SDR-recycling signal amplification for highly sensitive methyltransferase activity assay

Abstract

In this work, a new electrochemical method for rapid and sensitive evaluation of DNA methyltransferase (MTase) activity based on DNA strand displacement reaction (SDR) recycling signal amplification strategy was developed. Briefly, DNA adenine methyltransferase (Dam MTase) can catalyze the methylation of adenine into N6-methyladenine adenine (m6A) in the specific 5′-GATC-3′ of the hairpin DNA probe 1 (H1). Methylated H1 can be specifically recognized and cleaved by the restriction endonuclease DpnI, which releases a unstable loop DNA and quickly converted to a single-stranded DNA (S1). Then S1 can hybridize with hairpin DNA probe 2 (H2) on the electrode and open the hairpin structure of H2, and hairpin DNA probe 3 (H3) in the solution would hybridize with H2 to form a double-strand DNA (dsDNA), freeing S1 to trigger another reaction cycle and generating a significant response signal. The results demonstrated that the developed method could be used in highly sensitive detection of Dam MTase with a low detection limit of 0.03UmL−1. Furthermore, the proposed method suggested that 5-Fluorouracil could inhibit the Dam MTase activity with the half maximal inhibitory concentration (IC50) of 0.6μM, implying its promising application in screening of suitable inhibitors for Dam MTase.