A flow cytometry-based screen for synthetic riboswitches

Riboswitches regulate gene expression through direct, small molecule–mRNA interactions. The creation of new synthetic riboswitches from in vitro selected aptamers benefits from rapid, high-throughput methods for identifying switches capable of triggering dramatic changes in gene expression in the presence of a desired ligand. Here we present a flow cytometry-based screen for identifying synthetic riboswitches that induce robust increases in gene expression in the presence of theophylline. The performance characteristics of our newly identified riboswitches exceed those of previously described natural and synthetic riboswitches. Sequencing data and structure probing experiments reveal the ribosome binding site to be an important determinant of how well a switch performs and may provide insights into the design of new synthetic riboswitches.

[1]  Barbara Fink,et al.  Conditional gene expression by controlling translation with tetracycline-binding aptamers. , 2003, Nucleic acids research.

[2]  M Bjerknes,et al.  Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. , 1994, Nucleic acids research.

[3]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[4]  R. Breaker,et al.  Regulation of bacterial gene expression by riboswitches. , 2005, Annual review of microbiology.

[5]  Beatrix Suess,et al.  Screening for engineered neomycin riboswitches that control translation initiation. , 2007, RNA.

[6]  Dan S. Tawfik,et al.  High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. , 2005, Chemistry & biology.

[7]  Zasha Weinberg,et al.  A Glycine-Dependent Riboswitch That Uses Cooperative Binding to Control Gene Expression , 2004, Science.

[8]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[9]  S. K. Desai,et al.  A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. , 2007, Chemistry & biology.

[10]  Amir Aharoni,et al.  High-throughput screening methodology for the directed evolution of glycosyltransferases , 2006, Nature Methods.

[11]  A. Link,et al.  Beyond toothpicks: new methods for isolating mutant bacteria , 2007, Nature Reviews Microbiology.

[12]  M. Green,et al.  Controlling gene expression in living cells through small molecule-RNA interactions. , 1998, Science.

[13]  Markus Wieland,et al.  Improved aptazyme design and in vivo screening enable riboswitching in bacteria. , 2008, Angewandte Chemie.

[14]  Jerry Pelletier,et al.  Inhibition of translation by RNA-small molecule interactions. , 2002, RNA.

[15]  Shana Topp,et al.  Random Walks to Synthetic Riboswitches—A High‐Throughput Selection Based on Cell Motility , 2008, Chembiochem : a European journal of chemical biology.

[16]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[17]  Jeffrey W. Smith,et al.  Stochastic Gene Expression in a Single Cell , 2022 .

[18]  Grant R. Zimmermann,et al.  Interlocking structural motifs mediate molecular discrimination by a theophylline-binding RNA , 1997, Nature Structural Biology.

[19]  T. D. Schneider,et al.  Quantitative analysis of ribosome binding sites in E.coli. , 1994, Nucleic acids research.

[20]  A. Pardi,et al.  Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer. , 2000, RNA.

[21]  Jeffrey E. Barrick,et al.  The distributions, mechanisms, and structures of metabolite-binding riboswitches , 2007, Genome Biology.

[22]  C. Wilson,et al.  Inducible regulation of the S. cerevisiae cell cycle mediated by an RNA aptamer-ligand complex. , 2001, Bioorganic & medicinal chemistry.

[23]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[24]  B. Glick,et al.  Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed) , 2002, Nature Biotechnology.

[25]  Dan S. Tawfik,et al.  Flow cytometry: a new method to investigate the properties of water-in-oil-in-water emulsions. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[26]  Eric D Brown,et al.  A FACS‐Based Approach to Engineering Artificial Riboswitches , 2008, Chembiochem : a European journal of chemical biology.

[27]  J. Gallivan.  Toward reprogramming bacteria with small molecules and RNA. , 2007, Current opinion in chemical biology.

[28]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[29]  R. Breaker,et al.  Adenine riboswitches and gene activation by disruption of a transcription terminator , 2004, Nature Structural &Molecular Biology.

[30]  J. Belasco,et al.  Rapid genetic analysis of RNA-protein interactions by translational repression in Escherichia coli. , 2000, Methods in enzymology.

[31]  S. K. Desai,et al.  Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. , 2004, Journal of the American Chemical Society.

[32]  A. Pardi,et al.  A semiconserved residue inhibits complex formation by stabilizing interactions in the free state of a theophylline-binding RNA. , 1998, Biochemistry.