Micrococcal nuclease (MNase) is a naturally-secreted nucleic acid degrading enzyme with important role in the spread of the bacteria in an infected host. The content of MNase can be used to estimate the pathogenicity of Staphylococcus aureus (S. aureus). A fluorometric method is described here for determination of the activity of MNase and for identification of S. aureus using DNA templated fluorescent copper nanoclusters (CuNC). A double-stranded DNA (dsDNA) with AT-rich regions and protruding 3'-termini was identified as a high-selectivity substrate for MNase and as a template for CuNC. In the absence of MNase, the long AT-rich dsDNA templates the formation of CuNC that display bright yellow fluorescence, with excitation/emission peaks at 340/570 nm. However, the substrates are enzymatically digested to mononucleotides or short-oligonucleotide fragments, which fail to synthesize fluorescent CuNC. The method works in the 1.0 × 10-3 - 5.0 × 10-2 U mL-1 MNase activity range, has a 1.0 mU mL-1 detection limit, and is highly selective over other exonucleases. The assay was successfully applied to the detection of MNase secreted by S. aureus and to the identification of S. aureus. Graphical abstract A smart dsDNA, with AT-rich regions and 3'-protruding termini, is screened as the high-selectivity substrate for MNase and template for the formation of copper nanoclusters (CuNC). A method is described for determination of the activity of MNase and for identification of S. aureus via smart DNA templated formation of fluorescent CuNCs.
Keywords: Copper nanoclusters; High-selectivity; Nucleases; Staphylococcus aureus.