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

DNA rnethylation 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 HI 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 hairphi 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 FQprobe, 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 co/i cells, and we also demonstrated that the assay has the potential to screen suitable inhibitor drugs for DAM for disease(s) treatment.