Engineering a Programmable Split-Ribozyme Platform for Detecting RNA in Living Cells
Synthetic Biology Engineering Evolution Design SEED
2021
2021 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Poster Presenters - Accepted
Intracellular RNA detection technologies represent a powerful, yet relatively underexplored, area of synthetic biology. With the detection of viral infection, disease states, and antibiotic resistance critical to our ability to combat some of the greatest problems we are currently facing, methods for robust detection of specific RNA in cells are more important than ever. While a few technologies that link RNA signals to biomolecular outputs have emerged, the field has yet to converge on a tool that is easily customized to different endogenous RNAs and that can be paired with virtually any biomolecular output of choice. To address these issues, we have developed a novel plug-and-play RNA detection platform. This platform uses synthetic split ribozymes which, in the presence of a user-specified RNA target input, produces an mRNA output that is translated into protein. Because sensing is achieved through designable base-pairing interactions between target RNA and guide RNAs that are appended onto the split ribozyme, redesign of sensors for new targets is simple and straightforward. To deliver this system, I first identified the optimal ribozyme split site that yields a high-dynamic range of sensing, where we observed no detectable output protein expression in the absence of input RNA. I then established guide RNA design rules that allow for more predictable design of new sensors, and developed a modular approach whereby any protein output can be rapidly exchanged for another without system re-engineering. Finally, I demonstrated the ability of this system to sense cellular RNAs, thereby creating sensors for dynamic detection of cell state and phenotype. Future work will focus on improving generalizability by implementing this platform in different cell types and using it for diagnostic and therapeutic applications in biotechnology.