Structural effects of inosine substitution in telomeric DNA quadruplex.

The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG) n telomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG) 4 , (IGG) 4 , (GIG) 4 , (GGI) 4 , (IGI) 4 , (IIG) 4 , (GII) 4, and (III) 4 , bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na + and K + containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG) 4 and the terminal modified DNAs (IGG) 4 and (GGI) 4 in both Na + and K + containing buffers. Specifically, quadruplex hybrid was observed for (GGG) 4 in K + buffer. Among the other modified sequences, (GIG) 4 , (IGI) 4 and (GII) 4 show parallel features, while (IIG) 4 and (III) 4 show no detectable conformation in the presence of either Na + or K + . Our studies indicate that terminal lesions (IGG) 4 and (GGI) 4 may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI) 4 in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K + cation confers more thermodynamic stability compared to Na + . Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions.