A signal-on fluorescence based biosensing platform for highly sensitive detection of DNA methyltransferase enzyme activity and inhibition

摘要

DNA methylation mediated by DNA methyltransferase (MTase) enzyme is internal cell mechanism which regulate the expression or suppression of crucial genes involve in cancer early diagnosis. Herein, highly sensitive fluorescence biosensing platform was developed for monitoring of DNA Dam MTase enzyme activity and inhibition based on fluorescence signal on mechanism. The specific Au NP functionalized oligonucleotide probe with overhang end as a template for the synthesis of fluorescent silver nanoclusters (Ag NCs) was designed to provide the FRET occurrence. Following, methylation and cleavage processes by Dam MTAse and DpnI enzymes respectively at specific probe recognition site could resulted to release of AgNCs synthesizer DNA fragment and returned the platform to fluorescence signal-on state through interrupting in FRET. Subsequently, amplified fluorescence emission signals of Ag NCs showed increasing linear relationship with amount of Dam MTase enzyme at the range of 0.1-20 U/mL and the detection limit was estimated at 0.05 U/mL. Superior selectivity of experiment was illustrated among other tested MTase and restriction enzymes due to the specific recognition of MTase toward its substrate. Furthermore, the inhibition effect of applied Dam MTase drug inhibitors screened and evaluated with satisfactory results which would be helpful for discovery of antimicrobial drugs. The real sample assay also showed the applicability of proposed method in human serum condition. This novel strategy presented an efficient and cost effective platform for sensitive monitoring of DNA MTase activity and inhibition which illustrated its great potential for further application in medical diagnosis and drug discovery. (C) 2019 Elsevier B.V. All rights reserved.