Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.