r ec i pi t at ed w it h et h an ol and pu ri fi ed b y c ent ri fu g at i on. Th e pr ec i pi t at e w as w as h ed t w i c e and r edi sp er s ed in a 1M T MA O H s ol ut i on. Th en, t ri s odi um c it rat e (0. 0 4 g) w as add ed and th e pH w as adj u st ed t o 6. 5. Fin al l y, t h e s ol ut i on w as s oni c at ed f or 3 0 m inut es, and th e n an op art i c l es w er e c ol l ec t ed usi ng a m agn et an d r edi sp er s ed in p ur e w at er. Fu nct i ona l iz at i on of th e dif fer ent t ype of n an opar ti cl es : Gold na n opar ti cl es ( Au NP s) 2 -3 O Ds of TB A 1, -TB A 2 and O 6 - M e G- TB A 1 w er e r educ ed w it h 300 uL TE A A c 0. 1M and TCE P ? H Cl 34 uL (10 0 m M) at 55 ?C ov erni g ht , t o pr ev ent t he form at i on of disul fi d e bri d g es an d to br eak t h e alr ead y exi st i n g on es. N ext , 1 OD of th e d epr ot ec t ed oli g on uc l eot i d es w er e dil ut ed in 0. 5 m l of Mil l i Q w at er and w er e d es al t ed w it h a NA P -5 c ol um n. The r esul t i ng oli g on uc l eot i d e (1 m L) w as m ixed w it h 1 m L of A uNP s s ol ut i on an d l eft t o c onj ug at e ov erni g ht un d er agi t at i on. Th en, t h e s ol ut i ons w er e br ou ght t o a fin al c onc ent r at i on of 1 0 m M s odi um ph osp h at e (pH 7. 0). T h e m i xt ur es w er e all ow ed t o equi l i b r at e b ef or e b ei ng br oug ht t o a c onc ent r at i on of 0. 1 5 M N aC l st epw i s e ov er a 9 h p eri od. Th e s ol ut i on s w er e s oni c at ed f or 10 s b ef or e eac h addi t i on t o k eep t h e n an op art i c l es dis p er s ed d uri n g t h e s al t i n g pr oc edur e. Th e s al t i ng pr oc es s es w er e f ol l ow ed by an ov erni g ht inc ub at i on at r oom t em p er at ur e. Fin al l y, t h e n an op art i c l es w er e p uri fi ed by c ent ri f ug at i on at 132 00 rpm (16 10 0 x g) usi ng a b uf f er c ont ai ni n g 0. 15 M N aCl , 10 m M s odi um ph os ph at e pH 7 and N aN 3 0. 0 1% . Th e c ent ri fu g at i on pr oc ess w as p erf orm ed 4 tim es f or 45 m inut es at 15 -2 0? C. Th e TB A s -c onj u g at ed g ol d n an op art i c l es w er e an al yz ed b y UV -vi si bl e abs or pt i on. TB A s c onj ug at ed n an op art i c l es w er e st or ed at 4? C an d s oni c at ed du ri ng 5 m inut es b ef or e us e. S up erpar am agn e ti c ir on ox ide nan op art ic le s (S P I ON) : A l i qu ot s of thi ol at ed - apt am er s (TB A 1, TB A 2 and O 6 - M e -TB A 1 ) f or a fin al c onc ent r at i on of 245 ? M w er e m ixed w it h 200 ?L of 2. 4 m g Fe/ m L Fe 3 O 4 m agn et i c n an op art i c l es f unc t i on al i z ed w it h m al ei m i d o and 2-t hi op yri di ny l gr oup s [ 1 6 ] an d left t o r eac t ov er ni ght at room t em p er at ur e. Th e TB A - c onj ug at ed n an op art i c l es w er e pu ri fi ed b y it er at i v e c ent ri fug at i on - r es us p ensi on pr oc es s. Th e c ent ri f ug at i on w as r ep eat ed 3 tim es f or 3 0 m inut es at m axi m um sp eed at r oom t em per at ur e and w it h t he ad di t i on of sm al l am ount s of N aCl t o enh anc e p r ec i pi t at i on i f r equi r ed. Th e c ov al ent l y imm obi l i z ed TB A w as d et erm i ned by qu ant i fi c at i on of th e unb oun d TB A in the c ol l ec t ed s up ern at ant s. Th e TB A s c onj u g at ed n an op art i c l es w er e an al y s ed by UV -vi si bl e abs or pt i on an d st or ed at 4? C unt i l furt h er u s e. Gold sup erpar am agn et ic ir on oxid e nan opa rt ic les ( AuS PI ON ): E qu al v ol um e of g ol d c oat ed n an op art i c l es and thi ol at ed DNA s ol ut i on ( 1 OD ) w er e m i xed and l eft t o c onj u g at e u nd er agi t at i on and at r oom t em p er at ur e f or 48 h. Th e s ol ut i on f ol l ow ed t he s am e s al t agi n g pr oc es s t han AuNP s an d the r esul t i n g su sp ensi on w as c ent ri fu g ed und er t h e s am e c ondi t i ons u s ed f or th e AuNP s, resu sp en d ed in Mil l i Q w at er t o r em ov e n on-c onj ug at ed oli g on uc l eot i d es, and obs er v ed f or an y indi c at i on of ag gr eg at i on. Fi n al l y, t h e c onj ug at ed oli g on uc l eot i d es g ol d c oat ed s up erp ar am ag n et i c n an op art i c l es w er e an al y z ed b y UV -vi si bl e abs or pt i on. Th e c onj ug at ed n an op art i c l es w er e k ept at 4?C in Mil l i Q w at er unt i l furt h er us e, du e t o p r ec i pi t at i on w h en ad di ng s al t s t o th e s ol ut i on. 6tXG\ of tKe interaFtion EetZeen $XNPV anG $X6P,2NV anG ?- thr om bin by UV : B i ndi ng b et w een ? -t hr om bi n and TB A 1, TB A 2 and O 6 - M eG-TB A 1 n an op art i c l es w as m oni t or ed b y m easuri ng c h an g es in th e UV sp ec t rum (r ec ord ed fr om 650 t o 400 nm ) up on inc r easi n g c onc ent r at i ons of th e pr ot ei n. A m ixt u r e of NP -TB A 1 an d NP -TB A 2 w er e dil ut ed in bu f f er c ont ai ni ng 1 0 m M p h osph at e p H 7 and 5 m M KCl t o r eac h a c onc ent r at i on of 5 n M of n an op art i c l es in a v olum e of 50 0 ?L. A s c onj ug at i on w as app r oxi m at ed t o r ep r es ent a c oat i n g of 10 0 oli g on uc l eot i d es p er n an op art i c l e, t h e c onc ent r at i on of DNA in th e s am pl e w as c onsi d er ed t o b e ar oun d 500 n M. E ac h t im e, en oug h Tuantity 1 nM of human ? -t hr om bi n w as add ed. Aft er a quic k m anu al m ix, t he UV sp ec t r a w er e r ec ord ed . Th e UV sp ec t r a of th e m ixt ur e an d increasing concentrations of ? -t hr om bi n w as r ec ord ed unt i l r eac hi n g a fin al c onc ent r at i on of 1 u M, w hi c h repr es ent s a 2: 1 m ol ar r at i o b et w een ? -t hr om bi n and D NA . Thi s s am e exp eri m ent w as r ep eat ed u si n g t h e m odi fi ed NP - m e t h yl TB A 1 and NP -TB A 2. All s p ec t r a w er e ov erl ai d t og et h er t o st ud y t h e displ ac em ent of th e m axi m um peak fr om 520 nm in c as e of AuNP s and 54 5 nm for AuS P I ON s t o high er w av el engt h s, du e t o the form at i on of a n et w or k of TB A s - ? -t hr om bi n c om pl exes . N eg at i v e c ont r ol s h av e b een c arri ed on b y usi n g TB A 1, TB A 2 and O 6 - M eG-TB A 1 al ong w it h inc r easi ng c onc ent r at i ons of hA GT and sc r am bl ed D NA al on g with ? -t hrom bi n. Th e p r ot oc ol u s ed f or th es e st udi es w as th e s am e f or AuNP s and for AuS P I ON s. 6tXGieV of ?