Methylation-sensitive transcription-enhanced single-molecule biosensing of DNA methylation in cancer cells and tissues

As a major epigenetic modification, DNA methylation participates in diverse cellular functions and emerges as a promising biomarker for disease diagnosis and monitoring. Herein, we developed a methylation-sensitive transcription-enhanced single-molecule biosensor to detect DNA methylation in human cells and tissues. In this biosensor, a rationally designed transcription machine is split into two parts including a promoter sequence (probe-P) for initiating transcription and a template sequence (probe-T) for RNA synthesis. The presence of specific DNA methylation leads to the formation of full-length transcription machine through sequence-specific ligation of probe-P and probe-T, initiating the synthesis of abundant ssRNA transcripts. The resultant ssRNAs can activate CRISPR/Cas12a to catalyze cyclic cleavage of fluorophore- and quencher-dual labeled signal probes, resulting in the recovery of the fluorophore signal that can be quantified by single-molecule detection. Taking advantages of the high-fidelity ligation of split transcription machine and the high efficiency of transcription- and CRISPR/Cas12a cleavage-mediated dual signal amplification, this single-molecule biosensor achieves a low detection limit of 337 aM and high selectivity. Moreover, it can distinguish 0.01% methylation level, and even accurately detect genomic DNA methylation in single cell and clinical samples, providing a powerful tool for epigenetic researches and clinical diagnostics.