Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications.

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2'-deoxyribose, arabinose, and 2'-deoxy-2'-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications. An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3'-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.