(716a) A Plug-and-Play RNA Detection Platform for Sensing Cell Physiology and Phenotype

Cells respond to environmental and intracellular signals by modulating the levels of a myriad of proteins that ultimately determine cell physiology and phenotype. Regulatory mechanisms that operate at the transcriptional level (RNA) represent a major control point and provide blueprints to translate instructions contained in the genome into proteins. Detection of RNA levels thus provides a unique and critical readout of a cell’s physiologic status and phenotype. Moreover, integration of RNA sensing capabilities in the context of synthetic genetic programs, would enable advanced cellular programming in which engineered functions can be dynamically coupled with cell state to achieve feedback control to improve overall performance. However, technologies that link the detection of cellular RNAs in living cells, to the control of biomolecules are currently lacking. To address this, we present a genetically encoded plug-and-play RNA detection platform that can be programmed to sense user-specified cellular RNAs and in response, produce user-defined biomolecular outputs (i.e. RNAs or proteins). The basis for this system is a synthetically split-splicing ribozyme that has been engineered to be non-functional when present alone. However, in the presence of a user-specified RNA input, the ribozyme is able to bind to this RNA and reconstitute a functional ribozyme, resulting in splicing of the attached RNA exons. RNA exons can be designed so that splicing results in formation of a mature mRNA, resulting in protein production upon RNA detection. This talk will highlight our work in creating and optimizing this technology by harnessing concepts and technical approaches from protein engineering, to engineer a large and structurally complex catalytic RNA.