title: Label-free electrochemical detection of methyltransferase activity and inhibitor screening based on endonuclease HpaII and the deposition of polyaniline.
abstract: Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III, as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5-0.6 U mL(-1) with the detection limit of 0.12 U mL(-1). G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.
authors.affiliation: Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China; Analytical & Testing Center of Nanjing Normal University, Nanjing 210023, PR China.
authors.forename: Linqun
authors.initials: L
authors.lastname: Zhang
authors.affiliation: College of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Wei
authors.affiliation: Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Gao
authors.affiliation: Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China. Electronic address: wei_wei98@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
authors.forename: Yuanjian
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
authors.forename: Songqin
authors.initials: S
authors.lastname: Liu
mesh_terms: D000814:Aniline Compounds; D015374:Biosensing Techniques / Q000379:methods*; D019175:DNA Methylation; D015342:DNA Probes; D021881:DNA, Catalytic; D015257:DNA-Cytosine Methylases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000097:blood; D019003:Deoxyribonuclease HpaII; D004353:Drug Evaluation, Preclinical; D055664:Electrochemical Techniques / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D057230:Limit of Detection
chemical_list: D000814:Aniline Compounds; D015342:DNA Probes; D021881:DNA, Catalytic; D004791:Enzyme Inhibitors; C416807:polyaniline; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII
keywords: DNAzyme; Electrochemistry; Methylation; Methyltransferase activity; Polyaniline