Efficient Nucleic Acid Detection by Templated Reductive Quencher Release

RNA-templated fluorescence activation is a nucleic acid detection strategy that offers the possibility of direct visual detection of genetic information in living cells. Here we describe a new reaction strategy for fluorescence activation in which a phosphine on one DNA probe reduces an azide group in a linker on a second probe, resulting in linker cleavage and release of a fluorescence quenching group. These "Q-STAR" probes are shown to yield a strong fluorescence turn-on signal in approximately 20 min, with very low background and substantial amplification by turnover on the template. A green/red pair of such probes allowed the discrimination of two bacterial species by a single nucleotide difference in their 16S rRNA. The beneficial properties of the reductive quencher release design make these probes promising candidates for widespread application in the detection of nucleic acids in vitro and in cells.