Fluorescent Biosensor Based on Hairpin DNA Probe with Endonuclease Cleavage Inhibition for Rapid Detection of Dam Methyltransferase Activity

DNA adenine methyltransferase (Dam MTase) is a key epigenetic biomarker linked to bacterial pathogenicity and tumor progression, holding great value for disease diagnosis and drug discovery. Conventional Dam MTase detection approaches suffer from complex workflows, long assay times, limited sensitivity and unsatisfactory anti-interference ability, severely hindering their real-world implementation. Herein, a facile fluorescent biosensor was constructed for rapid Dam MTase activity detection by integrating a hairpin DNA probe, endonuclease cleavage inhibition and isothermal cyclic amplification. In the absence of Dam MTase, Nt.AlwI specifically cleaves the engineered hairpin probe to trigger amplification and generate intense fluorescence. Conversely, Dam-induced DNA methylation prevents enzymatic cleavage, suppresses amplification and reduces fluorescence output, where fluorescence intensity varies with Dam MTase activity in a concentration-dependent fashion. Experimental validation confirms high specificity and favorable anti-interference performance of this sensor under complex biological conditions. It enables inhibitor screening and reliable measurement of endogenous Dam MTase activity in bacterial samples. With simple configuration, easy manipulation and low cost, the biosensor addresses major drawbacks of classic assays and provides a robust analytical platform for epigenetic studies, clinical bacterial testing and high-throughput targeted-inhibitor screening.