Electrochemical Immunosensing Platform for DNA Methyltransferase Activity Analysis and Inhibitor Screening

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• 作者：Mo Wang ; Zhenning Xu ; Lijian Chen ; Huanshun Yin ; Shiyun Ai
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：November 6, 2012
• 年：2012
• 卷：84
• 期：21
• 页码：9072-9078
• 全文大小：309K
• 年卷期：v.84,no.21(November 6, 2012)
• ISSN：1520-6882

文摘

In this paper, we developed a novel electrochemical method to quantify DNA methyltransferase (MTase) and analyze DNA MTase activity. After the double DNA helix structure was assembled on the surface of gold nanoparticle modified glassy carbon electrode, it was first methylated by M. SssI MTase and then digested by restriction endonuclease HpaII, which could not recognize the methylated CpG site. Successively, anti-5-methylcytosine antibody was specifically conjugated on the CpG methylation site and horseradish peroxidase labeled goat antimouse IgG (HRP-IgG) was conjugated on anti-5-methylcytosine antibody. In the detection buffer solution containing H₂O₂ and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation by H₂O₂ to generate benzoquinone, resulting in a highly electrochemical reduction signal. Consequently, the activity of M. SssI MTase was assayed, and DNA methylation was detected using the signal change with and without methylation. Furthermore, the inhibition investigation demonstrated that, in the presence of 160 渭M S-adenosyl-l-methionine as methyl donor, 5-aza-2鈥?deoxycytidine, procaine, epicatechin, and caffeic acid could inhibit the M. SssI MTase activity with the IC₅₀ values of 45.77, 410.3, 129.03, and 124.2 渭M, respectively. Therefore, this study may provide a sensitive platform for screening DNA MTase inhibitors.