Engineering Riboswitches <i>in Vivo</i> Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3–5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3–5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch