Sensitive Detection of DNA Methyltransferase Using Hairpin Probe-Based Primer Generation Rolling Circle Amplification-Induced Chemiluminescence

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Abstract

DNA methyltransferases (MTases) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the 5-positon of cytosine in CpG islands, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Despite the development of various methods for the MTase assay, most of them are laborious and costly with poor sensitivity. Herein, we develop a highly sensitive chemiluminescence method for the MTase assay using hairpin probe-based primer generation rolling circle amplification (PG-RCA). In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate PG-RCA reaction by hybridizing with the circular DNA template, producing a large number of horseradish peroxidase-mimicking DNAzyme chains, which can catalyze the oxidation of luminal by H2O2 in the presence of hemin to yield distinct chemiluminescence signal. While in the absence of Dam MTase, neither methylation/cleavage nor PG-RCA reaction can be initiated and no chemiluminescence signal is observed. The proposed method exhibits a wide dynamic range from 0.025 to 400 U/mL and an extremely low detection limit of 1.29 × 10(-4) U/mL, which is superior to most conventional approaches for the MTase assay. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

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