-th r om bin in tera c t i on w i th T BAs n an opa rt ic les ( AuN PS , SPI ON s and AuS P I ONs) b y DLS : Binding between ? -t h r om bi n an d TB A 1, TB A 2 and O 6 -M eG- TB A 1 n an op art i c l es w as st udi ed by an al y zi n g t he av er ag e HD of th e p art i c l es at r oom t em p er at ur e. T h e m ol ec ul ar n et w ork ori gi n at ed f r om t h i s bindi ng w as m oni t or ed by m easuri ng t h e particles? radius enlargement upon increasing concentrations of the protein. 1 ?/ of a  nM mixture of 13 -TB A 1 and NP -TB A 2 in bu f f er c ont ai ni ng 10 m M ph osp h at e pH 7 and 5 m M KCl w er e m easu r ed du ri n g 100 s ec ond s usi n g a 3 D c ros s m et h od w it h a sc at t eri ng angl e of 90? at ?&. (nough Tuantity of human ? -t hr om bi n w as ad d ed t o r eac h a 0. 5: 1 molar ratio between ? -t h r om bi n and DNA . Aft er a quic k m anu al m ix, t h e r adi u s l en gt h w as r ec ord ed. Th e s am e exp eri m ent w as r ep eat ed u si n g t he m odi fi ed NP - O 6 - M eG-TB A 1 and NP -TB A 2. Th e p art i c l e r adi us w as c al c ul at ed by fit t i ng of th e fir st c um ul ant p ar am et er, an d th e r el at i v e int ensi t y of th e di ff er ent p art i c l e c om pl exes w as c al c ul at ed by fit t i ng t h e c ont i n p ar am et er. All the exp eri m en t s w er e p erf orm ed in tri pl i c at es. Th e pr oc edur e of th e DLS m easu r em ent s w as th e s am e f or AuNP s, SP I ON s and AuS P I ON s. 218 FULL PAPER 6tXGieV of ?-th r om bin in tera ct i on w i th T BAs n an opar t ic les (S PI ON s and AuS P I ONs) by MR I : NP s -TB A 1 ( or NP s -O 6 - M eG -TB A 1 ) and NP s- TB A 2 w er e m i xed in 1: 1 r at i o and dil ut ed in 10 0 m M N aCl , 25 m M KCl and 25 m M tri s-H Cl at pH 7. 4, t o reac h a fin al c onc ent r at i on of n an op art i c l es of 2. 4 ?g F e/ m L in the c as e of th e SP I ONs an d 2. 4 m g/m L f or th e AuS P I O Ns. Th en, t he s am pl es w er e i nc ub at ed w it h diff er ent concentrations , , 1 and  nM of ? -t hr om bi n. 1 00 ? L of th e m i xed s am pl e w er e l oad ed int o a m ic roc api l l ar an d T2 e xp eri m ent s w er e r ec or d ed. Th e m agn et i c fi el d st r engt h w as 1 4 T, c orr es p ondi n g t o a 1 H r es on anc e fr equ enc y of 6 00. 1 MHz. T h e im ag es w er e ac qui r ed at r oom t em p er at ur e, w it h t h e fol l ow i ng ac qui si t i on p ar am et ers: MS ME ( Mul t i Sli c e Mul t i Ec ho) ac q ui si t i on; num ber of sli c es: 1; thi c knes s: 1. 50 mm; FOV : 0. 8 mm; rep et i t i on t im e: 1500 m s; ec h o t im e 4. 5 m s; num ber of ec h oes: 10. Tw o sc an s w er e p erf orm ed, c orr es p on di ng t o a t ot al ac q ui si t i on t im e of 12 m in. T2 val u es w er e ext r ac t ed by a m ult i - p ar am et ri c n on -l i n ear fi t t i ng y =A +B _ e ?t / T2 of th e int en si t y d ec ays. All m eas ur em ent s w er e p erf orm ed t h r ee or four t im es in i nd ep end ent exp eri m ent s. Ac qui si t i on an d d at a pr oc es si n g w er e p er f orm ed w it h Par aV i si on v. 4. 0. The MRI m eas ur em ent s of th e AuS P I ON w er e p er f orm ed f ol l ow i n g t h e s am e pr ot oc ol as th e on e u s ed f or th e SP I ON. Ac k nowle dge me nts Th e C om mu ni t i es MU L TI F UN (c ont r ac t N MP 4 - L A - 20 1 1- 262943) and by ?Ministerio Ciencia e Innovaci?n? (grant s CT Q- 20 1 0- 2 0 54 1- C 0 3- 0 3 a n d C TQ 2 01 4- 5 2 5 88- R) ar e ac k n owl ed g e d for fi na nc i al s u pp or t . M. T. w as s up p or t e d by a pr e - d oc t or al fel l o ws hi p ( FP I) a n d a sh or t s t ay gr ant (E EB B) fr om MI NE C O. C. F is gr at ef ul to F un d ac i ? L a Mar at ? de T V 3 ( 20 1 3 20 3 2) for a res e ar c h c o nt r ac t . W e th a nk Dr . Sus a na Vi l c h e z fr o m t h e Sur f ac e Ch e mi s t r y gr ou p at IQ AC - CS I C for her h el p wi t h t h e DL S ex p er i m e nt s . LP th ank s RS P PT EC H ? 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W . W eber , R. H. Bouti n, M. A. Ne del m an, J . Li s ter - J am es , R. T . Dean, Bi oc on jug. Ch em. 1 990, 1 , 431- 4 37. 219 FULL PAPER FU L L PAPER Gold coated magnetic nanoparticles combine the magnetic properties of iron and the robust chemistry provided by the thiol functionalization of gold. Their good performance in the detection of a protein-aptamer complex formation by biophysical techniques make them suitable for the development of biosensors for biomedical applications. Mar i a Ti nt or ?, St ef a ni a Ma z zi ni , L a ur a Pol i t o, Mar c el l o Mar el l i , Al f o ns o L at or r e, ?l v ar o S om oz a, A n na Av i ? ?, C ar m e F? br eg a, a n d R am o n Er i t j a *. Page No. ? Page No. Molecular biosensing using gold- coated superparamagnetic nanoparticles functionalized with DNA aptamers. 