State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China.
Anal Chem. 2010 Mar 1;82(5):1935-41. doi: 10.1021/ac902670c.
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.
DNA 甲基化由甲基转移酶（MTase）催化，是调节基因表达的重要表观遗传过程。传统的 MTase 活性研究方法需要繁琐且昂贵的 DNA 标记过程。在本文中，我们报告了一种简单、比色且无需标记的甲基化响应 DNA 酶（MR-DNAzyme）策略，用于 MTase 活性分析。该新策略依赖辣根过氧化物酶（HRP）模拟 DNA 酶和 DNA 的甲基化响应序列（MRS），该序列可被 MTase/内切酶偶联反应甲基化和切割。MRS 的甲基化诱导断裂会激活 DNA 酶，从而可以催化产生颜色信号，用于增强检测甲基化事件。以 Dam MTase 和 DpnI 内切酶为例，我们基于 MR-DNAzyme 策略开发了两种比色方法。第一种方法是利用工程化的发夹-DNAzyme 杂交探针，用于简便的 Dam MTase 活性开式检测，具有较宽的线性范围（6-100 U/mL）和较低的检测限（6 U/mL）。此外，该方法可以轻松扩展以分析限制内切酶的活性和抑制作用。第二种方法涉及基于甲基化触发的 DNA 酶的 DNA 机器，通过两步信号放大级联实现了对 Dam MTase 活性的超高灵敏检测（检测限=0.25 U/mL）。
