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

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.

Improved method for chemiluminescent determination of peroxidase-mimicking DNAzyme activity

“…At the same time, many researchers carry out the determination of PMDNAzyme activity in Hepes buffer [11,22,37,38]. A comparison of PMDNAzyme activity determined in two buffers (Fig.…”

Circular Nucleic Acids: Discovery, Functions and Applications

“…Moreover, Zhang et al. develop a nicking enzyme‐assisted primer generation RCA method for highly sensitive chemiluminescence detection of DNA methyltransferases (Figure B), which catalyze the 5‐position methylation of cytosine in CpG islands in both prokaryotes and eukaryotes. In the presence of DNA methyltransferases, methylation‐responsive hairpin probes are methylated and cleaved by endonuclease Dpn I.…”

Electrogenerated chemiluminescence biosensing method for methyltransferase activity using tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide

“…Table S1. Among all the methods, the detection limit of the ECL strategy was lower than those of the previous methods [13,[38][39][40][41] for Dam MTase detection except Zhang et al's work [42]. The relative standard deviation for 0.5 U/mL Dam MTase was 4.65% (n = 5).…”