Ultrasensitive Detection of Active Turnover of DNA Methylation Using Single-Molecule Arrays with Clinical Implications

5-methylcytosine (5mC), the most well-known form of DNA methylation, is typically regulated by two types of enzymes: DNA methyltransferases (DNMT), "writers" responsible for the deposition of methylation marks, and ten-eleven translocation dioxygenases (TET), "erasers" controlling their active removal. Therefore, these two types of Enzymes play fundamental roles in regulating active turnover of 5mC modification. Given that 5mC-based approaches have been widely applied in diagnosis and treatment, and that a subtle imbalance in these enzyme activities often precedes aberrant 5mC deposition, we developed a single-molecule array-based method for sensitive detection of both Enzymes, enabling activity analysis at the single-cell level. Notably, in human peripheral blood mononuclear cells (PBMCs), DNMT activity is significantly higher in elders than in young individuals, while TET activity remains unchanged. Moreover, the relative active demethylation ratio (TET activity/DNMT activity) is significantly higher in young individuals. When applied to clinical disease samples, bone marrow mononuclear cells (BMNCs) from pediatric patients with active hematologic disease showed elevated TET activity and an increased active demethylation ratio. Overall, our approach provides a valuable tool for investigating diseases associated with aberrant 5mC modifications, with promising implications for biomarker development and disease monitoring.