220 General discussion 221 222 Cellular resistance to alkylating agents is a major drawback for the treatment of cancer patients. [1] One of the mechanisms responsible for this resistance is the overexpression of DNA repair enzymes , which revert the lesions produced by thes e chemotherapeutic drugs and lead to tumoral cell survival. [2] In particular, the human O 6 - alkylguanine-DNA-alkyltransferase [3] is a DNA repair protein that removes alky l groups from the O 6 position of guanines , restoring the DNA. For this reason, this protein represents a relevant pharmacological target in the fight against chemotherapy resistance for patients? survival. [4] In addition, the level of expression of h AGT has shown to be a prognosis biomarker of poor survival in patients who suffer from malignant gliomas. [5] Given the relevance of hAGT as a therapeutic target, several methods are available to characterise its repair activity. Moreover, they are also able to evaluate the capacity of small molecules of inhibiting hAGT. [6] Most of these methods involve radioactivity assays, [6b, 6e] while others are bas ed on multiple-step enzymatic reactions. [6a, 6c, 6d] However, the first methods are dangerous and require the use of rigorous safety procedures, and the others are discontinuous and time -consuming due to the neces sity of multiple step s . The lack of a straightforward, rapid and costless assay to study hAGT repair activity represents a limiting step in the search of potential inhibitors as chemotherapy enhancers. Over the last years, different small molecules with hAGT inhibitory capacity have been described , although all of them are pseudosubstrates. For example, O 6 -benzylguanine (O 6 -BG) inactivates hAGT in vitro and in vivo , [7] and has shown to improve the effectiveness of chloroethylating agents in clinical trials. [8] However, des pite its potential for therapeutic inhibition of hAGT, there are considerable limitations in current inhibitors such as O 6 -BG. First, O 6 -BG has little affinity for hAGT, if compared with the affinity of O 6 -alkylguanine incorporated into double stranded DNA. [9] Second, O 6 -BG has low bioavailability, very low water solubility and a very high plasma clearance. In addition, clinical trials phase I and II have shown that it is very likely to produce myelosuppression. [10] A new derivative of guanine, lomeguatrib [6- ( 4 -bromo-2-thienyl)methoxy]-purine-2-amino], was proven to be more active in vitro than O 6 - BG. [11] The O 6 -thienyl analogue was tested in phase I and II clinical trials in combination with temozolomide and, as well as O 6 - BG, did not significantly increase the cytotoxic effect associated with the chemotherapeutic agent per se , but also caused myelosuppression. [12] Later on, many compounds initially thought to be hAGT inhibitors have proven to be chec kpoint inhibitors instead [13] and only O 6 -BG is currently on the market. In vitro studies of O 6 - ( 4 -bromo-2- th ienyl)-guanine (PaTrin-2 or PAT), a pseudosubstrate inactivator of hAGT, show ed greater potency than O 6 -BG. However, it causes dose-limiting 223 toxicities when administered with TMZ. [14] Other novel approaches to hAGT inhibition are also being studied with promising results. [13, 15] The main obj ective of this thesis wa s the finding of new small molecules capable of inhibiting hAGT activity, to be used as chemotherapy enhancers . Due to the lack of a robust in vitro method to evaluate hAGT activity, we have devoted a substantial part of this thesis to the development of such method, trying to avoid radioactivity and compl ex multi- step reactions. For all the in vitro assays and complex formation exp eriments des cribed in this thesis, recombinant hAGT was used , which was overexpressed and purified following the protocols des cribed by Ruiz et al . [6e] Two different constructs of hAGT were used : hAGT- FL, the human full-length protein and hAGT ????-C145S, a mutant of hAGT where the cysteine of the active site was replaced by a serine, making hAGT loose its repair activity but maintaining its capacity to recognize and bind alkylguanine-DNA. The sequence of this mutant is 30 amino acids shorter than hAGT FL but preserves the folding. This modification was done to facilitate its overexpression and manipulation. This hAGT mutant was overexpressed and purified in order to us e it as a negative control for the different in vitro assays. Previously, a virtual screening for the selection of potential compounds candidates to interact with the active site of hAGT was realized in our group in collaboration with the Bioinformatics Unit of the CMBSO. A set of 10 compounds w as selected and purchased from commercial sources . The standard procedure to evaluate the compounds would be to start by an in vitro evaluation of their capacity to inhibit hAGT and subsequently asses s their toxicity and effectiveness in vivo. However, as detailed before, we do not have an adequate in vitro assay available currently and for this reason we started by the cell culture exp eriments and by the study of their ability to form a complex with hAGT by mass sp ectrometry. The mass exp eriments realized allowed us to detect the complex formation between hAGT and 5 out of the 10 compounds studied . These results also showed that one of them (compound 8) formed a specific complex and the other four (compounds 5, 6, 7 and 9) , non-specific ones. From the f ive compounds that showed no interaction with hAGT, one of them (compound 10) caused precipitation of hAGT immediately and did not permit the study of the complex formation. Another compound (4) showed an alteration in hAGT?s mass sp ectrum , indicating that they interact in such a way that it des tabilize d in some degree its stru c ture. 224 We then tested the toxicity and effectiveness of these potential inhibitors in cell culture through MTT assays in human colorectal adenocarcinoma cells . The results obtained are discussed extensively in chapter 1. Th e toxicity of the 10 potential inhibitors was studied in cell culture. Two out of the ten compounds (5 and 8) were found to be non-toxic, and the other eight compounds were toxic at different concentrations. From