Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, PR China; Division of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboraotory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, PR China.
Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, PR China.
Biosens Bioelectron. 2014 Apr 15;54:565-70. doi: 10.1016/j.bios.2013.11.055. Epub 2013 Nov 26.
DNA methyltransferase (MTase), catalyzing DNA methylation in both eukaryotes and prokaryotes, is closely related with cancer and bacterial diseases. Although there are various methods focusing on DNA MTase detection, most of them share common defects such as complicated setup, laborious operation and requirement of expensive analytical instruments. In this work, a simple strategy based on methylation-blocked cascade amplification is developed for label-free colorimetric assay of MTase activity. When DNA adenine methylation (Dam) MTase is introduced, the hairpin probe is methylated. This blocks the amplified generation of G-riched DNAzyme by nicking endonuclease and DNA polymerase, and inhibits the DNAzyme-catalyzed colorimetric reaction. Contrarily, an effective colorimetric reaction is initiated and high color signal is clearly observed by the naked eye in the absence of Dam MTase. A satisfying sensitivity and high selectivity are readily achieved within a short assay time of 77 min, which are superior to those of some existing approaches. Additionally, the application of the sensing system in human serum is successfully verified with good recovery and reproducibility, indicating great potential for the practicality in high concentrations of interfering species. By using several anticancer and antimicrobial drugs as model, the inhibition of Dam MTase is well investigated. Therefore, the proposed method is not only promising and convenient in visualized analysis of MTase, but also useful for further application in fundamental biological research, early clinical diagnosis and drug discovery.
DNA 甲基转移酶（MTase），能够催化真核生物和原核生物中的 DNA 甲基化，与癌症和细菌疾病密切相关。尽管有各种方法专注于 DNA MTase 的检测，但它们大多具有共同的缺陷，例如复杂的设置、繁琐的操作和昂贵的分析仪器的需求。在这项工作中，我们开发了一种基于甲基化阻断级联扩增的简单策略，用于 MTase 活性的无标记比色测定。当引入 DNA 腺嘌呤甲基化（Dam）MTase 时，发夹探针被甲基化。这阻止了内切核酸酶和 DNA 聚合酶对富含 G 的 DNA 酶的扩增生成，并抑制了 DNA 酶催化的比色反应。相反，在没有 Dam MTase 的情况下，有效起始比色反应，并通过肉眼清楚地观察到高颜色信号。在 77 分钟的短测定时间内，很容易实现令人满意的灵敏度和高选择性，优于一些现有方法。此外，该传感系统在人血清中的应用得到了很好的验证，具有良好的回收率和重现性，表明在高浓度干扰物质存在下具有很大的实用性。通过使用几种抗癌和抗菌药物作为模型，很好地研究了 Dam MTase 的抑制作用。因此，该方法不仅在 MTase 的可视化分析中具有很大的潜力和便利性，而且还可用于进一步应用于基础生物学研究、早期临床诊断和药物发现。
