An Evolutionary Approach for Development of Novel Riboswitches with Desired Dose-Response Curve

Various riboswitch-based genetic devices have been developed and applied to diverse fields such as live-cell imaging and screening for metabolite production. Since riboswitches should have distinct dose-response curves to meet the requirements for different applications, they have been redesigned by rational approaches based on sequence and structural information. However, rational approach is applicable only to few riboswitches whose structure and regulatory mechanism are well understood. Therefore, we developed an evolutionary approach for riboswitch engineering that is independent of preliminary knowledge about the riboswitch. First, we obtained naringenin (NRN) aptamer library by systematic evolution of ligands by exponential enrichment (SELEX), and constructed NRN riboswitch library composed of the aptamer library, random linker sequences, selection marker gene (tetA), and reporter gene (sgfp). Then we performed enrichments by dual selection in low, medium and high NRN concentrations. We chose enrichment conditions by investigation of the relationship between expression level of tetA and resistance to selection pressures (tetracycline or nickel ion concentration). We measured the dose-response curves of the enriched NRN riboswitches, and half maximal effective concentration (EC₅₀) of riboswitches gradually increased as the NRN concentration of enrichments. In conclusion, we established a novel approach for construction of riboswitches that have desired dose-response curves from scratch.