RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression

RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They consist solely of RNA and couple binding of a small molecule ligand to the so‐called “aptamer domain” with a conformational change in the downstream “expression platform” which then determines system output. The modular organization of riboswitches and the relative ease with which ligand‐binding RNA aptamers can be selected in vitro against almost any molecule have led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology over the past decade. This review highlights proof‐of‐principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro‐ and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in vivo, before finishing with a reflection on how to improve the regulatory properties of engineered riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully exploit their potential as control elements in regulating gene expression.

[1]  Beatrix Suess,et al.  Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. , 2013, Microbiology.

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

[3]  Daniel Karcher,et al.  Inducible gene expression from the plastid genome by a synthetic riboswitch , 2010, Proceedings of the National Academy of Sciences.

[4]  J. Gallivan,et al.  A family of synthetic riboswitches adopts a kinetic trapping mechanism , 2014, Nucleic acids research.

[5]  Shana Topp,et al.  Emerging applications of riboswitches in chemical biology. , 2010, ACS chemical biology.

[6]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[7]  Barbara Fink,et al.  Tetracycline‐aptamer‐mediated translational regulation in yeast , 2003, Molecular microbiology.

[8]  Beatrix Suess,et al.  Engineered riboswitches: Expanding researchers' toolbox with synthetic RNA regulators , 2012, FEBS letters.

[9]  Yohei Yokobayashi,et al.  Conditional RNA interference mediated by allosteric ribozyme. , 2009, Journal of the American Chemical Society.

[10]  H. Blau,et al.  Transcriptional control: rheostat converted to on/off switch. , 2000, Molecular cell.

[11]  F. Crick Central Dogma of Molecular Biology , 1970, Nature.

[12]  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.

[13]  B. Suess,et al.  Development of β-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans , 2014, Archaea.

[14]  R. Breaker,et al.  Riboswitches as versatile gene control elements. , 2005, Current opinion in structural biology.

[15]  Christof Fellmann,et al.  Stable RNA interference rules for silencing , 2013, Nature Cell Biology.

[16]  Pascale Cossart,et al.  Sequestration of a two-component response regulator by a riboswitch-regulated noncoding RNA , 2014, Science.

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

[18]  J. Collins,et al.  Construction of a genetic toggle switch in Escherichia coli , 2000, Nature.

[19]  Renee K Mosing,et al.  Isolating aptamers using capillary electrophoresis-SELEX (CE-SELEX). , 2009, Methods in molecular biology.

[20]  Y. Nakahira,et al.  Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in Cyanobacterium Synechococcus elongatus PCC 7942. , 2013, Plant & cell physiology.

[21]  K. Dery,et al.  Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing , 2008, BMC Molecular Biology.

[22]  J. Gallivan,et al.  A flow cytometry-based screen for synthetic riboswitches , 2008, Nucleic acids research.

[23]  M. McConnell,et al.  Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. , 2013, FEMS microbiology reviews.

[24]  Melissa M. Harrison,et al.  A CRISPR view of development , 2014, Genes & development.

[25]  R. Gaur,et al.  An artificial riboswitch for controlling pre-mRNA splicing. , 2005, RNA.

[26]  M. Fedor,et al.  The glmS Riboswitch Integrates Signals from Activating and Inhibitory Metabolites In Vivo , 2010, Nature Structural &Molecular Biology.

[27]  D. Patel,et al.  Adaptive recognition by nucleic acid aptamers. , 2000, Science.

[28]  F. Narberhaus,et al.  Thermozymes: Synthetic RNA thermometers based on ribozyme activity. , 2013, RNA biology.

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

[30]  Yohei Yokobayashi,et al.  Controlling Mammalian Gene Expression by Allosteric Hepatitis Delta Virus Ribozymes , 2013, ACS synthetic biology.

[31]  Rafael Silva-Rocha,et al.  Mining logic gates in prokaryotic transcriptional regulation networks , 2008, FEBS letters.

[32]  Robert T. Batey,et al.  Engineering modular ‘ON’ RNA switches using biological components , 2013, Nucleic acids research.

