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Sensitive Detection of DNA Methyltransferase Activity Based on Exonuclease-Mediated Target Recycling

Xi-Wen Xing, Feng Tang, Jun Wu, Jie-Mei Chu, Yu-Qi Feng, Xiang Zhou,* and Bi-Feng Yuan*

 

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, Hubei 430072, P.R. China


ABSTRACT: 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 N6- methyladenine (m6A). 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.


 

Sensitive Detection of DNA Methyltransferase Activity Based on Exonuclease-Mediated Target Recycling
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