Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters

摘要

S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of Si nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC(12)-Ag NCs). In this assay, dC(12) can function as both the template for the stabilization of Ag NCs and the substrate of the Si nuclease. Fluorescent Ag NCs could be effectively formed using dC(12) as the template without Si nuclease. In the presence of S1 nuclease, dC(12) is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. Si nuclease with an activity as low as 5 x 10(-8) U mu l(-1) (signaloise = 3) can be determined with a linear range of 5 x 10(-7) to 1 x 10(-3) U mu l(-1). The promising application of the proposed method in Si nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the Si nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis. (C) 2014 Elsevier Inc. All rights reserved.