Bifunctional Probe Propelling Multipath Strand Displacement Amplification Tandem CRISPR/Cas12a for Ultrasensitive and Robust Assay of DNA Methyltransferase Activity

Abstract

Background

DNA methylation catalyzed by various DNA methyltransferases (DNA MTases) is one of the important epigenetic regulations in both eukaryotes and prokaryotes. Therefore, the detection of DNA MTase activity is a vital target and direction in the study of methylation-related diseases.

Results

In this study, an ultrasensitive and robust strategy was developed for DNA MTase activity sensing based on bifunctional probe propelling multipath strand displacement amplification and CRISPR/Cas12a techniques. First, a bifunctional hairpin probe (bHpDNA) was designed instead of a conventional single-function probe. In the presence of DNA MTase, the bHpDNA was methylated and cleaved by a restriction endonuclease into two independent primers, both of which bind with the templates to trigger strand displacement amplification and produce the active DNA of CRISPR/Cas12a. Second, annealing-assisted binding instead of free diffusion adhesion was used to improve hybridization efficiency between the primers and templates. Finally, the CRISPR/Cas12a system was used to achieve fluorescence signal output to analyze DNA MTase activity. If targets were absent, there was no signal because no primers were released from the bHpDNA. To verify the reliability of the method, two key DNA MTases, Dam and M.SssI, were analyzed, and their limits of detection were 2.458 × 10⁻³ and 3.820 × 10⁻³ U/mL, respectively, which were lower than those of most reported fluorescence methods.

Significance

This method was successfully used in the evaluation of DNA MTase inhibitors and the detection of DNA MTase activity in complex biological systems with good recoveries and relative standard deviation at low spiked concentrations (0.1–1 U/mL), which all indicate that this method is an ultrasensitive and robust strategy in DNA MTase activity assay and has great potential in biomedical and clinical detection.