A Digestion-and-Turn-on Probe Based on DNA-Templated Silver Nanoclusters

DNA or RNA probes that fluoresce upon enzymatic digestion or cleavage, such as Taqman probes and DNaseAlert, play a key role in quantitative nucleic acid amplification tests and nuclease activity detection. However, relying on FRET as signal transduction mechanism, these short oligonucleotide probes require their two ends to be dually labeled with a fluorescence donor and a quencher. The need for purification at each labeling step makes these probes costly, and the required separation distance between the donor and the quencher (at least 5 nt) limits the fluorescence enhancement ratio of these probes upon digestion. Here we demonstrate a new class of “digestion-and-turn-on” probes based on DNA-templated silver nanoclusters (DNA/AgNCs). Not using FRET as fluorescence activation mechanism, our silver cluster probes are not only turned on upon nuclease cleavage, but display a significantly altered fluorescence color. We show that our silver cluster probes can replace DNaseAlert in the DNase I activity assay. The gel shift assay shows clear digestion of our silver cluster probes by DNase I and the digested products have a significantly enhanced fluorescence and altered emission color. In addition, our silver cluster probes can substitute the FRET probes in novel CRISPR-Cas assays (such as DETECTR and SHERLOCK) in the viral nucleic acid detection. Our fluorescent nanomaterial-based digestion-and-turn-on probes are easy to prepare (one-pot reaction at room temperature), low cost (no need of purification) and versatility (multicolor). Our results not only demonstrate that DNA-templated silver nanocluster probes can be a versatile nuclease activity probe, but also provide insights into the interactions between nucleases and metallo-DNA nanomaterials.