Highly sensitive and accurate detection of dam MTase activity via G-switch-activated AgNCs fluorescence

Abstract

DNA methyltransferases (MTases) play a critical role in epigenetic regulation and are closely associated with the occurrence and development of various diseases. To address the limitations of current detection methods, which often suffer from complicated operation and limited sensitivity we have developed a label-free fluorescent biosensing platform based on DNA-templated silver nanoclusters (DNA-AgNCs) for the detection of DNA adenine methyltransferase (Dam MTase) activity and the screening of its inhibitors. In this system, DNA-AgNCs signal probes are synthesized via a simple reduction reaction. Dumbbell-shaped DNA substrates are designed to be specifically methylated by Dam MTase in the presence of S-adenosylmethionine (SAM). The methylated substrates are subsequently recognized and cleaved by DpnI, yielding DNA fragments with 3'-OH termini. These termini are extended by terminal deoxynucleotidyl transferase (TdT) in the presence of dGTP, generating G-rich sequences, which hybridize with the DNA-AgNCs and significantly enhance the fluorescence intensity. This cascade reaction enables the sensitive detection of Dam MTase activity. Compared with the other approaches, the proposed method exhibits a wider linear range (0.5-10 U/mL) and a lower detection limit (0.165 U/mL). Furthermore, inhibition studies demonstrate that 5-fluorouracil and gentamicin effectively suppress Dam MTase activity. The method also shows excellent performance in complex biological samples. Overall, this fluorescence-based strategy offers high sensitivity and excellent specificity, demonstrating broad application prospects in epigenetic research and drug discovery.

Keywords: Biosensor; Dam MTase; Label-free detection; Silver nanoclusters.