Label-free fluorescent assay for real-time monitoring site-specific DNA cleavage by EcoRI endonuclease

Abstract

DNA cleavage reaction catalyzed by nucleases is essential in many important biological processes and medicinal chemistry. Therefore, it is important to develop reliable and facile methods to assay nuclease activity. With this goal in mind, we report a fluorescent assay for label-free, facile, and real-time monitoring of DNA cleavage by EcoRI endonuclease using SYBR Green I (SGI) as a signal probe. The fluorescence of SGI dramatically increased when the free SGI was mixed with double-stranded DNA (dsDNA) substrate. Upon interacting with EcoRI, which cleaves the dsDNA into small fragments, the weakened interaction between SGI and the shortened DNA fragments caused a decrease in fluorescence of SGI. EcoRI–DNA interaction was real-time studied by monitoring fluorescence change with the prolonging of interaction time. The important kinetic parameters, including Michaelis–Menten constant (K_M) and maximum initial velocity (V_max), were accurately calculated, which is consistent with previously reported studies. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis analysis, which indicated that this method is a simple and effective approach to assay DNA cleavage reaction. Specificity investigation demonstrated that EcoRI–DNA interactions can be studied with high selectivity. Compared with previously reported methods, this approach is selective, simple, convenient and cost-efficient without any labeling of the probe or of the target.