title: Protein-responsive ribozyme switches in eukaryotic cells.
abstract: Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers.
authors.affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Kennedy
authors.affiliation: Division of Chemistry and Chemical Engineering, 1200 E. California Boulevard, MC 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: James V
authors.initials: JV
authors.lastname: Vowles
authors.affiliation: Division of Chemistry and Chemical Engineering, 1200 E. California Boulevard, MC 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Leo
authors.initials: L
authors.lastname: d'Espaux
authors.affiliation: Department of Bioengineering, 443 Via Ortega, MC 4245 Stanford University, Stanford, CA 94305, USA csmolke@stanford.edu.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Smolke
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008274:Magnesium; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000032:analysis*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
chemical_list: D011506:Proteins; D016337:RNA, Catalytic; D008274:Magnesium