Label-free colorimetric assay for methyltransferase activity based on a novel methylation-responsive DNAzyme strategy.
DNA methylation catalyzed by methyltransferase (MTase) is a significant epigenetic process for modulating gene expression. Traditional methods to study MTase activity require a laborious and costly DNA labeling process. In this article, we report a simple, colorimetric, and label-free methylation-responsive DNAzyme (MR-DNAzyme) strategy for MTase activity analysis. This new strategy relies on horseradish peroxidase (HRP) mimicking DNAzyme and the methylation-responsive sequence (MRS) of DNA which can be methylated and cleaved by the MTase/endonuclease coupling reaction. Methylation-induced scission of MRS would activate the DNAzyme that can catalyze the generation of a color signal for the amplified detection of methylation events. Taking Dam MTase and DpnI endonuclease as examples, we have developed two colorimetric methods based on the MR-DNAzyme strategy. The first method is to utilize an engineered hairpin-DNAzyme hybrid probe for facile turn-on detection of Dam MTase activity, with a wide linear range (6-100 U/mL) and a low detection limit (6 U/mL). Furthermore, this method could be easily expanded to profile the activity and inhibition of restriction endonuclease. The second method involves a methylation-triggered DNAzyme-based DNA machine, which achieves the ultrahigh sensitive detection of Dam MTase activity (detection limit = 0.25 U/mL) by a two-step signal amplification cascade.
Li
Wang
W
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China.
Liu
Zhuoliang
Z
Lin
Hui
H
Nie
Zhou
Z
Chen
Jinhua
J
Xu
Xiahong
X
Yao
Shouzhuo
S
D001483:Base Sequence; D003124:Colorimetry / Q000379:methods*; D004247:DNA; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D021881:DNA, Catalytic / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel
D021881:DNA, Catalytic; D004247:DNA; D015254:DNA Modification Methylases