One-step digestion-ligation-activation universal strategy for ultrasensitive detection of DNA methylation

DNA methylation and its associated methyltransferases play pivotal roles in epigenetic regulation and are regarded as important biomarkers for early cancer diagnosis; however, the development of a universal platform capable of sensitively detecting both targets remains challenging. Herein, we report a one-step digestion-ligation-activation universal strategy integrating rolling circle extension-assisted loop-mediated isothermal amplification with CRISPR/Cas12a (DL-RLAMP/Cas12a) for ultrasensitive methylation analysis. In this design, a padlock probe hybridizes at CpG sites and undergoes simultaneous digestion and ligation, enabling methylation-dependent circular DNA formation. The use of methylation-sensitive restriction endonucleases (HhaI/BstUI) ensures selective survival of methylated or methyltransferase-modified targets, which subsequently initiate RLAMP amplification to generate abundant double stem-loop DNA structures for Cas12a-mediated trans-cleavage signal amplification. By integrating multistep reactions into a single workflow, the proposed strategy achieves efficient cascade amplification while maintaining an ultralow background signal. The assay discriminates methylation levels down to 0.1% and enables sensitive detection of CpG methyltransferase (M.SssI) with a detection limit of 6.92 × 10^-4 U mL^-1. Furthermore, the platform demonstrates applicability in cellular methylation analysis and methyltransferase inhibitor screening. This DL-RLAMP/Cas12a system provides a versatile and highly sensitive analytical framework for epigenetic biomarker detection and holds promise for early disease diagnostics.