Achieving a quantitative understanding of the transcriptome is critical to the development of RNA biology and RNA-based therapeutics. One of the main “road-blocks” to the advancement of these fields is the inability, at present, to dynamically track most RNA molecules in vivo. In order to achieve a quantitative understanding of RNA interactions in vivo, we propose to develop a new class of genetically encoded fluorescent probes that will be designed to dynamically track gene expression (mRNA), miRNA trans-interactions, riboswitch or hairpin-type cis-interactions, ribozyme, and any other class of ncRNA.
The probes will consist of large self-assembled RNA-FP complexes that will report RNA-RNA interaction dynamically via an engineered structural change that will be detected through a change in fluorescence. The signal will consist of a change in the light polarization, and will be detected by a novel implementation of a polarization Total Internal Reflection Fluorescence (polTIRF) microscope design, which will enable the detection of small structural changes within the nanoprobe at fast sampling rates.
In this proposal, I intend to develop, in bacteria, the first generation of these probes. Development of the probes and the imaging apparatus will take place simultaneously, in order to optimize both detection capability and the probe design. The development and demonstration of the probes' capabilities will be guided by three objectives, each divided into individual milestones and sub-projects. In particular, I intend to use this approach to quantitatively characterize synthetic hammerhead ribozyme trans interaction, cis-acting RNA thermosensors, and the Hfq RNA-protein complex. I believe that successful implementation of this combined molecular and imaging tool to quantitatively characterize the kinetics associated with RNA-RNA trans/cis-interactions will constitute a proof-of-principle to the broad applicability for our method to other RNA-based platforms.
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