Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.
Nucleic Acids Res
; 52(6): 3375-3389, 2024 Apr 12.
Article
em En
| MEDLINE
| ID: mdl-38366792
ABSTRACT
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 GCGC 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 tCOC+ 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Citosina
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2024
Tipo de documento:
Article