DNA-templated silver nanocluster-based cleavable probes for nucleic acid detection.

Abstract

DNA or RNA probes that fluoresce upon enzymatic digestion, such as TaqMan probes and DNaseAlert, allow for not only nuclease activity detection but also nucleic acid amplification assays. However, relying on FRET as a signal transduction mechanism, these short oligonucleotide probes need to be dually labeled with a donor and a quencher, thus suffering from the issues of high cost and singly labeled impurities. Here we demonstrate a new class of probe, based on DNA-templated silver nanoclusters (DNA/AgNCs). These nanoclusters exhibit fascinating photophysical properties that depend on the arrangement of the nucleobases in the DNA ligand. Not using FRET, our probes not only turn on upon nuclease cleavage but display a significantly altered fluorescence color. Although a variety of DNA/AgNC probes exist to detect proteins and nucleic acids, no AgNC probes have been designed to detect nuclease activity (enzymes that cleave the bonds between the nucleotides of nucleic acids such as DNA). Due to their multicolor nature, our probes also enable ratiometric sensing for the highly quantitative detection of nuclease activities. Here we show that our silver cluster probes can replace DNaseAlert for the enzymatic activity assay of DNase I. The electrophoresis gel shows clear digestion of our silver cluster probes by DNase I and the digested products have a significantly enhanced fluorescence with 700% increase in intensity and different emission color (90 nm red-shift). In addition, our silver cluster probes can substitute the FRET probes in novel CRISPR-Cas assays (such as DETECTR and SHERLOCK) for nucleic acid detection. We demonstrate that we can selectively detect SARS-CoV-2 cDNA in combination with CRISPR-Cas12a. 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.