them, three (2, 4 and 7) were toxic at LD 50 values lower than 100 ?M. Regarding their enhancement of carmustine ( BCNU) toxicity, compounds 5 and 8, which were non - toxic per se , were found to enhan ce significantly the effect of c armustine, as well as compounds 4, 6 and 7 but these three were toxic . Compounds 1, 2 and 3 exhibited a relative capacity to stimulate cell death caused by c armustin e and in contrast, compounds 9 and 10 were unable to increas e the effect of c armustine significantly. We performed colony formation assays staining with crystal v iolet with compounds 5 and 8 (non - toxic and enhance BCNU in the MTT assay ) and 6 and 7 (toxic at 1 ?M but good enhancement of carmustine). These exp eriments imply the study of the recovery of the cells after treatment with the compounds in pre sence or abse nce of c armustine, and their capacity to survive and grow during the following 10 days after treatment. The results obtained are more difficult to interpret and seem to contradict in some ways the exp eriments of MTT: all the tested compounds except compoun d 5 were found to be non - toxic, allowing the growing of co lonies to normal values after 7 days. In contrast, compound 5 presented a progressive reduction of colonies over concentration of potential inhibitor. These results indicate that cells are able to r ecover from the treatment after some days and redeem their rate of g rowing, except in the case of compoun d 5. However, their enhancement of Carmustine followed a different pattern: compounds 5, 7 and 8 improved the rate of cell death and poor survival, whi le compound 6 did not show a stimulating effect on c armustine . Even though, the colony formation assays are preliminary and should be reproduced b efore considering these results conclusive , as for example carmustine effect in the compound 6 assay did not s eem to be in the same range as shown in the other assays . From these results, we can pre-establish that compounds 8 seems to be the best candidate to enhance the effect of BCNU in cell culture, as it was found to be non-toxic at 100 ?M and improve d the rat e of cell death when incubated with Carmustine , both in the MTT and in the colony formation assay . In addition, compound 5 showed no toxicity and good enhancement of carmustine, but it was found to be toxic in colony formation assays. Combining the results obtained by ESI-MS and in cell culture, we can conclude that 2 compounds out of our first ten are able to form a specific (8) or non-specific (5) complex 225 with hAGT and are non-toxic. In addition, they seem to enhance the apoptotic effect of Carmustine in MTT and colony formation exp eriments , even if compound 5 was found to be toxic. These compounds represent good candidates for the study of their hAGT inhibition effect as potential chemotherapy enhancers. Following the line of this thesis, these potential inhibitors should be tested by the methods des cribed subsequently to reveal their active concentrations and their suitability to undergo a new optimization cycle to obtain a good candidate to start preclinical studies in animal models. At the end of this chapter, we include as an annex a work we have realized related to the search for inhibitors of another DNA repair protein, Ape 1, [16] involved in the base excision repair pathway. [17] This paper reports the identification of new compounds as potential Ape1 inhibitors through a docking -based virtual screening, following by a characterization of them by similar in vitro techniques and cytotoxic evaluating assays as the ones des cribed in chapter 1 for hAGT. Interaction of these compounds with the Ape1 protein was observed by mass sp ectrometry, and some of the compounds showed in vitro activities in the low - to-medium micromolar range . The ability of the candidates to inhibit the recombinant Ape1 activity in vitro was determined by a fluorescence -based assay des cribed by different groups . [18] These molecules also potentiated the cytotoxicity of the chemotherapeutic agent methylmethanesulfonate in fibrosarcoma cells. We then intended to evaluate the inhibitory capacity of the candidate molecules in vitro. The first intention was to employ the radioactivity assay used by Ruiz et. al. [6e] and firstly des cribed by Bresnick and coworkers [19] for th e in vitro evaluation of compounds. However, the radioactive compound used , 3 H - MNU ( tritiated methylnitrosourea), was no longer commercially available. Other radioactivity - based assays are des cribed , but in addition to being multi - enzymatic reactions , they require the use of rigorous safety procedures . [6b] Other assays, as ESI- MS- based exp eriments , [20] electrophoretic mobility shift assays [21] or HPLC assays [22] have also been conducted with the aim of studying hAGT repair. Mass sp ectrometry and elec trophoretic detection of repaired oligonucleotides were most efficient in the case of interstrand cross - linked DNA, due to the fact that the repaired oligonucleotide without a cross - link to the complementary strand ha s a lower mass than the two strands cov alently cross - linked , after forcing the denaturation of the duplex. [20b, 21b ] Other assay s reported the inhibition of restriction endonuclease followed by magnetic bead separation of products. [6a, 6d] Furthermore, a fluorescence assay was reported by Moser et al, proving useful and sensible detection of hAGT activity but it implied the use of a digestion step for the release of the fluoresc ent moiety. [6c ] All thes e methods are based on multi - step complex reactions and are discontinuous and time - 226 consuming and the neces sity to find a straightforward and sensible method for the detection of hAGT repair activity remains relevant. For this reason, we have devoted a great effort and the main part of this thesis to the development of new in vitro assays for the det ection of hAGT activity which allow to evaluate its inhibition by the candidate compounds. We have exploited all the resources and techniques of exp ertise available in our research group, whose field of interest is the chemistry and structure of nucleic acids and the application of DNA nanotechnology to biomedicine. Chapter 2 describes a method to evaluate hAGT activity that takes advantage of the conformational change of a single strand ed DNA quadruplex, [23 ] the thrombin binding aptamer (TBA), [24] an aptamer that is recog ni?e? by ?