| Abstract [eng] |
DNA modifications, particularly the epigenetic marks 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), play critical roles in gene regulation, embryonic and disease development, including cancer. Despite its biological importance, the detection and mapping of 5hmC remain technically challenging due to its chemical similarity to 5-methylcytosine and limitations of current sequencing methods. This study aimed to explore a novel chemoenzymatic strategy for 5hmC detection by evaluating the effect of G^C adduct formation, which is a product of 5hmC derivatization, on DNA polymerase fidelity and nanopore sequencing readouts. In this project we aimed to synthesize model DNA containing 5hmC with M.HhaI and formaldehyde; to derivatize 5hmC into a G^C adduct using a selenol-based chemoenzymatic modification; and to evaluate the impact of this modification on DNA polymerase DeepVent (exo-) and DreamTaq performance and sequencing accuracy. PCR analysis with polymerases revealed a 31% decrease in amplification efficiency from G^C-modified templates, indicating interference with polymerase activity. Nanopore sequencing further confirmed the functional impact of the G^C adduct. Soft-clipping was observed at the modification site, and downstream sequences showed complex, long-range deviations from the reference, supporting the hypothesis that the G^C adduct alters polymerase fidelity. These effects were not observed in the control samples. This study demonstrates that 5hmC can be converted into a unique intramolecular G^C adduct that significantly affects DNA polymerase behaviour and sequencing output. While the derivatization strategy shows potential as a novel method for 5hmC detection, the low modification yield and high error rate in sequencing readouts underscore the need for further optimization. Nonetheless, the approach provides a foundation for developing sensitive, modification-specific detection strategies in epigenetic research and diagnostics. |
