期刊
ANALYTICAL CHEMISTRY
卷 86, 期 4, 页码 2117-2123出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac403716g
关键词
-
资金
- Science and Engineering Research Council of the Agency for Science, Technology and Research (A*STAR) through the A*STAR-ANR program
- Ministry of Education, Republic of Singapore
A simple and highly sensitive electrochemical DNA methyltransferase (MTase) activity assay is presented in this report. The assay employs the electrocatalytic oxidation of ascorbic acid (AA) by a threading intercalator (N,N'-bis(3propylimidazole)-1,4,5,8-naphthalene diimide (PIND) functionalized with electrocatalytic redox Os(bpy)(2)Cl+ moieties (PIND-Os)). Briefly, a double-stranded DNA (ds-DNA) containing the symmetric sequence of 5'-CCGG-3' is first immobilized on a gold electrode. The electrode is then incubated with M.SssI CpG methyltransferase (M.SssI MTase) which catalyzes the methylation of the specific CpG dinucleotides, and the electrode is subsequently treated with a restriction endonuclease HpaII which recognizes the 5'-CCGG-3' sequence. Once the CpG site in the 5'-CCGG-3' is methylated, HpaII recognition is blocked. Higher M.SssI MTase activity leads to more CpG sites being methylated and consequently impedes more the restriction endonuclease HpaII digestion process. Thus, a larger amount of ds-DNA remains on the electrode surface after the HpaII. treatment. Thereafter, the electrode is incubated with PIND-Os during which PIND-Os specifically inserts itself between base pairs of ds-DNA and catalyzes the electrooxidation of AA. The methylation event corresponding to the MTase activity can therefore be monitored and amplified by the electrocatalytic oxidation of AA. A linear correlation between the catalytic oxidation current of AA and the activity of M.SssI MTase ranged from 0 to 120 U/mL with a current sensitivity of 0.046 mu A mL U-1 is obtained. The inhibitor screening ability of the developed MTase activity assay is also demonstrated.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据