A label-free fluorescence sensing strategy based on GlaI-assisted EXPAR for rapid and accurate quantification of human methyltranferase activity.

DNA methylation plays an important role in epigenetic modification. DNA methyltransferase (DNMT) is essential in the DNA methylation process, and its abnormal expression is closely related to cancer. In this study, we propose a novel biosensor platform (DS-GlaI-EXPAR) that combines hemi-methylated double-stranded DNA (dsDNA) as the substrate for DNMT1 with GlaI-assisted isothermal exponential amplification reaction (EXPAR) for rapid, simple, and sensitive detection of DNMT1 activity. The hemi-methylated dsDNA is fully methylated by DNMT1, and GlaI recognizes and cleaves the fully methylated sequence, generating terminal fragments that trigger EXPAR for efficient signal amplification. Whereas hemi-methylated dsDNA without DNMT1 will keep intact and cannot initiate EXPAR. DNMT1 activity can therefore be sensitively quantified by the real-time fluorescence signal of the DS-GlaI-EXPAR platform. The high-efficiency amplification of EXPAR and the recognition of GlaI enable the platform to overcome the inherent cumbersome and time-consuming shortcomings of traditional methods while meeting specificity and sensitivity. This DS-GlaI-EXPAR platform offers an impressively low limit of detection of 0.86 pg/μL and the entire detection process can be completed in a short time of 2.5 h in a single tube. Furthermore, DNMT1 activity detected by this platform in MCF-7 cells was significantly higher than that of HEK293 cells, and the inhibition of Apt. #9 was verified. This DNMT1 activity detection platform is very convenient and effective for the discovery of inhibitors and early cancer diagnosis.