Label-Free Colorimetric Assay for Methyltransferase Activity Based on a Novel Methylation-Responsive DNAzyme Strategy
Label-Free Colorimetric Assay for Methyltransferase Activity Based on a Novel Methylation-Responsive DNAzyme Strategy
复制标题
基于新型甲基化响应 DNAzyme 策略的甲基转移酶活性无标记比色测定
DOI：
10.1021/ac902670c
复制
发表时间：
2010-03-01
影响因子：
7.4
通讯作者：
Chen, Jinhua
中科院分区：
文献类型：
--
作者：
Li, Wang;Liu, Zhuoliang;Chen, Jinhua
甲基转移酶（MTase）催化的DNA甲基化是调控基因表达的重要表观遗传过程。研究MTase活性的传统方法需要费力且昂贵的DNA标记过程。在这篇文章中，我们报告了一个简单的，比色，和标记的甲基化反应的DNA酶（MR-DNA酶）的策略MTase活性分析。这种新的策略依赖于辣根过氧化物酶（HRP）模拟DNAzyme和DNA的甲基化反应序列（MRS），它可以被甲基化和切割的MTase/核酸内切酶偶联反应。甲基化诱导的MRS断裂将激活DNA酶，其可以催化产生用于甲基化事件的扩增检测的颜色信号。以Dam MTase和DpnI内切酶为例，我们建立了两种基于MR-DNAzyme策略的比色方法。第一种方法是利用工程化的发夹-DNAzyme杂交探针用于Dam MTase活性的简易开启检测，具有宽的线性范围（6-100 U/mL）和低的检测限（6 U/mL）。此外，该方法还可用于限制性内切酶活性和抑制的分析。第二种方法涉及甲基化触发的基于DNA酶的DNA机器，其通过两步信号放大级联实现Dam MTase活性的高灵敏度检测（检测限= 0.25 U/mL）。
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 it 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.