Study of DNA nanostructures with biomedical applications

Abstract

[eng] G-quadruplexes are non-canonical DNA structures that appear in Guanine-rich oligonucleotides. Four Guanines interact through Hoogsteen hydrogen bonds forming a tetrad which is stabilized by metallic ions. Two or more tetrads interact by pi-pi stacking forming the nanostructure. Some G-quadruplexes have the ability to be aptamers, which have the intrinsic property to interact with specific proteins expressed in the cancer cells membrane. In the first article, a library of tetramolecular parallel G-quadruplex modified with two different hydrocarbonated alkyl chains in the 3’ or 5’-end were designed as delivery systems of an antisense oligonucleotide. These “lipoquads” are expected to contribute on the cellular uptake process, endosomal scape or/and gene silencing machinery. None of the concentrations tested were toxic, internalization experiments with G-quadruplex were selective against cancer cells but, there was a similar internalization of the G-quadruplex and single strand oligonucleotide in cancer cells. Although, best luciferase inhibition was obtained from Luc_TG4T_C8 with or without lipofectamine at long times which can be explained by a positive contribution of lipoquads to endosomal scape and gene silencing. These results motivated the second article in which the therapeutic oligonucleotide is a floxuridine (FdU) oligomer. FdU is the 2’-deoxynucleoside of 5-fluorouracil (FU), a fluoropyridine used in chemotherapy for the treatment of different kinds of cancer. The activity of FU is produced once the drug is phosphorylated inside the cell and interfere in multiple critical cellular processes. However, a major limitation is that cancer cells become resistant to FU. So, we hypothesized that FU resistance may be overcome by using the aptamer properties of G- quadruplexes, to interact selectively with some overexpressed proteins in cancer cells membranes, followed by FdU oligomer degradation by endonucleases liberating multiple units of the active metabolite. The FdU oligomer did not disturb the G-quadruplex formation, the cellular uptake of the FdU oligomer was significantly improved by G-quadruplexes and consequently, a higher cytotoxicity was obtained in all cancer cells tested, especially with the (FdU)5TG6T. To expand the research about the possibilities of floxuridine, the third article was published by conjugating different small molecules that may enhance cellular uptake to improve pharmacological properties of FdU oligomer. We selected the introduction of Cholesterol, Palmitic acid, N-acetylgalactosamine (GalNAc), Folic acid and Polyethyleneglycol at the 5’ or 3’-ends of the FdU oligomers. Results confirmed the benefits of using small molecules to improve internalization efficiency. Surprisingly, Cholesterol conjugate achieved the best internalization values but, the Palmitic acid conjugate showed greater cytotoxicity by increasing late apoptosis events. To continue with the targeting properties of GalNAc, the fourth article involved the design of a new synthetic protocol to obtain an equivalent of the triantennary GalNAc by tetramerization of G-rich oligonucleotides monofunctionalized with one GalNAc. Three different libraries were designed with a Guanine-rich sequence functionalized with one unit of GalNAc at the 3’-end. The first library compared the effect of using phosphodiester or phosphothioate backbones as well as carrying a GalNAc with two different lengths. In the second library, a gapmer against the Renilla Luciferase mRNA was introduced at the 5’-end or between

the G-quadruplex and the GalNAc moiety. The third library introduced a single-stranded and a double-stranded RNA oligonucleotide anti-mTTR. Results demonstrate that the affinity to asialoglycoprotein receptor by tetrameric GalNAc was increased when compared with a monomeric GalNAc but, this affinity to the receptor it is reduced when longer oligonucleotides, like siRNAs, are used as the therapeutic cargo. Tetramolecular parallel G-quadruplex was confirmed by Circular Dichroism and RMN spectra. Internalization preferences of tetrameric GalNAc to HepG2 was observed, as well as, a better gene silencing was obtained from Luc-TG6T-GalNAc and TG6T-Luc -GalNAc.

[cat] El “G-quadruplex” es una estructura no-canònica que adopta l’ADN que comprèn seqüencies riques en Guanina formant tètrades de quatre Guanines a través d’enllaços d’hidrogen de tipus Hoogsteen. Varies tètrades s’apilen donant lloc a la nano-estructura. El primer article utilitza unes estructures anomenades “lipoquads”, compostes per una cadena alquil saturada situada en l’extrem 3’ o 5’, una seqüència rica en Guanines i un oligonucleòtid antisentit. Els resultats revelen que, tot i les modificacions, el “G-quadruplex” es forma, la internalització dels oligonucleòtids es veu incrementada en les línies cel·lulars de càncer i que la inhibició de la luciferasa a temps llargs millora amb el “lipoquad” Luc-TG4T-C8. El segon i tercer article estudia diferents estratègies per a la millora de la internalització d’un oligòmer de floxuridina (FdU). Floxuridina es el 2’-desoxinucleòsid del 5-fluorouracil (FU), un antimetabòlit utilitzat com antineoplàstic. Per evitar els inconvenients de resistència a FU i la toxicitat en teixits sans , s’ha unit un oligòmer de FdU amb un “G-quadruplex” produint una major internalització i efecte citotòxic en cèl·lules cancerígenes resistents a FU, sent (FdU)5TG6T l’oligonucleòtid mes actiu. Per altra banda, s’han unit diverses molècules per millorar la internalització: colesterol, àcid palmític, àcid fòlic, polietilenglicol i N-acetilgalactosamina. El conjugat amb colesterol millora la entrada però el conjugat amb àcid palmític es el més citotòxic. El quart article proposa una nova ruta per a la síntesi d’oligonucleòtids funcionalitzats amb la molècula tridentada de N-acetilgalactosamina. En el nostre cas, una seqüencia formadora de “G-quadruplex” unida a una molècula de N-acetilgalactosamina busca el reclutament dels receptors de l’asialoglicoproteina per introduir el oligonucleòtid terapèutic a l’interior de la cèl·lula hepàtica. L’afinitat amb el receptor millora gràcies a l’estructura de “G-quadruplex” al igual que una internalització selectiva als hepatòcits. La inhibició de la expressió gènica es veu afavorida gràcies a la construcció Luc-TG6T-GalNAc.