Structural effects of inosine substitution in telomeric DNA quadruplex

Abstract

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.

Keywords: CD spectra; G-I mutation; G-quadruplex; inosine; telomeric repeats.