title: An electrochemical assay for DNA methylation, methyltransferase activity and inhibitor screening based on methyl binding domain protein.
abstract: DNA methylation is one of important epigenetics events, and responsible to transcription, genomic imprinting and cellular differentiation. Aberrant DNA methylation is always contacted with various diseases. Methyl binding domain (MBD) proteins can specifically bind to the methylated CpG dinucleotides. Conventional assay for DNA methylation normally need bisulfide treatment, methylated nucleotide labeling or PCR amplification. Here, we fabricated a novel electrochemical biosensor for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor based on MBD protein and coomassie brilliant blue G250 (CBB-G250), where the electrochemical signal of CBB-G250 was used to monitor the methylation event. After the hybrids of DNA S1 and DNA S2 were treated with M. SssI MTase in the presence of S-adenosylmethionine, the MBD proteins were specifically conjugated to the methylation site of CpG dinucleotides, and then, the MBD proteins were stained with CBB-G250. The electrochemical signal of CBB-G250 increased linearly with increasing M. SssI MTase concentration in the range from 0.1 to 40 unit/mL. Furthermore, the inhibition investigation demonstrates that fisetin and chlorogenic acid can inhibit the M. SssI MTase activity with the IC(50) value of 153.12 and 137.07 μM, respectively. Therefore, we think that this study may provide a sensitive platform for screening of DNA MTase inhibitors.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.forename: Zhenning
authors.initials: Z
authors.lastname: Xu
authors.forename: Lijian
authors.initials: L
authors.lastname: Chen
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA; D015254:DNA Modification Methylases