Li Jun, Yan Hongfei, Wang Kemin, Tan Weihong, Zhou Xingwang
Biomedical Engineering Center, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha 410082, P R China.
Anal Chem. 2007 Feb 1;79(3):1050-6. doi: 10.1021/ac061694i.
DNA methylation catalyzed by methylase plays an important role in many biological events. However, traditional methods of methylase activity analysis by gel electrophoresis were laborious and discontinuous. In this paper, we report a new strategy to study methylase activity using fluorescent probes coupled with enzyme-linkage reactions. A hairpin DNA probe is prepared with a fluorophore and a quencher linked at the 5'- and 3'-terminus of the probe. A disturbance of the stem sequence by DNA methylation would cause the separation of the fluorophore and the quencher, resulting in the restoration of the fluorescence. We used DNA adenine methylation (Dam) methyltransferase (MTase) and Dpn I endonuclease, both having a 5'-G-A-T-C-3' recognition sequence. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and Dpn I cut the sequence of 5'-G-Am-T-C-3'. The fluorescence of the hairpin probe was restored when it was cleaved by Dpn I endonuclease during the course of methylation. Unlike traditional methods, this assay was done in real time and could be used to monitor the dynamic process of methylation. Our method is easy, simple, and nonradioactive, yet as efficient as gel electrophoresis in detecting the activity of methylase. It also had the potential to screen suitable inhibitor drugs for Dam methylase.
由甲基化酶催化的DNA甲基化在许多生物事件中起着重要作用。然而，传统的通过凝胶电泳分析甲基化酶活性的方法既费力又不连续。在本文中，我们报道了一种使用荧光探针结合酶联反应来研究甲基化酶活性的新策略。制备一种发夹DNA探针，在探针的5'端和3'端连接一个荧光团和一个猝灭剂。DNA甲基化对茎序列的干扰会导致荧光团和猝灭剂分离，从而使荧光恢复。我们使用了DNA腺嘌呤甲基化（Dam）甲基转移酶（MTase）和Dpn I核酸内切酶，它们都具有5'-G-A-T-C-3'识别序列。Dam MTase催化5'-GATC-3'序列的甲基化，而Dpn I切割5'-G-Am-T-C-3'序列。在甲基化过程中，当发夹探针被Dpn I核酸内切酶切割时，其荧光恢复。与传统方法不同，该检测是实时进行的，可用于监测甲基化的动态过程。我们的方法简便、简单且无放射性，在检测甲基化酶活性方面与凝胶电泳一样有效。它还有筛选适合Dam甲基化酶的抑制剂药物的潜力。
