title: Automated physics-based design of synthetic riboswitches from diverse RNA aptamers.
abstract: Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications.
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Amin
authors.initials: A
authors.lastname: Espah Borujeni
authors.affiliation: Department of Biological Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Mishler
authors.affiliation: Department of Biological Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Jingzhi
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Walker
authors.initials: W
authors.lastname: Huso
authors.affiliation: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA Department of Chemistry, Emory University, Atlanta, GA 30322, USA salis@psu.edu.
authors.forename: Howard M
authors.initials: HM
authors.lastname: Salis
mesh_terms: D000465:Algorithms; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry*; D015374:Biosensing Techniques; D004298:Dopamine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008163:Luminescent Measurements / Q000379:methods; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D059370:RNA Folding; D015203:Reproducibility of Results; D058928:Riboswitch / Q000235:genetics*; D052156:SELEX Aptamer Technique; D013974:Thyroxine / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D052157:Aptamers, Nucleotide; D058928:Riboswitch; D013974:Thyroxine; D004298:Dopamine