Probing telomeric-like G4 structures with full or partial 2'-deoxy-5-hydroxyuridine substitutions

Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by non canonical G-G base tetrads. We synthesised the nucleoside analogue 2'-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2'-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligandbound states.(c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Automated summary

In this study, a nucleoside analogue was synthesized and inserted into oligonucleotide structures for investigating the stability and helicase activity of guanine quadruplexes (G4s). The results showed that the modified structures maintained partial stability in G4s, but had significant effects on fully replaced structures. Additionally, the study revealed that other analogues might also influence the stability of cellular DNA and RNA quadruplexes.