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Multiplex suppression of four quadruplet codons via tRNA directed evolution
- Source :
- Nature Communications, Nature Communications, Vol 12, Iss 1, Pp 1-13 (2021)
- Publication Year :
- 2020
-
Abstract
- Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon–anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.<br />Genetic code expansion strategies are limited to specific codons that can be reassigned to new amino acids. Here the authors show that quadruplet-decoding tRNAs (qtRNAs) can be rapidly discovered and evolved to decode new quadruplet codons, enabling four independent decoding events in a single protein in living cells.
- Subjects :
- Base pair
Science
General Physics and Astronomy
Computational biology
Biology
General Biochemistry, Genetics and Molecular Biology
Article
Amino Acyl-tRNA Synthetases
Synthetic biology
RNA, Transfer
Anticodon
Escherichia coli
Multiplex
Amino Acids
Cloning, Molecular
Codon
chemistry.chemical_classification
Multidisciplinary
Escherichia coli Proteins
fungi
food and beverages
Translation (biology)
General Chemistry
Genetic code
Directed evolution
tRNAs
Amino acid
RNA, Bacterial
chemistry
Protein Biosynthesis
Transfer RNA
Directed Molecular Evolution
Subjects
Details
- ISSN :
- 20411723
- Volume :
- 12
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Nature communications
- Accession number :
- edsair.doi.dedup.....2f997acc3afab70e7a5379247b92f731