Amplified Detection of microRNA with a Novel Fluorescent Oligonucleotide Probe: microRNA:Argonaute Cleavage (miRACle) Beacons

This thesis concerns the development and exploration of a new class of fluorogenic microRNA sensors which exploit cellular processes to enable new capabilities. These sensors consist of a fluorophore and matching quencher, tethered to opposite ends of an oligonucleotide strand. Previously, miRNA sensors were simple adaptations from DNA-targeting sensors and relied on simple hybridization to achieve their specificity. MicroRNAs, however, are genetic sequences that are only one component of the multi-protein complex called the RNA-induced Silencing Complex (RISC), a major regulatory nexus for gene expression present in eukaryotic cells. This new class of sensor binds to a specific microRNA within RISC and undergoes enzymatic processing through the endonuclease Argonaute-2 to permanently activate fluorescence. This microRNA:Argonaute Cleavage (miRACle) beacon was investigated in a recombinant system and validated in solution with immunoprecipitated RISC. Fluorescence upon miRACle beacon activation increased 13-fold above hybridization and showed single nucleotide specificity. After validating the concept, the project split into two avenues of research. The first avenue was evaluating if the system could discern between frame-shifted miRNAs, termed isomiRs. To investigate this, common nuclease resistance modifications were shifted along the oligonucleotide strand to assess their impact on assay performance. Modifications within the catalytic site abrogated activity, demonstrating that miRACle beacons may detect frame shifted isomiRs correctly. The second avenue for miRACle beacons focused on bringing the assay into cells and resisting the processes that give false positive signals. Established strategies to prevent non-specific nuclear accumulation or nuclease degradation were screened to determine compatible modifications to the assay. A porphyrin dye, pyropheophorbide-a, could exclude the beacons from the nucleus in vitro, however no tested strategy definitively showed miRNA specific activation and stability to non-specific nucleases in vitroPh.D