-thrombin when it is folded into its quadruplex structure. First, we demonstrated by UV monitoring of the thermal stability of the quadruplex and by circular dichroism that the replacem ent of a single guanine by an O6 -methylguanine disrupts the G-quadruplex native structure of TBA, because it diminishes the amount of chemical groups available for the Hoogsteen hydrogen bonding that forms the G-quartets. Th e conformational change of this G-quadruplex was used to develop a fluoresc ence-quencher system that reduces the e mission of fluorescence when hAGT repairs the methylation introduced previously. The fluorophor and the quencher molecule were placed in opposite ends of the sequence, and the distance between them dep ends on its conformation: the native TBA is folded into its quadruplex structure, forcing the proximity of the fluorophor and the quencher whilst the methylated TBA, being unable to fold the quadruplex, maintains its ends in opposite senses and the fluorophor and the quencher are k ept apart from each other. The reaction of hAGT represents the key to the folding and unfolding of TBA and thus to the emission or suppression of fluorescence . Even if the methodology is rapid, simple and avoids the use of radioactivity, it had an unexpected drawback : the measurement of the loss of fluoresc ence was slightly delicate, as it is always more difficult to detect and reproduce a particular loss of signal than an increase of it from basal conditions. These deliberations led us to consider the robustness of this new methodology in terms of be ing able to implement it for the search of more potent inhibitors of hAGT activity. For this reason, taking advantage of the exp ertise of other me mbers of the lab oratory in DNA Nanotechnology and making use of the conclusion obtained in chapter 2 about the distortion of the G-quadruplex when a guanine is replaced by a methylguanine , 227 w e d esigned a DNA origami platform [25] as a nanotechnological device to detect hAGT activity. The DNA origami had previously been applied as platform to follow single molecule reactions, [26] even for the detection of conformational changes of a G- quadruplexes , [27] ?or A repair proteins? reaction tracking [28] and for spatial organization of proteins [29] or other nanomaterials. [ 30] All these advances in DNA nanotechnology lead us to envisage a new platform for hAGT repair study using DNA origami. This m ethod is des cribed in chapter 3 . We exploited the spatial addressability of DNA origami in combination with the con formational change of a DNA G - quadruplex , TBA, to detect by AFM in real - time the complex formation with its substrate ?- thrombin. This structural change is caused by a single methylation in the central guanines , as reported in chapter 2, and it causes that TBA is no longer recognize d by ?- thrombin . This fact was demonstrated by fluorescence and EMSA assays, and both resul ts confirmed that the introduction of a methylated guanine prevented ?- thrombin interaction, and besides , that a concentration of 10 fold of ?- thrombin can be sufficient to appreciate a different pattern of union of this protein to the TBAs and methyl - TBAs - containing origami . The different beha?io?r o? ?- thrombin towards the TBA and the methylated TBA can be utilised to detect the DNA repair activity of hAGT, given that methylguanine is the substrate of this protein. In this case, we used two different type s of TBA, ?hich bin? at opposite e?osites o? ?- thrombin in a cooperative way [24] and improve d the attachment of the proteins over the DNA origami, preventing the sw eeping away by the AFM tip. The arrangement of ?- th rombin over the surface of the DNA origami follows the distance - d ep endent design described by Rinker et. a l , [29b] who found that upon placing the two aptamers at a specific distance of 5.8 nm from each other, the recognition of the protein increased by 10 - fold. The introduction of several methylated TBAs in specific positions of the origami allowed to observe the loss of thrombin binding. We proved that only one of the aptamers of the pair being methylated ?as eno??h to ?isr?pt the bin?in?? an? no ?- thrombin interaction was observed by AFM while the control pairs of unmodified aptamers exhibited high rate of binding. W hen the O6 - methy???anine o? the mo?i?ie? TA ?as repaire? by hAGT? ?