Generation of Orthogonally Selective Bacterial Riboswitches by Targeted Mutagenesis and In Vivo Screening

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Abstract

Riboswitches are naturally occurring RNA-based genetic switches that control gene expression in response to the binding of small-molecule ligands, typically through modulation of transcription or translation. Their simple mechanism of action and the expanding diversity of riboswitch classes make them attractive targets for the development of novel gene expression tools. The essential first step in realizing this potential is to generate artificial riboswitches that respond to nonnatural, synthetic ligands, thereby avoiding disruption of normal cellular function. Here we describe a strategy for engineering orthogonally selective riboswitches based on natural switches. The approach begins with saturation mutagenesis of the ligand-binding pocket of a naturally occurring riboswitch to generate a library of riboswitch mutants. These mutants are then screened in vivo against a synthetic compound library to identify functional riboswitch–ligand combinations. Promising riboswitch–ligand pairs are then further characterized both in vivo and in vitro. Using this method, a series of artificial riboswitches can be generated that are versatile synthetic biology tools for use in protein production, gene functional analysis, metabolic engineering, and other biotechnological applications.