title: Electrochemical immunosensing platform for DNA methyltransferase activity analysis and inhibitor screening.
abstract: 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 H(2)O(2) and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation by H(2)O(2) 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 μM S-adenosyl-L-methionine as methyl donor, 5-aza-2'-deoxycytidine, procaine, epicatechin, and caffeic acid could inhibit the M. SssI MTase activity with the IC(50) values of 45.77, 410.3, 129.03, and 124.2 μM, respectively. Therefore, this study may provide a sensitive platform for screening DNA MTase inhibitors.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, Taian, 271018, PR China.
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.forename: Zhenning
authors.initials: Z
authors.lastname: Xu
authors.forename: Lijian
authors.initials: L
authors.lastname: Chen
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004563:Electrochemistry / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases