title: Design criteria for synthetic riboswitches acting on transcription.
abstract: Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.
authors.affiliation: a University of Leipzig ; Institute for Biochemistry ; Leipzig , Germany.
authors.forename: Manja
authors.initials: M
authors.lastname: Wachsmuth
authors.forename: Gesine
authors.initials: G
authors.lastname: Domin
authors.forename: Ronny
authors.initials: R
authors.lastname: Lorenz
authors.forename: Robert
authors.initials: R
authors.lastname: Serfling
authors.forename: Sven
authors.initials: S
authors.lastname: Findeiß
authors.forename: Peter F
authors.initials: PF
authors.lastname: Stadler
authors.forename: Mario
authors.initials: M
authors.lastname: Mörl
mesh_terms: D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D058928:Riboswitch; D013329:Structure-Activity Relationship; D058615:Synthetic Biology; D013806:Theophylline / Q000737:chemistry; D013816:Thermodynamics; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D058928:Riboswitch; D049452:Green Fluorescent Proteins; D013806:Theophylline; D001616:beta-Galactosidase
keywords: GFP, green fluorescent protein; MU, Miller units; PCR, polymerase chain reaction; RS, riboswitch; bgaB, B. stearothermophilus-derived beta-galactosidase; riboswitch design; synthetic biology; synthetic riboswitch; tandem riboswitch; theophylline aptamer; transcription regulation
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