Year: 2018
Type: preprint
Abstract: 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.
DOI: 10.1101/501783
Institutions: Pohang University of Science and Technology, Harvard University, Wyss Institute for Biologically Inspired Engineering, Arizona State University, Northwestern University, Cornell University, Technical University of Munich, Nanosystems Initiative Munich, Broad Institute, Massachusetts Institute of Technology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: National Science Foundation, U.S. Department of Defense, Bill and Melinda Gates Foundation, Gordon and Betty Moore Foundation, Arizona State University, Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Arizona Biomedical Research Commission, National Institutes of Health, Defense Advanced Research Projects Agency, Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University, Office of Naval Research, National Institute of General Medical Sciences
Awards: CAREER: Uncovering Quantitative Design Principles of RNA Regulators For Synthetic Biology, ERASynBio - Runde 2 - UNACS - Ein vereinheitlichtes System der nukleinsäurebasierten Informationsverarbeitung in lebenden Organismen, Arizona State University Foundation for A New American University - to create point-of-care diagnostics for multiple diseases at low costs by developing molecular technologies hosted by cell-free systems embedded in..., G2628423846, G2661221081, G3877071125, Collaborative Research: Molecular Programming Architectures, Abstractions, Algorithms, and Applications, G4805816520, G5166045897, G5622811596, G6098521345, G6251665261, G6671297155, ERA SynBio: A Unified Nucleic Acid Computation System (UNACS) for Organisms, G7184121498, G7547220548, Molecular Fuses for Real-Time, Label-Free, Multiplexed Imaging of RNAs in Living Cells, G8876996369, G9000124568
OpenAlex ID: W2903594480