State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China.
J Mater Chem B. 2019 Jan 7;7(1):157-162. doi: 10.1039/c8tb02096e. Epub 2018 Dec 7.
DNA methyltransferase (MTase) is an important epigenetic modification enzyme responsible for DNA methylation, and the dysregulation of DNA MTase activity is associated with various diseases in humans. Herein, we take advantage of the DNA lesion repair mechanism in vivo to develop a new fluorescence approach for the specific and sensitive detection of DNA methyltransferase (DNA MTase) on the basis of the DNA lesion repair-directed cascade isothermal amplification. Due to the high amplification efficiency of the uracil repair-mediated exponential isothermal amplification reaction (EXPAR), the efficient cleavage of endonuclease IV (Endo IV)-induced cyclic catalysis, and the low background signal caused by single uracil repair-mediated inhibition of nonspecific amplification, this approach exhibits high sensitivity with a detection limit of 0.014 U mL for pure Dam MTase and 0.61 × 10 mg mL for Dam MTase in E. coli cells and it can be further applied for the screening of DNA MTase inhibitors. More importantly, this approach can be applied to detect other DNA MTases by designing appropriate substrates, showing great potential in biomedical research and clinical diagnosis.
DNA 甲基转移酶（MTase）是一种重要的表观遗传修饰酶，负责 DNA 甲基化，而 DNA MTase 活性的失调与人类的各种疾病有关。在此，我们利用体内 DNA 损伤修复机制，基于 DNA 损伤修复导向级联等温扩增，开发了一种新的荧光方法，用于特异性和灵敏地检测 DNA 甲基转移酶（DNA MTase）。由于尿嘧啶修复介导的指数等温扩增反应（EXPAR）的高扩增效率、内切酶 IV（Endo IV）诱导的循环催化的有效切割以及单尿嘧啶修复介导的非特异性扩增抑制引起的低背景信号，该方法具有很高的灵敏度，对纯 Dam MTase 的检测限为 0.014 U mL，对大肠杆菌细胞中的 Dam MTase 的检测限为 0.61×10 mg mL，并且可以进一步用于筛选 DNA MTase 抑制剂。更重要的是，通过设计适当的底物，该方法可用于检测其他 DNA MTases，在生物医学研究和临床诊断中具有很大的应用潜力。
