Creating small transcription activating RNAs.

We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression.

[1]  E. Wagner,et al.  Antisense RNA‐mediated transcriptional attenuation: an in vitro study of plasmid pT181 , 2000, Molecular microbiology.

[2]  Richard M. Murray,et al.  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology , 2013, Journal of visualized experiments : JoVE.

[3]  Manolis Kellis,et al.  Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo , 2013, Nature.

[4]  H. Isambert,et al.  RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation , 2009, Physical biology.

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

[6]  J. Collins,et al.  DIVERSITY-BASED, MODEL-GUIDED CONSTRUCTION OF SYNTHETIC GENE NETWORKS WITH PREDICTED FUNCTIONS , 2009, Nature Biotechnology.

[7]  David H. Mathews,et al.  RNAstructure: web servers for RNA secondary structure prediction and analysis , 2013, Nucleic Acids Res..

[8]  R. Weiss,et al.  CRISPR transcriptional repression devices and layered circuits in mammalian cells , 2014, Nature Methods.

[9]  Richard M. Murray,et al.  Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems , 2014, ACS synthetic biology.

[10]  V. Viasnoff,et al.  Encoding folding paths of RNA switches , 2006, Nucleic acids research.

[11]  Engineering artificial small RNAs for conditional gene silencing in escherichia coli , 2014 .

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

[13]  Feng Zhang,et al.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system , 2013, Nucleic acids research.

[14]  J. Park,et al.  Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs , 2013, Nature Biotechnology.

[15]  K. Dill,et al.  Molecular driving forces : statistical thermodynamics in chemistry and biology , 2002 .

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

[17]  V. Noireaux,et al.  An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. , 2012, ACS synthetic biology.

[18]  Christopher A. Voigt,et al.  Automated design of synthetic ribosome binding sites to control protein expression , 2016 .

[19]  Christopher A. Voigt,et al.  Environmental signal integration by a modular AND gate , 2007, Molecular systems biology.

[20]  G. Church,et al.  Synthetic Gene Networks That Count , 2009, Science.

[21]  David H Mathews,et al.  Prediction of RNA secondary structure by free energy minimization. , 2006, Current opinion in structural biology.

[22]  Julius B. Lucks,et al.  Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals , 2012, Nucleic acids research.

[23]  T. Lu,et al.  Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas , 2013, ACS synthetic biology.

[24]  G. Storz,et al.  Regulation by small RNAs in bacteria: expanding frontiers. , 2011, Molecular cell.

[25]  E Westhof,et al.  Progression of a loop–loop complex to a four‐way junction is crucial for the activity of a regulatory antisense RNA , 2000, The EMBO journal.

[26]  Koichi Abe,et al.  Improving the gene-regulation ability of small RNAs by scaffold engineering in Escherichia coli. , 2014, ACS synthetic biology.

[27]  Jay D Keasling,et al.  Model-Driven Engineering of RNA Devices to Quantitatively Program Gene Expression , 2011, Science.

[28]  Farren J. Isaacs,et al.  Engineered riboregulators enable post-transcriptional control of gene expression , 2004, Nature Biotechnology.

[29]  Thomas E. Landrain,et al.  De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells , 2012, Proceedings of the National Academy of Sciences.

[30]  Vincent Noireaux,et al.  Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. , 2014, ACS synthetic biology.

[31]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[32]  Adam P Arkin,et al.  Supplementary information for Rationally designed families of orthogonal RNA regulators of translation , 2012 .

[33]  Adam P. Arkin,et al.  A versatile framework for microbial engineering using synthetic non-coding RNAs , 2014, Nature Reviews Microbiology.

[34]  Adam P Arkin,et al.  Versatile RNA-sensing transcriptional regulators for engineering genetic networks , 2011, Proceedings of the National Academy of Sciences.

[35]  Paul S. Freemont,et al.  Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology , 2013, Nucleic acids research.

[36]  Adam P Arkin,et al.  An adaptor from translational to transcriptional control enables predictable assembly of complex regulation , 2012, Nature Methods.

[37]  Cole Trapnell,et al.  Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) , 2011, Proceedings of the National Academy of Sciences.

[38]  J. Collins,et al.  Toehold Switches: De-Novo-Designed Regulators of Gene Expression , 2014, Cell.

[39]  S. Brantl.  Regulatory mechanisms employed by cis-encoded antisense RNAs. , 2007, Current opinion in microbiology.

[40]  Julius B. Lucks,et al.  A modular strategy for engineering orthogonal chimeric RNA transcription regulators , 2013, Nucleic acids research.

[41]  James J. Collins,et al.  Genetic switchboard for synthetic biology applications , 2012, Proceedings of the National Academy of Sciences.

[42]  Kyle E. Watters,et al.  The centrality of RNA for engineering gene expression , 2013, Biotechnology journal.

[43]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[44]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[45]  James J Collins,et al.  Programmable bacteria detect and record an environmental signal in the mammalian gut , 2014, Proceedings of the National Academy of Sciences.

[46]  L. Nissim,et al.  Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. , 2014, Molecular cell.

[47]  B Lucks Julius,et al.  プライマー伸長塩基配列決定法(SHAPE‐Seq)を用いて分析した選択的2′‐ヒドロキシルアシル化による多重RNA構造の特徴化 , 2011 .