Breaking Riboswitch Speed Limits by Systematic Mutation and Orthogonal Base Pair Incorporation

Engineered RNAs have applications in diverse fields, from biomedical to environmental. In many cases, folding of the RNA is critical to its function. Here we describe a strategy to improve the folding kinetics of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal unnatural base pairs (UBP). Both natural and unnatural base pair mutants were found to improve biosensor response kinetics without compromising fold turn-on or ligand affinity. These strategies can be transferred to improve the performance kinetics of other RNA-based tools.