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

详细信息    查看全文

• 作者：Wang Li ; Zhuoliang Liu ; Hui Lin ; Zhou Nie ; Jinhua Chen ; Xiahong Xu ; Shouzhuo Yao
• 刊名：Analytical Chemistry
• 出版年：2010
• 出版时间：March 1, 2010
• 年：2010
• 卷：82
• 期：5
• 页码：1935-1941
• 全文大小：275K
• 年卷期：v.82,no.5(March 1, 2010)
• ISSN：1520-6882

文摘

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.