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Creating small transcription activating RNAs

Abstract

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.

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Figure 1: Design and characterization of the anti-anti-terminator STAR mechanism.
Figure 2: Design and characterization of the direct anti-terminator STAR mechanism.
Figure 3: STAR design principles.
Figure 4: Determining the orthogonality of STAR regulators and transcriptional attenuators.
Figure 5: Characterization of novel RNA-only transcriptional logic gates.

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Acknowledgements

The authors acknowledge J. Roberts, E. Strobel, A. Stroock and the Lucks Lab members for helpful discussions. We would also like to thank C. Trapnell for help with RNA-seq experimental design and analysis. We also thank J. Peters (Department of Microbiology, Cornell University) for providing E. coli strain K12 MG1655. Finally, we would like to thank D. Tapias-Rojas for preliminary work on targeting naturally occurring intrinsic terminators. This material is based on work supported by the National Science Foundation Graduate Research Fellowship Program (grant no. DGE-1144153 to M.K.T.), the Defense Advanced Research Projects Agency Young Faculty Award (DARPA YFA; no. N66001-12-1-4254 to J.B.L.) and an Office of Naval Research Young Investigators Program Award (ONR YIP; no. N00014-13-1-0531 to J.B. L.). J.B.L. is an Alfred P. Sloan Research Fellow.

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J.C., M.K.T. and J.B.L. conceived the ideas, designed the experiments and wrote the manuscript. J.C. and M.K.T. performed the experiments.

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Correspondence to Julius B Lucks.

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The authors have submitted a provisional patent application (No. 61/981,241) for the technologically important developments included in this Article.

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Supplementary Results, Supplementary Tables 1–5, Supplementary Figures 1–14 and Supplementary Note. (PDF 2448 kb)

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Chappell, J., Takahashi, M. & Lucks, J. Creating small transcription activating RNAs. Nat Chem Biol 11, 214–220 (2015). https://doi.org/10.1038/nchembio.1737

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