De novo-designed translation-repressing riboregulators for multi-input cellular logic

Jongmin Kim; Yu Zhou; Paul D. Carlson; Mario Teichmann; Soma Chaudhary; Friedrich C. Simmel; Pamela A. Silver; James J. Collins; Julius B. Lucks; Peng Yin; Alexander A. Green

doi:10.1038/s41589-019-0388-1

De novo-designed translation-repressing riboregulators for multi-input cellular logic

Jongmin Kim, Yu Zhou, Paul D. Carlson, Mario Teichmann, Soma Chaudhary, Friedrich C. Simmel, Pamela A. Silver, James J. Collins, Julius B. Lucks, Peng Yin^*, Alexander A. Green

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

100 Scopus citations

Abstract

Efforts to construct synthetic biological circuits with more complex functions have often been hindered by the idiosyncratic behavior, limited dynamic range and crosstalk of commonly utilized parts. Here, we employ de novo RNA design to develop two high-performance translational repressors with sensing and logic capabilities. These synthetic riboregulators, termed toehold repressors and three-way junction (3WJ) repressors, detect transcripts with nearly arbitrary sequences, repress gene expression by up to 300-fold and yield orthogonal sets of up to 15 devices. Automated forward engineering is used to improve toehold repressor dynamic range and SHAPE-Seq is applied to confirm the designed switching mechanism of 3WJ repressors in living cells. We integrate the modular repressors into biological circuits that execute universal NAND and NOR logic and evaluate the four-input expression NOT ((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These capabilities make toehold and 3WJ repressors valuable new tools for biotechnological applications.

Original language	English (US)
Pages (from-to)	1173-1182
Number of pages	10
Journal	Nature Chemical Biology
Volume	15
Issue number	12
DOIs	https://doi.org/10.1038/s41589-019-0388-1
State	Published - Dec 1 2019

Funding

This work was supported by an NIH Director\u2019s New Innovator Award (1DP2GM126892), an Alfred P. Sloan Research Fellowship (FG-2017-9108), Gates Foundation funds (OPP1160667), an Arizona Biomedical Research Commission New Investigator Award (ADHS16-162400), a DARPA Young Faculty Award (D17AP00026), Gordon and Betty Moore Foundation funds (#6984), NIH funds (1R21AI136571) and Arizona State University funds to A.A.G.; an NIH Director\u2019s Pioneer Award (1DP1GM133052-01), Office of Naval Research funds (N00014-16-1-2410) and NSF funds (CCF-1317291, MCB-1540214) to P.Y.; an NSF CAREER award to J.B.L. (1452441); Defense Threat Reduction Agency funds (HDTRA1-14-1-0006), Air Force Office of Scientific Research funds (FA9550-14-1-0060) and Paul G. Allen Frontiers Group funds to J.J.C.; and BMBF funds (Erasynbio project UNACS -031L0011) and DFG funds (SFB 10327TPA2) to F.C.S. J.K. acknowledges a Wyss Institute Director\u2019s Cross-Platform Fellowship. The views, opinions and/or findings contained in this article are those of the authors and should not be interpreted as representing the official views or policies, either expressed or implied, of the NIH, DARPA or the Department of Defense.

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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abstract = "Efforts to construct synthetic biological circuits with more complex functions have often been hindered by the idiosyncratic behavior, limited dynamic range and crosstalk of commonly utilized parts. Here, we employ de novo RNA design to develop two high-performance translational repressors with sensing and logic capabilities. These synthetic riboregulators, termed toehold repressors and three-way junction (3WJ) repressors, detect transcripts with nearly arbitrary sequences, repress gene expression by up to 300-fold and yield orthogonal sets of up to 15 devices. Automated forward engineering is used to improve toehold repressor dynamic range and SHAPE-Seq is applied to confirm the designed switching mechanism of 3WJ repressors in living cells. We integrate the modular repressors into biological circuits that execute universal NAND and NOR logic and evaluate the four-input expression NOT ((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These capabilities make toehold and 3WJ repressors valuable new tools for biotechnological applications.",

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De novo-designed translation-repressing riboregulators for multi-input cellular logic

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