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 noncanonical 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 ligand-bound states.

Analytical and Structural Studies for the Investigation of Oxidative Stress in Guanine Oligonucleotides.

DNA damage plays a decisive role in epigenetic effects. The detection and analysis of DNA damages, like the most common change of guanine (G) to 8-oxo-7,8-dihydroguanine (OG), is a key factor in cancer research. It is especially true for G quadruplex structure (GQ), which is one of the best-known examples of a non-canonical DNA arrangement. In the present work, we provided an overview on analytical methods in connection with the detection of OG in oligonucleotides with GQ-forming capacity. Focusing on the last five years, novel electrochemical tools, like dedicated electrodes, were overviewed, as well as different optical methods (fluorometric assays, resonance light scattering or UV radiation) along with hyphenated detection and structural analysis methods (CD, NMR, melting temperature analysis and nanopore detection) were also applied for OG detection. Additionally, GQ-related computational simulations were also summarized. All these results emphasize that OG detection and the analysis of the effect of its presence in higher ordered structures like GQ is still a state-of-the-art research line with continuously increasing interest.