Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University , Wuhan, Hubei 430072, P.R. China.
Anal Chem. 2014 Nov 18;86(22):11269-74. doi: 10.1021/ac502845b. Epub 2014 Oct 30.
DNA methylation plays vital roles in various biological processes in both prokaryotes and eukaryotes. In bacteria, modification of adenine at N6 can protect bacterial DNA against cleavage by restriction enzymes, and bacterial DNA adenine methyltransferases are essential for bacterial virulence and viability. DNA adenine methyltransferase (DAM) targets the sequence of 5'-GATC-3' and can convert adenine into N(6)-methyladenine (m(6)A). Because mammals do not methylate DNA at adenine, bacterial DAM represents an excellent candidate for antibiotic development. Here, we developed an exonuclease III-aided target recycling strategy to sensitively assay activity of DAM. In this method, a hairpin probe labeled with a donor fluorophore (FAM) at the 5' end and a quencher (BHQ) close to the 3' end (FQ probe) was employed as reporter. Another hairpin substrate containing sequence of GATC was used as the methylation substrate of DAM. Once the hairpin substrate was methylated by DAM, it could be recognized and cleaved by Dpn I, which allows the release of a single-stranded oligodeoxynucleotide (ssODN). The ssODN can then hybridize to the 3' protruding terminus of FQ probe, which subsequently triggers the exonuclease III-mediated target recycling reaction and therefore can significantly improve the detection sensitivity of DAM. The exonuclease-mediated target recycling strategy is extremely sensitive and as low as 0.01 U/mL DAM can be distinctly determined. Using this developed method, we evaluated DAM activity in different growth stages of E. coli cells, and we also demonstrated that the assay has the potential to screen suitable inhibitor drugs for DAM for disease(s) treatment.
DNA 甲基化在原核生物和真核生物的各种生物过程中都起着至关重要的作用。在细菌中，腺嘌呤在 N6 位的修饰可以保护细菌 DNA 免受限制性内切酶的切割，而细菌 DNA 腺嘌呤甲基转移酶对于细菌的毒力和生存能力是必不可少的。DNA 腺嘌呤甲基转移酶（DAM）靶向 5'-GATC-3'序列，并能将腺嘌呤转化为 N(6)-甲基腺嘌呤（m(6)A）。由于哺乳动物不会在腺嘌呤上甲基化 DNA，因此细菌 DAM 是开发抗生素的理想候选物。在这里，我们开发了一种外切酶 III 辅助的靶标回收策略来灵敏地检测 DAM 的活性。在该方法中，发夹探针在 5'端标记供体荧光团（FAM），在 3'端附近标记淬灭剂（BHQ）（FQ 探针）作为报告分子。另一个发夹底物包含 GATC 序列，作为 DAM 的甲基化底物。一旦 DAM 将发夹底物甲基化，它就可以被 Dpn I 识别和切割，这允许释放单链寡脱氧核苷酸（ssODN）。ssODN 可以与 FQ 探针的 3'突出末端杂交，随后触发外切酶 III 介导的靶标回收反应，从而显著提高 DAM 的检测灵敏度。外切酶介导的靶标回收策略非常灵敏，低至 0.01 U/mL 的 DAM 也可以被明显检测到。使用这种开发的方法，我们评估了 DAM 在大肠杆菌不同生长阶段的活性，并且还证明该测定法有可能筛选出适合用于 DAM 疾病治疗的抑制剂药物。
