Homogeneous fluorescent biosensing method for DNA methyltransferase activity analysis and inhibitor screening based on highly efficient isothermal amplification

DNA methyltransferase (MTase) activity assay and its inhibitor screening are important for the diagnosis and treatment of methylation-related diseases. Herein, a label-free and highly sensitive biosensing method based on entropy-driven reaction and toehold-initiated rolling circle amplification (TIRCA) was developed for DNA MTase activity detection and inhibitor screening. Briefly, in the presence of MTase, the triple-stranded complex (TSC) could be methylated to avoid cleaving by MboI endonuclease. The complete TSC was able to initiate downstream entropy-driven reaction and TIRCA to produce G-quadruplex that can bind with Thioflavin T (ThT) to output fluorescent signal. Under the optimal experimental conditions, the established biosensing strategy could detect Dam MTase down to 0.06 U/mL with a linear range from 0.1 U/mL to 40 U/mL. Importantly, the signal-to-noise (S/N) of this biosensing strategy was extremely high. This strategy could discriminate target Dam MTase from another two MTases efficiently. Moreover, this developed biosensing method was applied to screen DNA MTase inhibitors. Therefore, we anticipate this unique strategy will serve as an alternative tool to detect DNA MTase activity and screen its inhibitors in clinical diagnosis and therapeutics.