Impact of a single 8-oxoguanine lesion on Watson–Crick and Hoogsteen base pair hybridization in telomeric DNA quantified using single-molecule force spectroscopy

8-Oxoguanine (8-oxoG) is a common DNA oxidative lesion prevalent in telomeric regions. However, the impact of 8-oxoG modification on the Watson-Crick base pairing energy remains controversial, potentially due to the formation of partially folded intermediates. Here, we used single-molecule magnetic tweezers to characterize the mechanical stability and equilibrium folding/unfolding transitions of human telomeric hairpins containing a single 8-oxoG lesion at different positions. Our results reveal that fully folded hairpins with a centrally located 8-oxoG exhibit similar hybridization energy (ΔΔG ∼ 1 kBT) and folding/unfolding rates to the wild type. This provides valuable data for refining the energy contribution of 8-oxoG-C base pair. In contrast, a single 8-oxoG lesion near the unzipping termini (5'-end or 3'-end) significantly enhances end fraying and hinders the complete folding of the hairpin under force. A 5'-end 8-oxoG lesion increased the unfolding rates by a 130-fold compared to the wild type at 10.1 pN. These findings provide insights into the unzipping dynamics of DNA duplexes containing 8-oxoG lesions at replication and transcription forks. Furthermore, we observed that an 8-oxoG at 5'-end of telomeric G-quadruplexes (G4s) significantly decreases folding rates and folding free energy (from 5.9 kBT to 2.3 kBT), shedding light on the dynamics of G4s under oxidative stress.