- thrombin was able to bind again to the repaired aptamers, as they can form the quadruplex structure required for interaction. The system is extremely effective and reliable, and the resul ts are clearly followed by AFM. Their consistency suggests that our system could be further evolved for the study of other DNA repair enzymes . Even though, the complexity and high cost of the AFM technique hinders the application of this technology for the high throughput evaluation of potential inhibitors of hAGT. As a result of the high impact achieved by the publication of this work, our group was invited to write a review on the state of the art of DNA nanotechnolog y. This work is 228 included as an annex of this chapter. In this review we examine recent progresses towards the potential use of DNA nanostructures for molecular and cellular biology . In parallel, we developed a second fluorescence as say which tried to address the limitations found before (c hapter 4 ) . Studying the mechanism of action of hAGT and some reports in the literature where it is des cribed that hAGT is more active over double stranded DNA, [31] we decided to use a double stranded oligonucleotide with a labelled alkylated guanine which could b e transferred to the active site of hAGT upon DNA repair and could be quantified . This would represent a direct measurement of the activity of the protein and could be used to evaluate the effect of potential inhibitors. We synthesized a double stranded oligonucleotide where one of the two strands incorporated an alkyl- guan os ine chemical ly modifi ed in the O 6 position with an aminobenzyl group. W e used this group because O 6 -benzylguanine is reported to be the most efficiently repaired bulky adduct by hAGT [31- 32] and the free amino group in the para po sition allowed the post- synthetic chemical introduction of a fluorophore (fluoresc ein isothiocyanate, FITC). This work implied the design of a novel synthetic route for the properly protected O 6 - benzylguanosine to incorporate it to the automatized DNA synt hesis cycle. This O 6 - benzylguanosine ?as prepare? ?rom ??- d eoxyguanosine and the corresponding protected 4 - aminomethylbenzyl alcohol through a Mitsunobu reaction . [33 ] Prior to the setting up of the fluorescence assa y, we conducted HPLC studies of the repair capability of hAGT of this O6 - benzylguanine with the attached fluorescein, to ensure that this bulky substituent can be accommodated in the active site of the protein. Post - synthetically , w e attached a fluorophore to this amin obenzyl group to follow the repa ir reaction by means of fluores c ence. W e incorporated a nucleotide modified with a quencher group in the complementary strand to reach v ery low basal fluorescence when the two strands are annealed . Two options wer e explored to ensure the maximal quenching of fluoresc ence for the background conditions of the assay : in one case, the quencher was placed in the exact complementary position , and in the other, at one nucleotide shift in the complementary sequence . The pl acement of the quencher in the complementary base of the modified guanosine provokes a mismatch in the sequence , due to the fact that there is no commercially available cytidine covalently modified with the dabcyl group and the use of a thymidine was requi red. In the case of the complementary duplex, we exp ected that the fluoresc ence extinction would remain efficient even if the quencher was not placed in the complementary position . As spatial orientation of this bulky substituents remains unclear, both wer e st??ie? in terms o? therma? stabi?ity an? capacity to enter hAGT?s acti?e site, and finally the complementary duplex was used for the fluoresc ence ass ays b ecause it remained stable at the temp erature range required for the efficient reaction of hAGT . As 229 exp ected , once hAGT repaired the O 6 alkylguanine, it transferred the fluorophore to its active site together with the alkyl group, bringing it apart from the quencher. This resulted in an immediate increase in fluorescence that could be detected and correlated to hAGT activity. In contrast with the previous method s , and as it has been mentioned before, this system uses a double stranded oligonucleotide, which is the real substrate for the activity of hAGT in vivo and thus increases the efficiency of the repair reaction. In addition, as the variation in fluorescence is increasing upon activity, it is easier to quantify than the loss of fluoresc ence measured before. This method is currently ready to be used for the study of potential hAGT inhibitors, and some preliminary results are shown in the annex 4 . These results point to compound 8 as a promising candidate to inhibit hAGT in vitro, w hich is in consonance with the results obtained in chapter 1, where it was found to form a specific complex with hAGT, to be non-toxic per se and to produce a significant enhancement of carmustine toxicity. A similar approach had b een described for the preparation of a fluorescent derivative of O 6 -benzylguanine to be used in the detection of the hAGT activity in cells. [34 ] In these exp eriments, the O 6 -alkylnucleobase is allowed to enter the cells b y passive diffusion and after a certain time the exces s of the fluoresc ent nucleobase is washed away and the remaining fluorescence m easured. This remaining fluorescence inside the cells corresponds to the hAGT which has reacted, becoming fluorescently labeled . In our case, we need to evaluate in real-time the activity of hAGT and for this reason, we had to introduce the fluorescently labeled nucleoside in a DNA sequence, as well as we require the quencher molecule to be in the complementary sequence to enhance the change of fluoresc ence when the fluorescein molecule is transferred to hAGT. Our approach represents a more complex system which allow s to measure in vitro the activity of hAGT in real-time. Following with the nanotechnology, and during a short stay in the Department of Food, Environmental and Nutrition Sciences of the University of Milan, we studied the use of gold-coated magnetic nanoparticles for molecular biosen sin?? in?in? o? ?-thr mbin to these nanoparticles conjugated with two aptamers which recognize different sites of the protein causes aggregation, [35] a ph enomenon that can be detected by three different techniques: UV, DLS and MRI. These techniques discriminate even a single methylation in one of the aptamers, prev enting aggrega tion ??e to the inabi?ity o? ?