Single-Molecule Fluorescence Imaging for UltrasensitiveDNA Methyltransferase Activity Measurement and Inhibitor Screening

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently the streptavidin-labelled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labelled methylated dsDNA molecules, the single molecule imaging of methylated dsDNA molecules was recorded to quantify th...