A sensitive strategy for the fluorescence detection of DNA methyltransferase activity based on the graphene oxide platform and T7 exonuclease-assisted cyclic signal amplification

In this work, a simple fluorescence strategy based on the graphene oxide (GO) platform and T7 exonuclease (T7 Exo)-assisted cyclic signal amplification is developed for the fast and sensitive detection of DNA methyltransferase (MTase) activity and inhibition. In the sensing design, Dam MTase was used as a model analyte. In the presence of Dam MTase, a hairpin probe (HP) was methylated, and then specially recognized and cleaved by Dpn I endonuclease, releasing a ssDNA fragment. The released ssDNA subsequently hybridized with a FAM-labeled signal probe (DP) to form a duplex with a blunt 5'-terminal of DP and a 4-mer overhang at the 5'-end of the released ssDNA. This would trigger the T7 Exo-assisted cyclic signal amplification by repeating the hybridization and digestion of DP, liberating the fluorophore. The liberated fluorophore could not be adsorbed on the GO surface due to low affinity and the fluorescence signal was retained. In contrast, no enzymatic degradation of the DP occurred in the absence of Dam MTase. Thus the intact DP was then adsorbed on the GO surface, resulting in fluorescence quenching. By combining the efficient digestion ability of T7 Exo and the super fluorescence quenching efficiency of GO, the present strategy exhibits a high signal-to-background ratio, providing a satisfying sensitivity for the Dam MTase activity assay. In addition, this method does not require a specific recognition sequence for enzymatic cyclic amplification and dual labels with fluorophore/quencher pairs, making the design easy and low cost. Furthermore, the proposed method was also applied to assay the inhibition of Dam MTase activity. This approach may offer potential applications in clinical diagnostics, drug screening and some other related biomedical research.