By introducing fluorescent cytosine derivatives into key positions of an i-DNA structure, their formation inside the cell has been observed by using fluorescence microscopy.
The i-motif (or i-DNA) 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, induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39 ºC 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-DNA folding in cells.
B. Mir, I. Serrano-Chacón, P. Medina, V. Macaluso, M. Terrazas, A. Gandioso, M. Garavís, Modesto Orozco,* Núria Escaja,* and Carlos González*. Site-Specific Incorporation of a Fluorescent Nucleobase Analog Enhances I-Motif Stability and allows Monitoring its Folding Inside the Cell. Nucleic Acids Res., in press, 2024.
