• 文献标题:   Methyltransferase activity assay based on the use of exonuclease III, the hemin/G-quadruplex system and reduced graphene oxide on a gold electrode, and a study on enzyme inhibition
  • 文献类型:   Article
  • 作  者:   YANG ZQ, XIE LY, YIN HS, ZHOU YL, AI SY
  • 作者关键词:   electrochemical bioassay, differential pulse voltammetry, hemin, gquadruplex, dna adenine methyltransferase, dna methylation
  • 出版物名称:   MICROCHIMICA ACTA
  • ISSN:   0026-3672 EI 1436-5073
  • 通讯作者地址:   Shandong Agr Univ
  • 被引频次:   10
  • DOI:   10.1007/s00604-015-1645-y
  • 出版年:   2015

▎ 摘  要

We describe an electrochemical bioassay for the detection of the activity of methyltransferase (MTase), and for screening this enzyme's inhibitors. The assay is based on the conjugation of a hemin to a G-quadruplex that enables enzymatic signal amplification with the aid of exonuclease III (ExoIII). In the first step, double-stranded DNA containing the quadruplex-forming oligomer is assembled on the surface of a gold electrode and then methylated by DNA adenine methyltransferase (DAM). After cleaved by endonuclease DpnI, the methylated DNA is digested by ExoIII and the quadruplex-forming oligomers are liberated. This leads to the formation of a hemin/G-quadruplex (in presence of hemin and of potassium ions). The hemin/G-quadruplex catalyzes the oxidization of hydroquinone by H2O2 and the benzoquinone was formed to generate electrochemical signal. Finally, the gold electrode modified with reduced graphene oxide was used as working electrode for performing differential pulse voltammetry. The method has a detection limit of 0.31 unit center dot mL(-1). A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 mu M.