Abstract
The engineering of synthetic biological systems requires precise, modular, and scalable components for sensing and responding to intracellular metabolic fluxes. While natural riboswitches provide a robust framework for gene regulation, their limited repertoire of ligands necessitates the development of de novo design strategies. This study presents a comprehensive computational pipeline for the creation of synthetic riboswitches tailored for metabolite sensing in Saccharomyces cerevisiae. By integrating automated RNA design algorithms with ligand-docking simulations, we generated a library of small-molecule-responsive aptamers targeting non-native metabolites. Our methodology utilizes the 'Capture-SELEX' framework combined with thermodynamic modeling to ensure high-affinity binding and conformational switching. We successfully identified several de novo candidates that exhibit significant fold-induction in response to exogenous ligands, with binding affinities in the low micromolar range. These synthetic switches were integrated into the yeast genome to control the expression of fluorescent reporters, demonstrating their utility as real-time biosensors. The results indicate that computational de novo design can effectively bypass the limitations of natural evolution, offering a versatile platform for metabolic engineering and dynamic pathway control. This work establishes a foundation for the systematic development of RNA-based sensors for a wide array of industrial and therapeutic metabolites, providing a scalable alternative to protein-based sensing systems in eukaryotic hosts.