Q-STAR Fluorogenic Probes

About

Abstract Stanford researchers have developed a novel linker that can be cleaved in a chemoselective manner under particularly mild conditions. This linker makes possible new, high-performance fluorogenic probes ("QSTAR probes") for the detection of nucleic acids in vitro and directly inside cells. The linker contains an alpha-azidoether functionality, which allows cleavage by mild reducing agents and terminal functional groups enabling derivatization and bioconjugation. For nucleic acids detection, a fluorophore-labeled oligonucleotide probe is modified with a quencher attached through the described azidoether linker. A second probe containing a reducing agent cleaves the linker when the two probes are hybridized at adjacent positions of the analyte nucleic acid. These probes enable the sensing and imaging of genetic targets with specificity sufficient for the discrimination of single nucleotide polymorphisms. Furthermore, the probes are amenable to multi-color detection and provide an amplified signal under isothermal conditions. This enables them to be used in complex mixtures of cells, and removes the need for nucleic acid isolation and amplification. The genetic sensing requires only one step and a few minutes' time. These probes are particularly suitable for detection of genetic markers directly in cells because the robustness of the azidoether-linker minimizes background signal. Cellular detection of RNAs opens numerous opportunities in molecular biology and clinical diagnostics: for example the direct genetic discrimination of bacterial pathogens, genetic analysis of fixed tissue specimens, and even the sorting of cells by genetic differences. A green/red pair of such probes allowed the discrimination of two bacterial species by a single nucleotide difference in their 16S rRNA. The described linker may find applications beyond the detection of RNA/DNA, in applications where gene-selective release of a reporter or a drug is required. Stage of Development This technology was a finalist in the Gates Foundation Grand Challenges competition. Technical details are provided in the Publications section below. Applications Food safety Genetic discrimination of pathogenic bacteria by direct visualization in one step Medical Diagnostics Discrimination of pathogens in patient samples Analysis of pathological tissue samples Genetic identification of specific alleles in vivo Molecular Biology Nucleic acid detection in PCR Identification and imaging of specific cellular RNAs with single nucleotide selectivity Chemically cleavable peptide-conjugates Reversible surface-attachment of molecules Fluorescence activated cell sorting based on genetic variations Advantages Much easier to use than PCR Analysis does not require purification or prior cell fixation Provides amplified detection Minimal background signal Rapid detection time Applicable in living cells Use of standard fluorescence equipment Cost-effective preparation