Administrative Supplement for a Cytosurge FluidFM OMNIUM instrument: The RNA nanomachines of the gene expression machinery dissected at the single molecule level

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PROJECT SUMMARY: This supplement will critically update the instrumentation available for several aspects of the parent R35 MIRA award, entitled The RNA nanomachines of gene expression dissected at the single molecule level. The most critical features of the proposed instrumentation, the Cytosurge FluidFM OMNIUM, are its versatility, turnkey readiness, and ease of use for injecting, sampling and manipulating the content of live mammalian cells. These features will dramatically facilitate access by the diverse group of postdoctoral fellows, graduate students and undergraduate students in the PI's group to especially our intracellular single molecule fluorescence microscopy tools, while also empowering for the first time parallel single live cell transcriptome analyses. The injection capability of the FluidFM OMNIUM will be critical for upgrading our more difficult, aging single mammalian cell microinjector for use in the parent grant, soon to be renewed. Our MIRA grant aims to dissect the mechanisms of the nanoscale RNA machines of gene expression at the single molecule level, so far focusing on bacterial riboswitches and the yeast spliceosome. Acquisition of the FluidFM OMNIUM will empower us to include the intracellular miRNA-guided silencing machinery for the renewal. Building on our group's 23-year expertise in this space, we aim to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new functional RNAs. 2.) Push the limits of our approaches to be able to probe increasingly complex biological contexts and mechanisms since unexpected discoveries often await where individual RNA nanomachines interact. In pursuit of these aims, we will address the unifying hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, as exemplified by the fact that transient miRNA:mRNA interactions lead to the regulation of protein translation in mammalian cells. Demonstrating the power of our scientific approach to address this hypothesis, we have developed a suite of intracellular single particle tracking approaches that depend on microinjecting fluorophore labeled RNAs at defined time points, which will be uniquely enabled by the FluidFM OMNIUM. To reveal the RNA-guided gene regulatory processes of the cell, we will mechanistically probe the dynamics of miRNA-guided gene silencing complexes using a tailored combination of single cell microinjection and intracellular single molecule fluorescence microscopy. A major bottleneck in these pursuits so far has been the steep learning curve associated with our microinjection-equipped microscopes that keep new group members from making significant contributions until they have completed 1-2 years of training. We anticipate that addition of the Cytosurge FluidFM OMNIUM system to our microscopy arsenal will transform the speed of our progress in intracellular single particle tracking and functional analysis by introducing an easy-to-use cell microinjection and sampling instrument that postdocs, graduate and undergraduate students can quickly use independently.