Molecular tools for controlling gene expression are essential for manipulating biological systems. One class of tools includes RNA switches that incorporate RNA-based sensors, known as aptamers. However, most switches reported to date are responsive to toxic molecules or to endogenous metabolites. For effective conditional control, switches must incorporate RNA aptamers that exhibit selectivity against such endogenous metabolites. We report a systematic approach which combines a rapid in vitro assay and an in silico model to support an efficient, streamlined application of aptamers into RNA switches. Model predictions were validated in vivo and demonstrate that the RNA switches enable selective and programmable gene regulation. We demonstrate the method using aptamers that bind the FDA-approved small molecule (6R)-folinic acid, providing access to new molecular targets for gene expression control and much-needed clinically relevant tools for advancing RNA-based therapeutics.