A sensitive cyclic signal amplification fluorescence strategy for determination of methyltransferase activity based on graphene oxide and RNase H

Abstract

This study describes a novel fluorometric method for the determination of methyltransferase (MTase) activity in DNA adenine methylation (Dam) by using graphene oxide (GO) and ribonuclease H (RNase H)-assisted signal amplification. In the presence of DNA adenine methylation methyltransferase (Dam MTase) and DpnI, the hairpin probe containing 5′-GATC-3′ as the recognition site is methylated by Dam MTase. Then, it is cleaved by methylation-sensitive restriction enzyme DpnI, and a single-stranded DNA (ssDNA) is released. In the next step, the released ssDNA hybridizes with FAM-labeled RNA (F-RNA) to generate DNA/RNA duplexes. Due to the specific ability of RNase H to hydrolyze RNA in RNA/DNA double strands, the F-RNA is digested by RNase H and the FAM fluorophore is released. Because this molecule is adsorbed on the GO surface, the fluorescence signal remains high. RNase H-assisted cyclic signal amplification is achieved by repeating the hybridization of ssDNA/F-RNA and digestion of F-RNA, a strategy allowing the fluorescence signal to be significantly amplified. The hairpin probe cannot be cleaved by DpnI and individual F-RNA cannot be degraded by RNase H without Dam MTase. As a result, the F-RNA is adsorbed by GO, resulting in quenching the fluorescence. Under the optimum conditions, the fluorescence intensity is linearly correlated in a concentration range of 0.1 to 10 U mL⁻¹ with a low detection limit of 0.06 U mL⁻¹. In addition, this approach proved to be trustworthy based on a successful application in inhibitor screening and determination of Dam MTase activity in human serum samples.