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Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion

Abstract

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1–mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNACUA-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.

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Figure 1: Diverging the decoding properties of natural and orthogonal ribosomes.
Figure 2: Design of ribosome-decoding libraries.
Figure 3: Selection and phenotypic characterization of ribo-X.
Figure 4: The translational fidelity of ribo-X is comparable to that of the natural ribosome.
Figure 5: Ribo-X enhances the efficiency of BpaRS/tRNACUA-dependent unnatural amino acid incorporation in response to single and double UAG codons.

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Acknowledgements

J.W.C. is an EMBO Young Investigator. K.W. is grateful for a Medical Research Council-Laboratory of Molecular Biology (MRC-LMB) Cambridge Scholarship, an Honorary External Research Studentship from Trinity College, Cambridge, and an Overseas Research Studentship Award. H.N. is an MRC Career Development Fellow. We are grateful to O. Barrett and W. An for sharing unpublished materials and assisting in early stages of this project. We are grateful to M. Babu for extracting E. coli amber codon usage, P.G. Schultz (TSRI) for the pSUP Bpa vector. This work was funded by The Medical Research Council.

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Correspondence to Jason W Chin.

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Supplementary information

Supplementary Fig. 1

The anticodon stem loops of the tRNAs used. (PDF 40 kb)

Supplementary Fig. 2

The context of UAGA and UAG selector codons in cat reporter genes. (PDF 43 kb)

Supplementary Fig. 3

The linker region of the gst-malE expression construct. (PDF 25 kb)

Supplementary Fig. 4

35S misincorporation in GST-MBP. (PDF 796 kb)

Supplementary Fig. 5

The ribosome dependence of O-DLR derived Renilla luciferase (O-R-luc) activity. (PDF 108 kb)

Supplementary Fig. 6

The Ribo-X mediated enhanced unnatural amino acid incorporation efficiency is robust in minimal medium. (PDF 297 kb)

Supplementary Table 1

528/36 16S rDNA Libraries Construction. (PDF 32 kb)

Supplementary Table 2

Primers Used. (PDF 48 kb)

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Wang, K., Neumann, H., Peak-Chew, S. et al. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nat Biotechnol 25, 770–777 (2007). https://doi.org/10.1038/nbt1314

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