In vitro ribosome synthesis and evolution through ribosome display
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Abstract
Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 10 7 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 10 3 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.
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In vitro affinity maturation methods, such as random mutagenesis with display technologies, improve antibody binding against target proteins but are time consuming and labor intensive.
“In vitro affinity maturation methods, such as random mutagenesis with display technologies, has been shown to improve antibody binding against target proteins, but such approaches are time consuming and labor intensive (27–32).”
From Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization (2022) · cites this paper as [28]
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References 50 · Cited by 78
- Authors
- Hammerling, M. J., Fritz, B. R., Yoesep, D. J., Kim, D. S., Carlson, E. D., & Jewett, M. C.
- Affiliations
- Northwestern University (US)
- Corresponding author
- Jewett, Michael C.
- Venue
- Nature Communications
- Published
- 2020-02-28
- Volume
- 11
- Issue
- 1
- Pages
- 1108-1108
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC7048773
- OpenAlex
- W3009890745
- Cited by
- 78
- References
- 50
- Citations by year
- 2026: 6 · 2025: 5 · 2024: 16 · 2023: 9 · 2022: 19 · 2021: 13 · 2020: 8 · 2019: 1
- FWCI
- 4.29
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- United States Department of Defense | United States Army | U.S. Army Research, Development and Engineering Command | Army Research Office (W911NF-11-1-0445, W911NF-16-1-0372)
- National Science Foundation (MCB-1716766)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
- Physical Sciences › Physics and Astronomy › Astronomy and Astrophysics › Origins and Evolution of Life
- Keywords
- ribosome evolution, ribosome display, clindamycin resistance, epistasis, translation (biology), ribosome assembly, directed evolution
- MeSH terms
- Anti-Bacterial Agents, Clindamycin, Epistasis, Genetic, Escherichia coli, Genotype, Mutation, Peptidyl Transferases, Protein Synthesis Inhibitors, Ribosomal Proteins, Ribosomes, RNA, Ribosomal, Gene Library, Directed Molecular Evolution, Drug Resistance, Bacterial, Synthetic Biology
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