Highly sensitive electrochemical methyltransferase activity assay

Abstract

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(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND) functionalized with electrocatalytic redox Os(bpy)2Cl+ 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 μA mL U-1 is obtained. The inhibitor screening ability of the developed MTase activity assay is also demonstrated. © 2014 American Chemical Society.