Simple and convenient G-quadruplex-based fluorescence assay of micrococcal nuclease activity

Abstract

As the extracellular nuclease of Staphylococcus aureus (S. aureus), micrococcal nuclease (MNase), which preferentially digests single-stranded nucleic acids, can be used as the standard to identify S. aureus and can be used to evaluate the pathogenicity of S. aureus. So the assay of MNase is of high importance. However, traditional methods for the assay of MNase activity have intrinsic limitations such as sophisticated synthesis processes, the need for functionalizing thiol (or dye)-modified oligonucleotide probes or critical operation conditions. Herein, a simple and convenient fluorescence sensing platform for MNase activity has been developed based on N-methyl mesoporphyrin IX (NMM)/G-quadruplexes. In the absence of MNase, the G-rich single stranded DNA (ssDNA) folds into a quadruplex in the presence of monovalent ions, thus greatly enhancing the fluorescence of NMM (a specific G-quadruplex binder). In the presence of MNase, the G-rich ssDNA was digested into small fragments. As a result, the fluorescence of the solution decreases with increase of MNase activity. Under the optimized conditions, the fluorescence intensity exhibits a linear correlation to MNase concentration in a wide range of 1.2 × 10⁻⁴–2.4 × 10⁻³ units per mL with a detection limit of 7.1 × 10⁻⁵ units per mL and good selectivity. The detection limit is much better or at least comparable to previous reports. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays.