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De-Novo-Designed Translational Repressors for Multi-Input Cellular Logic

Jongmin Kim, Yu Zhou, View ORCID ProfilePaul Carlson, Mario Teichmann, View ORCID ProfileFriedrich C. Simmel, View ORCID ProfilePamela A. Silver, James J. Collins, View ORCID ProfileJulius B. Lucks, View ORCID ProfilePeng Yin, View ORCID ProfileAlexander A. Green
doi: https://doi.org/10.1101/501783
Jongmin Kim
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
2Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Republic of Korea
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Yu Zhou
3Biodesign Center for Molecular Design and Biomimetics, The Biodesign Institute and School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
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Paul Carlson
4Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, 14853, USA
5Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
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Mario Teichmann
6Physics Department E14 and ZNN/WSI, Technische Universität München, Garching 85748, Germany
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Friedrich C. Simmel
6Physics Department E14 and ZNN/WSI, Technische Universität München, Garching 85748, Germany
7Nanosystems Initiative Munich, Munich 80799, Germany
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Pamela A. Silver
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
8Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
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James J. Collins
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
9Institute for Medical Engineering and Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
10Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
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Julius B. Lucks
5Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
11Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA
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Peng Yin
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
8Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA
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Alexander A. Green
3Biodesign Center for Molecular Design and Biomimetics, The Biodesign Institute and School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
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ABSTRACT

Synthetic biology aims to apply engineering principles toward the development of novel biological systems for biotechnology and medicine. Despite efforts to expand the set of high-performing parts for genetic circuits, achieving more complex circuit functions has often been limited by the idiosyncratic nature and crosstalk of commonly utilized parts. Here, we present a molecular programming strategy that implements RNA-based repression of translation using de-novo-designed RNAs to realize high-performance orthogonal parts with mRNA detection and multi-input logic capabilities. These synthetic post-transcriptional regulators, termed toehold repressors and three-way junction (3WJ) repressors, efficiently suppress translation in response to cognate trigger RNAs with nearly arbitrary sequences using thermodynamically and kinetically favorable linear-linear RNA interactions. Automated in silico optimization of thermodynamic parameters yields improved toehold repressors with up to 300-fold repression, while in-cell SHAPE-Seq measurements of 3WJ repressors confirm their designed switching mechanism in living cells. Leveraging the absence of sequence constraints, we identify eight- and 15-component sets of toehold and 3WJ repressors, respectively, that provide high orthogonality. The modularity, wide dynamic range, and low crosstalk of the repressors enable their direct integration into ribocomputing devices that provide universal NAND and NOR logic capabilities and can perform multi-input RNA-based logic. We demonstrate these capabilities by implementing a four-input NAND gate and the expression NOT((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These features make toehold and 3WJ repressors important new classes of translational regulators for biotechnological applications.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 19, 2018.
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De-Novo-Designed Translational Repressors for Multi-Input Cellular Logic
Jongmin Kim, Yu Zhou, Paul Carlson, Mario Teichmann, Friedrich C. Simmel, Pamela A. Silver, James J. Collins, Julius B. Lucks, Peng Yin, Alexander A. Green
bioRxiv 501783; doi: https://doi.org/10.1101/501783
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De-Novo-Designed Translational Repressors for Multi-Input Cellular Logic
Jongmin Kim, Yu Zhou, Paul Carlson, Mario Teichmann, Friedrich C. Simmel, Pamela A. Silver, James J. Collins, Julius B. Lucks, Peng Yin, Alexander A. Green
bioRxiv 501783; doi: https://doi.org/10.1101/501783

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