T7 Endonuclease I-Mediated Single-Base Mismatch Biosensing Strategy for High-Resolution Quantitative Analysis of...

5-hydroxymethylcytosine (5hmC) plays a pivotal role in the DNA demethylation pathway and transcriptional regulation. While sequencing-based methods such as TET-assisted bisulfite sequencing offer single-base resolution, they are not ideal for dynamic, time-sensitive quantification. Here, we present a novel enzymatic biosensing strategy leveraging T7 endonuclease I for rapid and locus-specific 5hmC detection with a single-base resolution. This electrochemical platform captures double-tagged dsDNA and detects 5hmC by monitoring the signal reduction upon T7 endonuclease cleavage of A-C mismatches. The method achieved high sensitivity, detecting as little as 10 pg of hydroxymethylated DNA amid a 100,000-fold excess of methylated or unmethylated DNA. Furthermore, we demonstrated its ability to quantify real-time 5hmC variation during umbilical cord mesenchymal stem cell differentiation. This approach offers a powerful tool for 5hmC analysis in dynamic biological processes.