Label-Free Colorimetric Assay for Methyltransferase Activity Based on a Novel Methylation-Responsive DNAzyme Strategy

• Published in: Analytical chemistry (Washington) Vol. 82; no. 5; pp. 1935 - 1941
• Main Authors: Li, Wang, Liu, Zhuoliang, Lin, Hui, Nie, Zhou, Chen, Jinhua, Xu, Xiahong, Yao, Shouzhuo
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.03.2010
• Subjects: Analytical chemistry; Base Sequence; Chemistry; Colorimetry - methods; Deoxyribonucleic acid; DNA; DNA Methylation; DNA Modification Methylases - metabolism; DNA, Catalytic - metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme kinetics; Exact sciences and technology; Spectrometric and optical methods; Studies; Tissue engineering; Catalytic reaction; Chemical analysis; Nucleotide sequence; Enzyme; Color; Activation; Colorimetry; Method; Gene expression; Probe; Gene amplification; Signal; Peroxidases; DNA; Detection limit; Restriction endonuclease; Strategy; Peroxidase; Oxidoreductases; Coupling; Inhibition; Detection; Methylation; Methyl transferases
• DOI: 10.1021/ac902670c
• ISSN: 0003-2700; 1520-6882

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.