Reactive Quantum Dot-Based FRET Systems for Target-Catalyzed Detection of RNA

Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or an RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters such as semiconductor quantum dots (QDs) has the potential to improve the sensitivity of detection by enhancing fluorescence signals and allowing for very low probe concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye forming a thioester motif. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.