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

Abstract

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-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001–1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Genome Mining and Biosynthesis Study of a Type B Linaridin Reveals a Highly Versatile α- N -Methyltransferase

“…The fluorescence from allyl-nisin-Tz was collected by the same objective and captured by an EMCCD camera. 19…”

Advances in single‐molecule fluorescent nanosensors

“…(b) QD‐based single‐molecule fluorescent nanosensor for M.SssI activity detection. Reprinted with permission from H. Zhang, Zhang, Yao, et al (2019). Copyright 2019 American Chemical Society.…”

“…The above nanosensors require specific restriction endonuclease for target DNA methyltransferase signal recognition, which is relatively complicated and expensive. Alternatively, Liu et al introduced a QD‐based single‐molecule fluorescent nanosensor to directly detect M.SssI using antibody for target recognition (H. Zhang, Zhang, Yao, et al, 2019). As shown in Figure 4(b), a double‐strand DNA (dsDNA) is designed as a detection probe and it contains the M.SssI recognition sites.…”

Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay

“…The assays based on restriction enzymes are not sensitive to DNA hemi-methylation because the cleavage efficiency by methylation-dependent nucleases such as DpnI is compromised for hemi-methylated DNA substrate [ 36 ]. Methylated cytosines (methylcytosines) can be recognized alternatively by methyl-specific antibodies [ 31 , 37 , 38 ], but the antibody-based methods are dependent upon the sensitivity and specificity of antibodies.…”

“…Moreover, the preparation of special nanoparticles, enzymes, and a battery of reagents, often required for such assays, add complexity to these methods. Several single-molecule approaches such as solid-state nanopore technology and single-molecule fluorescence microscopy have been applied and have exhibited the fast and precise detection of methylcytosine in a CpG site with antibodies or methyl-binding proteins [ 38 , 43 , 44 , 45 ].…”