Single-Molecule Nanopore Sequencing of the TERT Core Promoter CpG Island Reveals PQS-Associated Strand Asymmetry of De Novo Methylation and Its Modulation by Photo-Oxidation

DNA methylation of the human telomerase reverse transcriptase (hTERT) promoter contributes to telomerase regulation in cancer. We examined how strand identity and sequence context relate to methylation by the Dnmt3a catalytic domain (Dnmt3a-CD). Strand-resolved nanopore sequencing compared four promoter variants (wild type, C228T, C250T, and C228T/C250T) in amplicons and plasmids. Amplicons were also photo-oxidized with methylene blue before methylation. Methylation was higher on the cytosine-rich than on the guanine-rich strand. Defined putative quadruplex-forming sequence (PQS) cores showed reduced strand asymmetry and lower site-to-site variability; grouping sites by high G4Hunter scores did not reproduce this pattern. Variants and plasmid substrates largely retained the spatial profile. Photo-oxidized amplicons showed lower measured 5mC, particularly on the guanine-rich strand, while broadly retaining the profile. Photo-oxidation reduced sequencing quality. Bisulfite analysis of the C-rich strand of Raji DNA revealed a PQS-associated hypomethylation trough in both Dnmt3a-CD-treated DNA and an untreated aliquot. Together, these results highlight the importance of PQS sequence context for hTERT promoter methylation and the challenges of accurately sequencing guanine-rich, oxidation-prone DNA.