Quantitation of DNA methyltransferase activity via chronocoulometry in combination with rolling chain amplification

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. Sssl MTase, the methylated CpG site in the unmethylated double DNA helix structure could be specifically recognized and cleaved by Hpall, 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. Sssl MTase was assayed in the range of 0.5-60 U/mL with a detection limit of 0.09 U/mL (S/N=3). In addition, the inhibition of procaine and epicatechin on M. Sssl 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. (C) 2016 Elsevier B.V. All rights reserved.