Zhao Zhihan, Chen Shixing, Liu Zhixiao, Su Jing, Lü Junhong, Hao Lihong, Dou Yanzhi, Wang Lihua, Song Shiping
Institute of Materiobiology, College of Science, Shanghai University, Shanghai 200444, China.
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China.
JACS Au. 2025 Feb 17;5(3):1320-1327. doi: 10.1021/jacsau.4c01184. eCollection 2025 Mar 24.
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.
5-羟甲基胞嘧啶（5hmC）在DNA去甲基化途径和转录调控中起着关键作用。虽然基于测序的方法，如TET辅助亚硫酸氢盐测序提供单碱基分辨率，但它们对于动态、对时间敏感的定量并不理想。在此，我们提出了一种新颖的酶促生物传感策略，利用T7核酸内切酶I实现具有单碱基分辨率的快速且位点特异性的5hmC检测。这个电化学平台捕获双标记的双链DNA，并通过监测T7核酸内切酶切割A-C错配后信号的降低来检测5hmC。该方法具有高灵敏度，在甲基化或未甲基化DNA过量100,000倍的情况下，能检测低至10 pg的羟甲基化DNA。此外，我们证明了其在脐带间充质干细胞分化过程中实时定量5hmC变化的能力。这种方法为动态生物学过程中的5hmC分析提供了一个强大的工具。