-thrombin to recognize it. This system can be further developed for the detection of the activity of DNA repair proteins which have alkylated guanines as substrate. 230 We conjugated the thrombin binding aptamers 1 and 2 to gold nanoparticles (AuNPs) iron-oxide nanoparticles (SPION) and gold-coated iron-oxide (AuSPION) nanoparticles , and then comparatively studied the ability of ?-thrombin to recognize a mixture of methylated and unmethylated TBA-conjugated nanoparticles . The mixture containing unmodified TBAs was able to bind ?-thrombin in its both recognizing sites, forming a molecular network. In contrast, the methylated TBA1 together with the unmodified TBA2 was unable to form the network, and the aggregation process did not oc cur. SPION and AuSPION allow the detection of ?-thrombin by means of Magnetic Resonance Imagining, because they are contrast agents for image enhancement. The unmodified mixture of TBAs is able to interact with ?-thrombin and a molecular network is formed , increasing the image contrast and its T 2 value, as reported previously. [35] AuSPION and AuNP have a surface plasmon maximum at 520 nm that shifts to higher wavelengths when precipitation occurs, allowing the use of UV-spectroscopy as a detection method. Other works have previously exploited this feature to develop biosensors for lipase activity, [36] to monitor oxidative stress [37] or to detect blood cholesterol, [ 38] b etween others. And finally, precipitation of the three types of nanoparticles can be detected by Dynamic Light Scattering, measuring the main diameter of the nanoparticles before and after thrombin addition and finding a huge increase in the case of the unmodified mixture of nanoparticles . Aggregation detected by DLS has also been used before to develop sensors for cancer biomarkers, [39 ] or for the detection of small molecules in solution. 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A. Alsager, S. Kumar, G. R. Willmott, K. P. McNatty, J. M. Hodgkiss, Biosens Bioelectron 2014, 57, 262-268. 235 236 Conclusions 237 238 CONCLUSIONS: - 10 compounds selected by a virtual screening study with potential inhibitory activity against hAGT have been analysed in vitro and in cell culture assays : x The analysis by mass spectrometry (ESI-MS) confirmed the complex formation of hAGT with 5 of them: compound 8 formed a specific complex and compounds 5, 6, 7 and 9 an unspecific one. x MTT cytotoxicity studies showed that 2 compounds (5 and 8) were non-toxic and the other 8 compounds presented different levels of toxicity per se. In addition, 5 compounds showed enhancement of carmustine toxicity (4, 5, 6, 7 and 8). Compounds 5 and 8 seem to be the best candidates for hAGT inhibition, as they enhance BCNU without being toxic by themselves. x Compounds 5, 6, 7 and 8 were further characterized by colony formation assays, which confirmed that compound 8 was non-toxic at long-term experiments and exhibited a stimulation effect of carmustine. - A new method to detect hAGT activity based on the conformational change of the thrombin binding aptamer and followed by FRET has been designed and set up successfully. This technology allows the quantification of hAGT activity in a single step and in a straightforward manner, avoiding radioactivity and reaching a detection limit of 0.5 pmols of hAGT. Furthermore, this method can be easily transferred to a high throughput experiment for the evaluation of small molecules as potential hAGT inhibitors. - The DNA origami platform has been exploited to design a new methodology to follow in real-time the repair activity of hAGT by Atomic Force Microscopy. This study combines the recognition capacity o? ?-thrombin, able to discriminate methyl-TBA and TBA, and the single-molecule features of the DNA origami technique, applied to the detection of DNA repair. The system is extremely effective and reliable, and the results are clearly followed by AFM, allowing the detection of single-molecule reactions, the highest detection limit possible. Their consistency suggests that our system could be further evolved to design hAGT activity assays for the identification of potential inhibitors as chemotherapy enhancers and for the study of other DNA repair enzymes. - A new assay to detect hAGT activity by the transfer of a fluorescent moiety from the substrate oligonucleotide to the active site of the protein has been described. For this purpose: 239 x We have described a novel route for synthesizing an O6-benzyl-2?-deoxyguanosine precursor which incorporated to an oligonucleotide sequence, can become a fluorescently labelled substrate for measuring hAGT repair activity. x Annealing with a complementary quencher strand produces a stable duplex confirmed by physicochemical methods which presents a low basal fluorescence. x We have tested the efficiency of the fluorescence substrate transfer to hAGT. This system allows the detection of 0.5 pmols of hAGT, the same obtained for the fluorescence assay based on the conformational change of a G-quadruplex. x On theses bases, we have developed a new rapid and straightforward assay to detect hAGT activity. The system uses a double-stranded oligonucleotide, the natural substrate for hAGT activity, improving efficiency and reliability due to its easy quantification of the fluorescence increase upon activity. x We have tested 9 compounds with potential inhibitory activity of hAGT, finding that compound 8 inhibits significantly the DNA repair activity of hAGT in vitro. This represents a proof of concept of the usability of our method, as well as confirms the results previously obtained in chapter 1. - Three types of nanoparticles (AuNPs, SPIONs and AuSPIONs) have been used to detect by different techniques (UV, DLS and MRI) a single methylation in G-quadruplex. We have proven the detection capacity of AuSPION as biosensors, demonstrating that their performance is comparable to AuNPs and SPIONs. The bases for setting up a new detection platform for hAGT activity over a nanoparticle system have been accomplished. 