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Artificial Riboswitches

Volume 1111 of the series Methods in Molecular Biology pp 107-129

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

  • Helen A. VincentAffiliated withManchester Institute of Biotechnology and School of Chemistry, The University of Manchester
  • , Christopher J. RobinsonAffiliated withManchester Institute of Biotechnology and School of Chemistry, The University of Manchester
  • , Ming-Cheng WuAffiliated withManchester Institute of Biotechnology and School of Chemistry, The University of Manchester
  • , Neil DixonAffiliated withManchester Institute of Biotechnology and School of Chemistry, The University of Manchester
  • , Jason MicklefieldAffiliated withManchester Institute of Biotechnology and School of Chemistry, The University of Manchester

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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.

Key words

Orthogonal riboswitches Artificial riboswitches Aptamer domain Synthetic ligands Gene expression tools In vivo screening In vitro transcription Isothermal titration calorimetry Synthetic biology