title: Applicability of a computational design approach for synthetic riboswitches.
abstract: Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand-dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.
authors.affiliation: Leipzig University, Institute for Biochemistry, 04103 Leipzig, Germany.
authors.forename: Gesine
authors.initials: G
authors.lastname: Domin
authors.affiliation: University of Vienna, Research Group Bioinformatics and Computational Biology, Faculty of Computer Science, A-1090 Vienna, Austria.|University of Vienna, Institute for Theoretical Chemistry, A-1090 Vienna, Austria.
authors.forename: Sven
authors.initials: S
authors.lastname: Findeiß
authors.affiliation: Leipzig University, Institute for Biochemistry, 04103 Leipzig, Germany.
authors.forename: Manja
authors.initials: M
authors.lastname: Wachsmuth
authors.affiliation: Leipzig University, Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, 04107 Leipzig, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Will
authors.affiliation: University of Vienna, Institute for Theoretical Chemistry, A-1090 Vienna, Austria.|Leipzig University, Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, 04107 Leipzig, Germany.|Max Planck Institute for Mathematics in the Science, 04103 Leipzig, Germany.|Fraunhofer Institute for Cell Therapy and Immunology, 04103 Leipzig, Germany.|Santa Fe Institute, Santa Fe NM 87501, USA.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Stadler
authors.affiliation: Leipzig University, Institute for Biochemistry, 04103 Leipzig, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Mörl
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D009690:Nucleic Acid Conformation; D058928:Riboswitch / Q000187:drug effects; D013307:Streptomycin / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology; D061806:Transcription Termination, Genetic
chemical_list: D052157:Aptamers, Nucleotide; D058928:Riboswitch; D013752:Tetracycline; D013307:Streptomycin