(582n) Quantitative Tailoring of RNA Device Activity for Conditional Control of Cell Fate

RNA is a versatile substrate for encoding sensing, processing, and control functions within cells. Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions. The ability to quantitatively tailor device regulatory activities will support broader implementation in cellular networks by effectively matching activities of circuit components.

Strategic genes candidates to regulate using programmable controllers possess activities that function as key regulation points in cellular decision making, including threshold activities that trigger phenotypic switching. Modulating gene expression across this threshold using a ligand-responsive RNA switch provides access to both phenotypes, based solely on ligand availability.

We developed a high-throughput, quantitative, cell-based screening strategy to support rapid generation of ribozyme-based RNA switches with user-specified regulatory activities. A dual reporter system was implemented to resolve device regulatory activity from cellular noise, enabling clean isolation of devices with targeted activities. Device libraries were generated through sequence randomization of individual components, from which we isolated switches with enhanced regulatory activities, including greater than tenfold increases in catalytic activity. Furthermore, our screening strategy is capable of enriching a single functional sequence from over a million variants to near purity in two sorting rounds.

We targeted a negative regulator of the yeast mating MAPK pathway to conditionally direct cellular behavior to either a mating or nonmating fate in response to an environmental trigger molecule. A series of RNA devices with varying regulatory activities were tested to locate the negative regulator’s threshold activity. Implementation of an RNA switch with improved regulatory stringency that was isolated using our cell-based screen resulted in distinct switching in gene expression, mating pathway activation, and cell cycle arrest phenotype. The ability to match regulatory activities of genetic control elements to thresholds activating phenotypic switching enables rewiring of biological networks and control of cellular decision making in response to user-defined molecular signals.