School of Pharmaceutical and Materials Engineering, Taizhou University, Jiaojiang 318000, Zhejiang, China.
State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Anal Chem. 2022 Mar 15;94(10):4407-4416. doi: 10.1021/acs.analchem.1c05332. Epub 2022 Mar 2.
DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (H) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin H was methylated and specifically cleaved by I endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.
DNA 甲基化由一类 DNA 甲基转移酶催化，这些酶在各种生物过程中起着至关重要的作用。因此，在生物医学研究和临床诊断中，非常需要超灵敏的甲基转移酶检测方法。然而，传统的 DNA 甲基转移酶活性检测方法通常涉及放射性标记、昂贵的设备和繁琐的操作。在这项研究中，我们开发了一种使用纳米孔技术结合 DNA 级联信号放大反应的超灵敏、无标记方法来检测 DNA 腺嘌呤甲基转移酶（Dam）和 CpG 甲基转移酶（M.SssI）。巧妙设计了一个包含甲基化反应序列的发夹 DNA（H）。在 Dam 甲基转移酶存在下，发夹 H 的相应识别序列被甲基化，并被 I 内切酶特异性切割，从而形成一个诱导催化发夹组装和杂交链式反应（CHA-HCR）的 DNA 片段。生成的产物可以被吸附到 Zr 涂层的纳米孔上，导致离子电流整流信号发生变化。考虑到纳米孔的高灵敏度和对甲基转移酶/内切酶识别的优异特异性，我们开发的方法可以在同一传感平台上同时检测 Dam 和 M.SssI 甲基转移酶。此外，经过级联 INHIBIT-AND 逻辑门的集成，设计的纳米孔传感器可以实现 Dam 和 M.SssI 甲基转移酶的多重检测。这种超灵敏的甲基转移酶检测方法在癌症诊断领域具有广阔的应用前景。