[33]  A. Serganov,et al.  A Decade of Riboswitches , 2013, Cell.

[34]  P. Sharp,et al.  The role of miRNAs in regulating gene expression networks. , 2013, Journal of molecular biology.

[35]  J. Micklefield,et al.  Reengineering orthogonally selective riboswitches , 2010, Proceedings of the National Academy of Sciences.

[36]  Ali Nahvi,et al.  A Parsimonious Model for Gene Regulation by miRNAs , 2011, Science.

[37]  S. Sabbioni,et al.  MicroRNAs in liver cancer: a model for investigating pathogenesis and novel therapeutic approaches , 2014, Cell Death and Differentiation.

[38]  R. Batey,et al.  Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices. , 2013, ACS synthetic biology.

[39]  R. Breaker Riboswitches and the RNA world. , 2012, Cold Spring Harbor perspectives in biology.

[40]  J. Gallivan,et al.  Guiding bacteria with small molecules and RNA. , 2007, Journal of the American Chemical Society.

[41]  B. Suess,et al.  Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch. , 2010, Angewandte Chemie.

[42]  Ali Nahvi,et al.  Genetic control by a metabolite binding mRNA. , 2002, Chemistry & biology.

[43]  J. Kurreck RNA Interference: From Basic Research to Therapeutic Applications , 2009, Angewandte Chemie.

[44]  B. Suess,et al.  Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes. , 2015, ACS synthetic biology.

[45]  Yohei Yokobayashi,et al.  A synthetic riboswitch with chemical band-pass response. , 2010, Chemical communications.

[46]  G. Balázsi,et al.  Linearizer gene circuits with negative feedback regulation. , 2011, Methods in molecular biology.

[47]  R R Breaker,et al.  Rational design of allosteric ribozymes. , 1997, Chemistry & biology.

[48]  J. Burnett,et al.  RNA-based therapeutics: current progress and future prospects. , 2012, Chemistry & biology.

[49]  Pascale Cossart,et al.  A riboswitch-regulated antisense RNA in Listeria monocytogenes , 2013, Proceedings of the National Academy of Sciences.

[50]  D. Ahmadvand,et al.  Biological targeting and innovative therapeutic interventions with phage-displayed peptides and structured nucleic acids (aptamers). , 2011, Current opinion in biotechnology.

[51]  M. Win,et al.  Higher-Order Cellular Information Processing with Synthetic RNA Devices , 2008, Science.

[52]  Charles Wilson,et al.  Recognition of Planar and Nonplanar Ligands in the Malachite Green–RNA Aptamer Complex , 2004, Chembiochem : a European journal of chemical biology.

[53]  Mark S Dunstan,et al.  Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species. , 2014, Journal of the American Chemical Society.

[54]  S. Haas,et al.  Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses , 2012, Nucleic acids research.

[55]  M. Win,et al.  A modular and extensible RNA-based gene-regulatory platform for engineering cellular function , 2007, Proceedings of the National Academy of Sciences.

[56]  R. Batey,et al.  A disconnect between high-affinity binding and efficient regulation by antifolates and purines in the tetrahydrofolate riboswitch. , 2014, Chemistry & biology.

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

[58]  Michael Müller,et al.  Thermodynamic characterization of an engineered tetracycline-binding riboswitch , 2006, Nucleic acids research.

[59]  Beatrix Suess,et al.  Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast. , 2011, Molecular bioSystems.

[60]  Michael E Webb,et al.  Thiamine biosynthesis in algae is regulated by riboswitches , 2007, Proceedings of the National Academy of Sciences.

[61]  Ronald R. Breaker,et al.  Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression , 2002, Nature.

[62]  Markus Wieland,et al.  Artificial Riboswitches: Synthetic mRNA‐Based Regulators of Gene Expression , 2008, Chembiochem : a European journal of chemical biology.

[63]  J. Gallivan,et al.  A Riboswitch-Based Inducible Gene Expression System for Mycobacteria , 2012, PloS one.

