Dumbbell-shaped DNA topology drives self-sustaining CRISPR/Cas12a exponential amplification for ultrasensitive monitoring of DNA methyltransferase activity

DNA methylation is an essential epigenetic mechanism, and abnormal methylation has been linked to the onset and progression of many diseases, representing a potential threat to health. Monitoring DNA methyltransferase (MTase) activity is essential for understanding DNA methylation regulation and developing MTase-targeted inhibitors. To address this challenge, we developed a dumbbell-shaped DNA topology that drives self-sustaining (autocatalytic) CRISPR/Cas12a system for exponential signal amplification, enabling ultrasensitive fluorescent detection of DNA MTase activity. In this strategy, a DNA dumbbell topological structure (DDTS) is designed, in which two double-strand DNA (dsDNA) loops effectively block the activity of CRISPR/Cas12a. Upon Dam MTase presence, DpnI endonuclease cleaves the methylated recognition sites in the DNA probe, disrupting the DDTS topology to generate linear dsDNA activators. These activators restore the trans -cleavage of CRISPR/Cas12a, which further cleaves the single-stranded DNA (ssDNA) domain in DDTS probes to produce additional activators, creating an exponential amplification loop through autocatalysis. The system achieves a detection limit of 6.37 × 10 −4 U/mL for Dam MTase, with a linear range of 1 × 10 −3 to 15 U/mL, and shows excellent selectivity over other MTases and nucleases. It also enables inhibitor screening, with the half-maximal inhibitory concentration (IC 50 ) value of 1.84 μM for 5-fluorouracil. Therefore, the method has great potential for application in the early diagnosis of diseases and drug discovery.