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

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 H 2 O 2 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 · mL −1 . A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 μM. We describe an electrochemical bioassay for the detection of the activity of methyltransferase and for screening for its inhibitors. Due to the conjugation of a hemin to a G-quadruplex, strong enzymatic signal amplification is enabled with the aid of exonuclease III.