[64]  R. D'Amato,et al.  Exogenous control of mammalian gene expression through modulation of RNA self-cleavage , 2004, Nature.

[65]  N. Sharpless,et al.  Detecting and characterizing circular RNAs , 2014, Nature Biotechnology.

[66]  Wade C. Winkler,et al.  A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator , 2014, Science.

[67]  M. Elowitz,et al.  A synthetic oscillatory network of transcriptional regulators , 2000, Nature.

[68]  Florian Groher,et al.  Synthetic riboswitches - A tool comes of age. , 2014, Biochimica et biophysica acta.

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

[70]  Sergey N Krylov,et al.  Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides , 2006, Nature Protocols.

[71]  Henning Ulrich,et al.  Nucleic acid aptamers as high affinity ligands in biotechnology and biosensorics. , 2013, Journal of pharmaceutical and biomedical analysis.

[72]  G. Hannon RNA interference : RNA , 2002 .

[73]  Markus Wieland,et al.  Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation. , 2012, Molecular bioSystems.

[74]  Samuel Bocobza,et al.  Riboswitch-dependent gene regulation and its evolution in the plant kingdom. , 2007, Genes & development.

[75]  Mark A. Miller,et al.  Riboswitches for Intracellular Study of Genes Involved in Francisella Pathogenesis , 2012, mBio.

[76]  Chase L. Beisel,et al.  Model-guided design of ligand-regulated RNAi for programmable control of gene expression , 2008, Molecular systems biology.

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

[78]  K. Flärdh,et al.  Signals and regulators that govern Streptomyces development. , 2012, FEMS microbiology reviews.

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

[80]  Travis S. Bayer,et al.  Programmable ligand-controlled riboregulators of eukaryotic gene expression , 2005, Nature Biotechnology.

[81]  P. Stadler,et al.  De novo design of a synthetic riboswitch that regulates transcription termination , 2012, Nucleic acids research.

[82]  Howard Y. Chang,et al.  Physiological roles of long noncoding RNAs: insight from knockout mice. , 2014, Trends in cell biology.

[83]  Evgeny Nudler,et al.  Sensing Small Molecules by Nascent RNA A Mechanism to Control Transcription in Bacteria , 2002, Cell.

[84]  Jeffrey E. Barrick,et al.  New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[85]  S. K. Desai,et al.  Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species , 2010, Applied and Environmental Microbiology.

[86]  Atsushi Ogawa,et al.  Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors. , 2011, RNA.

[87]  Oliver Spadiut,et al.  Microbials for the production of monoclonal antibodies and antibody fragments , 2014, Trends in biotechnology.

[88]  W. Filipowicz,et al.  The widespread regulation of microRNA biogenesis, function and decay , 2010, Nature Reviews Genetics.

[89]  B. Suess,et al.  A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. , 2004, Nucleic acids research.

[90]  Michael Musheev,et al.  Non-SELEX selection of aptamers. , 2006, Journal of the American Chemical Society.

[91]  A. Ferré-D’Amaré,et al.  Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch. , 2008, Chemistry & biology.

[92]  Zasha Weinberg,et al.  An Allosteric Self-Splicing Ribozyme Triggered by a Bacterial Second Messenger , 2010, Science.

[93]  R. Rosenfeld Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[94]  J. Oost,et al.  Unravelling the structural and mechanistic basis of CRISPR–Cas systems , 2014, Nature Reviews Microbiology.

[95]  R. Breaker,et al.  Control of gene expression by a natural metabolite-responsive ribozyme , 2004, Nature.

[96]  C. Berens,et al.  A tetracycline-binding RNA aptamer. , 2001, Bioorganic & medicinal chemistry.

[97]  A. Pardi,et al.  NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer. , 2009, Journal of the American Chemical Society.

[98]  R. Breaker,et al.  Riboswitch Control of Gene Expression in Plants by Splicing and Alternative 3′ End Processing of mRNAs[W][OA] , 2007, The Plant Cell Online.

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

[100]  R. Breaker,et al.  Engineering precision RNA molecular switches. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[101]  Alexander Revzin,et al.  Modulating endogenous gene expression of mammalian cells via RNA-small molecule interaction. , 2008, Biochemical and biophysical research communications.

