DNA methylation is a predominant epigenetic modification that plays crucial roles in various cellular processes. DNA methyltransferase (MTase) is responsible for DNA methylation, and its dysregulation may induce aberrant methylation patterns that are closely related to cancers. Conventional methods for DNA MTase assay are usually cumbersome and laborious with poor sensitivity. Alternatively, some signal amplification strategies are employed to improve the sensitivity, but they suffer from poor specificity and consequently limited sensitivity due to the nonspecific amplification. Herein, we develop for the first time a new fluorescence method to specifically and sensitively detect DNA MTase activity on the basis of single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. In the presence of DNA MTase, the hairpin substrate is methylated and cleaved by endonuclease Dpn I, releasing a 24-nt cleavage product. The 24-nt cleavage product may function as a primer and adjacently hybridize with the ligation probes (LP1 and LP2) to form the template (LP1–LP2) for strand displacement amplification (SDA), initiating the single-ribonucleotide repair-mediated cyclic ligation-dependent SDA to produce a large number of reporter probes. The reporter probe can subsequently hybridize with the signal probe that is modified with FAM and BHQ1 to form a stable double-stranded DNA (dsDNA) duplex with a ribonucleotide mismatch. Ribonuclease HII (RNase HII) can excise the single ribonucleotide, resulting in the cyclic cleavage of signal probes and the generation of an enhanced fluorescence signal. Taking advantage of the high specificity of RNase HII-catalyzed single-ribonucleotide excision and the high amplification efficiency of cyclic ligation-dependent SDA, this assay exhibits the highest sensitivity reported so far with a detection limit of 4.8 × 10⁻⁶ U mL⁻¹ and a large dynamic range of 5 orders of magnitude. Moreover, this method can be used for the discrimination of Dam MTase from other DNA MTases, the accurate quantification of Dam MTase activity in E. coli cells, and the screening of Dam MTase inhibitors, providing a new paradigm for biomedical research and clinical diagnosis.

中文翻译：

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单核糖核苷酸修复介导的依赖连接的循环信号放大，可灵敏而特异地检测DNA甲基转移酶†

DNA甲基化是一种主要的表观遗传修饰，在各种细胞过程中起着至关重要的作用。DNA甲基转移酶（MTase）负责DNA甲基化，其失调可能会诱导与癌症密切相关的异常甲基化模式。用于DNA MTase测定的常规方法通常麻烦且费力且灵敏度低。可替代地，一些信号放大策略被用于提高灵敏度，但是由于非特异性放大，它们具有差的特异性以及因此灵敏度有限的缺点。在本文中，我们首次开发了一种新的荧光方法，可以在单核糖核苷酸修复介导的依赖连接的循环信号扩增的基础上特异性地和灵敏地检测DNA MTase活性。在DNA MTase存在的情况下，发夹底物被甲基化并被核酸内切酶Dpn I裂解，释放出24 nt裂解产物。24-nt裂解产物可充当引物，并与连接探针（LP1和LP2）相邻杂交，形成用于链置换扩增（SDA）的模板（LP1-LP2），从而启动单核糖核苷酸修复介导的环状连接依赖的SDA产生大量的报告探针。报告探针随后可与经FAM和BHQ1修饰的信号探针杂交，形成具有核糖核苷酸错配的稳定双链DNA（dsDNA）双链体。核糖核酸酶HII（RNase HII）可以切除单个核糖核苷酸，从而导致信号探针的环状切割和增强的荧光信号的产生。^-6 U mL ^-1和5个数量级的大动态范围。此外，该方法可用于区分Dam MTase与其他DNA MTase，准确定量大肠杆菌细胞中Dam MTase活性，以及筛选Dam MTase抑制剂，为生物医学研究和临床诊断提供了新的范例。