title: Quantitation of DNA methyltransferase activity via chronocoulometry in combination with rolling chain amplification.
abstract: In this paper, a rolling chain amplification (RCA) strategy was proposed for chronocoulometric detection of DNA methyltransferase (MTase) activity. Briefly, after the double DNA helix structure was assembled on the surface of gold electrode, it was first methylated by M. SssI MTase and then RCA was realized in the presence of E. coli and phi29 DNA polymerase. Successively, numerous hexaammineruthenium (III) chloride ([Ru(NH3)6)(3+), RuHex) were adsorbed on replicons by electrostatic interaction and generated a large electrochemical readout, the signal was "on". On the contrary, in the absence of M. SssI MTase, the methylated CpG site in the unmethylated double DNA helix structure could be specifically recognized and cleaved by HpaII, resulting in a disconnection of RCA from the electrode. This led seldom RuHex to be absorbed onto the surface of electrode, the signal was "off". Based on the proposed strategy, the activity of M. SssI MTase was assayed in the range of 0.5-60U/mL with a detection limit of 0.09U/mL (S/N=3). In addition, the inhibition of procaine and epicatechin on M. SssI MTase activity was evaluated. When the proposed method was applied in complex matrix such as human serum samples, acceptable accuracy, precision and high sensitivity were achieved. Therefore, the proposed method was a potential useful mean for clinical diagnosis and drug development.
authors.affiliation: Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Ji
authors.affiliation: Key Laboratory of Environmental Medicine Engineering, Ministry of Education, 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: Liu
authors.affiliation: Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Key Laboratory of Environmental Medicine Engineering, Ministry of Education, 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: Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China. Electronic address: liusq@seu.edu.cn.
authors.forename: Songqin
authors.initials: S
authors.lastname: Liu
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans
chemical_list: D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases
keywords: Chronocoulometry; DNA methylation; Inhibition; Methyltransferase activity; Rolling chain amplification