Mir Morro, BartomeuSerrano Chacón, IsraelMedina, PedroMacaluso, VeronicaTerrazas Martínez, MontserratGandioso, AlbertGaravís, MiguelOrozco López, ModestoEscaja Sánchez, NuriaGonzález, Carlos2024-11-042024-11-042024-02-160305-1048https://hdl.handle.net/2445/216188The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.1 p.application/pdfengcc-by-nc (c) Mir, B. et al., 2024http://creativecommons.org/licenses/by-nc/4.0/Àcids nucleicsADNDinàmica molecularNucleic acidsDNAMolecular dynamicsinfo:eu-repo/semantics/article7446712024-11-04info:eu-repo/semantics/openAccess