ArticleJACS Au2025
T7 Endonuclease I-Mediated Single-Base Mismatch Biosensing Strategy for High-Resolution Quantitative Analysis of 5-Hydroxymethylcytosine in Genomic DNA.
Article in JACS Au, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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9 authors.
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Abstract
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.
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