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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/216188
Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells
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The 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.
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MIR MORRO, Bartomeu, et al. Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells. Nucleic Acids Research. 2024. ISSN 0305-1048. [consulted: 8 of August of 2026]. Available at: https://hdl.handle.net/2445/216188