240 Summary 241 242 The O6-alkylguanine DNA alkyltransferase (hAGT or MGMT) is a DNA repair protein in charge of removing alkyl adducts from the O6 position of guanines, blocking their cytotoxic effects and playing an important role as a resistance mechanism to chemotherapy in cancer patients. For these reasons, it is considered relevant as a prognosis marker of cancer and represents a potential therapeutic target. Intense research efforts have been devoted to the identification of small molecules capable of inhibiting hAGT activity and enhancing the cytotoxic effect of the alkylating agents in tumour cells. In this doctoral thesis, we have explored 10 compounds with potential inhibitory activity against hAGT. The analysis by mass spectrometry (ESI-MS) confirmed the complex formation of hAGT with 5 of them (compounds 5, 6, 7, 8 and 9). MTT cytotoxicity studies in cell culture showed that 2 compounds (5 and 8) were non-toxic and showed enhancement of carmustine toxicity. This compounds were further analysed by colony formation assays, which confirmed that compound 8 was non-toxic at long-term experiments and exhibited a stimulation effect of carmustine. Compound 8 seem to be the best candidates for hAGT inhibition, as it forms a complex with hAGT and it enhances BCNU without being toxic in MTT and colony formation assays. Due to the lack of a consistent in vitro assay for the activity of hAGT, we have devoted part of this doctoral thesis to the search of bio and nanotechnologies to detect hAGT activity which enable the evaluation of potential inhibitors of the protein. Chapter 2 describes the development of a new fluorescence method using the conformational change of a DNA G- quadruplex, the thrombin binding aptamer (TBA), as a molecular beacon for the detection of hAGT activity and the development of new inhibitor compounds. The conformational change of TBA is further explored to develop a detection platform on DNA origami which allows de quantification of the repair activity of hAGT on a single molecules basis, through the direct visualization by AFM of the interaction of TBA ?ith its target protein ?-thrombin when its G-quadruplex structure is restored. In addition, this work reports the synthesis of guanine derivatives modified at position 6 and properly functionalized for their incorporation into double stranded oligonucleotides that are used for the development of another novel fluorescence methodology to evaluate hAGT activity and to assess potential inhibitors as enhancers of chemotherapy. Finally, during a short stay in the University of Milan, we have developed a new sensor to detect a methylation in TBA using three types of nanoparticles: AuNPs, SPIONs and AuSPIONs. AuSPIONs combine the features of the gold coating and the magnetic core, 243 and exhibit similar performance as AuNPs and SPIONs in UV, DLS and MRI assays to detect thrombin and a single methylation in TBA. These results provide the basis for the development of a new straightforward method to study hAGT activity and to evaluate potential inhibitors. 244 RESUM a prote?na ?e reparaci? ?e ??A a???i???anina-ADN-O 6 - alquiltransferasa (hAGT) elimina productes d'alquilaci? en la posici? O 6 de les guanines , bloquejant la citotoxicitat dels agents alquilants i produint resist?ncia a la quimioterapia . Es consider a rellevant com a marcador de pron?stic en c?ncer i representa una po tencial diana terap?utica . ?ob?ecti? a ??ar? termini ??a??esta tesi ?octora? ?s trobar inhibi?ors ?e ??acti?itat ??hAGT per millorar l' efecte de la quimioter?pia en pacients amb c?ncer. En primer lloc, es va avaluar la capacitat de 10 compostos, potencials inhibidors ??hAGT, d e formar un complexe amb hAGT utilitzant esp ectrometria de masses , i es va estudiar la seva toxicitat en cultius cel?lulars a trav?s d'assajos de MTT i de formaci? de col?nies. A continuaci?, es des envolupen diferents m?todes per a la detecci? ?e ??activitat ??hAGT, per a avaluar inhibidors ??a??esta prote?na in vitro? os ??a??ests m?todes utilitzen el canvi conformacional que es produeix en l ?apt?mer ???ni? a ?a trombin (TBA) en introduir una O 6 -metilguanina, substrat ??hAGT? en ?na ?e ?es se?es t?trades centrals. En el primer m?tode es va emprar el TBA per generar un sensor de fluorescencia incorporant un fluor?f or i un inhibidor de fluores c?ncia a cadascun de ls seus extrems . Aquest sensor permet la detecci? de la disminuci? en la fluoresc ?ncia deguda al canvi conformacional del TBA produit per ??acti?itat ??hAGT. Posteriorment, el canvi conformacional del TBA va permetre dissenyar un biosensor de l'activitat d' hAGT a nivell unimolecular sobre ?a s?per?ice ???n origami d'ADN. La interacci? del TBA amb l a se?a proteina ?iana? ???-trombina, es va seguir per AFM per detectar que ??estructura de G-qu?druplex metilada es restableix per la reparaci? ??hAGT. El tercer m?tode es basa en la transfer?ncia de fluoresc?ncia a? centre acti? ??hAGT d egut a la reparaci? ???n o?i?on?c?e?ti? que cont? una guanina modificada amb un grup alquil marcat amb un fluor?for. Amb aquest obj ectiu, es va portar a terme l a s?ntesi qu?mica ??aquesta guanina modificada. n e? marc ???na esta?a en ?a ni?ersitat ?e i??? es d es criu ??est??i de nanopart?cules funcionalitzades amb TBAs per a detectar una metilaci? en una guanina utilitzant espectroscopia ???  o  ? amb ??objectiu de d es envolupar un nou assaig ?e ??acti?itat repara?ora ??A ??hAGT. 245 246