Principle of quantum dot (QD)-based target-templated RNA detection assay.
Source: BAM, dvision Biophotonics and Humboldt-Universität zu Berlin, Institute for Chemistry
Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes permit sensitive detection of nucleic acids by using DNA or RNA target as a catalyst of chemical reaction. OTRs are typically designed so that the reaction products fluoresce and can be detected fluorometrically. The overwhelming majority of OTR systems employ organic chromophores as fluorescent reporters. This allows straightforward synthesis of fluorescently labeled probes using a toolbox of commercially available dyes. However, such dyes have only moderate brightness, thereby limiting the achievable fluorescence output when the probes are applied at low concentrations, which leads to moderate limits of detection. The use of exceptionally bright semiconductor nanoparticles (quantum dots, QDs) in place of molecular emitters has the potential to boost the sensitivity of nucleic acid detection by providing higher signals at low probe concentration, thereby allowing the use of probes at ultralow concentrations. Herein we present an RNA-templated reaction between fluorescently labeled peptide nucleic acid (PNA) probes, which takes place on the surface of a polymer-functionalized CdSe/ZnS QD. The label acceptor PNA probe is immobilized on the QD and bears a cysteine residue at the N-terminus, while the label donor PNA probe is modified with a Cy5 dye attached via a thioester bond. We selected the QD and Cy5 dye so that the emission band of the QD overlaps with the absorption band of Cy5. The PNA probes are designed to hybridize adjacently with an RNA target, which brings the cysteine and thioester groups into proximity and triggers a native chemical ligation-like reaction between them. This results in Cy5 transfer onto the QD-immobilized PNA and indicates the presence of the RNA target. The progress of the RNA-catalyzed dye transfer can be conveniently monitored by measuring the intensity of the Cy5 emission resulting from the fluorescence resonance or Förster energy transfer (FRET) from the QD to the Cy5, which occurs upon excitation of the QD with visible light. This QD-based OTR can be performed in a conventional microtiter plate and read out using a fluorescence reader. Due to its simplicity and robustness the assay has the potential to be implemented in point-of-care testing strategies.
Reactive Quantum Dot-Based FRET Systems for Target-Catalyzed Detection of RNA
Oleksandr Zavoiura, Ute Resch-Genger, Oliver Seitz, published in Peptide Nucleic Acids. Methods in Molecular Biology, vol. 2105, pp 187-189, 2020
BAM Division Biophotonics