[102]  Tina M. Henkin,et al.  Natural Variability in S-Adenosylmethionine (SAM)-Dependent Riboswitches: S-Box Elements in Bacillus subtilis Exhibit Differential Sensitivity to SAM In Vivo and In Vitro , 2007, Journal of bacteriology.

[103]  James W. Golden,et al.  Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches , 2014, Applied and Environmental Microbiology.

[104]  Katherine E Deigan,et al.  Riboswitches: discovery of drugs that target bacterial gene-regulatory RNAs. , 2011, Accounts of chemical research.

[105]  Charlotte Rehm,et al.  In vivo screening for aptazyme-based bacterial riboswitches. , 2014, Methods in molecular biology.

[106]  Thomas Scheper,et al.  Aptamers: versatile probes for flow cytometry , 2013, Applied Microbiology and Biotechnology.

[107]  Benedikt Klauser,et al.  Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae. , 2015, ACS synthetic biology.

[108]  Susie Choi,et al.  Rational design of SAM analogues targeting SAM-II riboswitch aptamer. , 2011, Bioorganic & medicinal chemistry letters.

[109]  C. Buchrieser,et al.  A trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria monocytogenes , 2009, Cell.

[110]  V. Wendisch Microbial production of amino acids and derived chemicals: synthetic biology approaches to strain development. , 2014, Current opinion in biotechnology.

[111]  Beatrix Suess,et al.  Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast , 2007, Nucleic acids research.

[112]  M. Famulok,et al.  A novel RNA motif for neomycin recognition. , 1995, Chemistry & biology.

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

[114]  G. Mayer,et al.  Carba-sugars activate the glmS-riboswitch of Staphylococcus aureus. , 2011, ACS chemical biology.

[115]  Beatrix Suess,et al.  Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity , 2010, Nucleic acids research.

[116]  P. Viollier,et al.  Decoding Caulobacter development. , 2012, FEMS microbiology reviews.

[117]  Benedikt Klauser,et al.  In vivo screening of ligand-dependent hammerhead ribozymes. , 2012, Methods in molecular biology.

[118]  Christof von Kalle,et al.  Artificial riboswitches for gene expression and replication control of DNA and RNA viruses , 2014, Proceedings of the National Academy of Sciences.

[119]  B. Suess,et al.  Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch , 2014, Chembiochem : a European journal of chemical biology.

[120]  Christina D Smolke,et al.  Synthetic RNA switches as a tool for temporal and spatial control over gene expression. , 2012, Current opinion in biotechnology.

[121]  Svetlana V. Harbaugh,et al.  Development of a 2,4-dinitrotoluene-responsive synthetic riboswitch in E. coli cells. , 2013, ACS chemical biology.

[122]  Jeffrey E. Barrick,et al.  Tandem Riboswitch Architectures Exhibit Complex Gene Control Functions , 2006, Science.

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

[124]  B. Séraphin,et al.  Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion , 2001, The EMBO journal.

[125]  R. Breaker,et al.  Control of alternative RNA splicing and gene expression by eukaryotic riboswitches , 2007, Nature.

[126]  R. Knight,et al.  Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch. , 2013, Journal of molecular biology.

[127]  D. Chakravortty,et al.  Salmonella enterica serovars Typhimurium and Typhi as model organisms , 2012, Virulence.

[128]  L. Serrano,et al.  Engineering stability in gene networks by autoregulation , 2000, Nature.

[129]  Harald Schwalbe,et al.  Three-state mechanism couples ligand and temperature sensing in riboswitches , 2013, Nature.

[130]  Simon Ausländer,et al.  A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression. , 2010, Molecular bioSystems.

[131]  Yohei Yokobayashi,et al.  Engineering complex riboswitch regulation by dual genetic selection. , 2008, Journal of the American Chemical Society.

[132]  G. F. Joyce,et al.  The expanding view of RNA and DNA function. , 2014, Chemistry & biology.