A High-Affinity Methyl-CpG-Binding Protein for Endonuclease-Free and Label-Free DNA Methyltransferase Activity Detection

DNA methyltransferase (MTase) activity detection has received increasing attention as a promising biomarker and therapeutic target. However, most of these detection methods rely on endonuclease digestion and signal groups labeling. Herein, we present a novel platform for sensing DNA MTase activity that overcomes these limitations. Our approach is both endonuclease-free and label-free, utilizing a combination of a high-affinity streptavidin-methyl-CpG-binding domain (SA-MBD) protein and surface plasmon resonance (SPR) technology. The SA-MBD protein specifically recognizes a hairpin probe containing methylated CpG sites, which is treated with M.SssI MTase. This recognition event generates a corresponding SPR response signal. The limit of detection is as low as 0.016 U/mL, owing to the high-affinity of the SA-MBD protein. Notably, we have demonstrated the feasibility of our method for M.SssI MTase activity analysis in serum and inhibitor screening, which implies the potential prospects for biomedical research.