Sensitive and versatile fluorescent enzymatic assay of nucleases and DNA methyltransferase based on a supercharged fluorescent protein

Abstract

DNA-related enzymes, including nucleases and DNA modifying enzymes, play key roles in DNA metabolism. Herein, a versatile platform using supercharged green fluorescence protein (ScGFP) as a recognition and signal output element was developed for homogeneous, turn-on, rapid, and sensitive detection of various DNA-related enzymes, including endonucleases, restriction nucleases, as well as DNA methyltransferase (MTase). This platform takes advantage of the DNA length-dependent binding affinity between ScGFP and DNA for assaying the cleavage activity of nucleases. The long negatively charged DNA probe (labelled with a quencher) can tightly bind to the positively charged ScGFP, and efficiently quench the fluorescence of ScGFP through fluorescence resonance energy transfer (FRET). After nuclease cleavage or the coupling reaction of DNA MTase/nuclease, the resulting short DNA segment with the quencher with significantly less negative charges loses the ability to be absorbed on ScGFP, causing the restored fluorescence and signal on detection. This method is a simple mix-and-read platform capable of screening enzyme inhibitors. Furthermore, the multiplex detection of various enzymes was realized by using a microplate reader. In principle, our platform presents a promising method for DNA-related enzymes-targeted biochemical research.