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A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design


Orthogonal (O) ribosome-mediated translation of O-mRNAs enables the incorporation of up to three distinct non-canonical amino acids (ncAAs) into proteins in Escherichia coli (E. coli). However, the general and efficient incorporation of multiple distinct ncAAs by O-ribosomes requires scalable strategies for both creating efficiently and specifically translated O-mRNAs, and the compact expression of multiple O-aminoacyl-tRNA synthetase (O-aaRS)/O-tRNA pairs. We automate the discovery of O-mRNAs that lead to up to 40 times more protein, and are up to 50-fold more orthogonal, than previous O-mRNAs; protein yields from our O-mRNAs match or exceed those from wild-type mRNAs. These advances enable a 33-fold increase in yield for incorporating three distinct ncAAs. We automate the creation of operons for O-tRNA genes, and develop operons for O-aaRS genes. Combining our advances creates a 68-codon, 24-amino-acid genetic code to efficiently incorporate four distinct ncAAs into a single protein in response to four distinct quadruplet codons.

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Fig. 1: A thermodynamic model for the initiation of protein synthesis by wt and O-ribosomes on an mRNA.
Fig. 2: Automated design of O-mRNA sequences that are specifically and efficiently translated by O-ribosomes.
Fig. 3: Efficient production of proteins containing three distinct ncAAs is enabled by new O-mRNAs.
Fig. 4: Four orthogonal aaRS/tRNA pairs decoding four orthogonal quadruplet codons are expressed from aaRS operons and computationally generated tRNA operons and are mutually orthogonal in their aminoacylation specificity, recognize distinct ncAAs and decode distinct orthogonal codons.
Fig. 5: Genetically encoding four distinct ncAAs into a protein using a 24-amino-acid, 68-codon genetic code.

Data availability

All relevant data are included in the article and its Supplementary Information. Materials generated or analysed in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The code for the O-mRNA design method and the tRNA operon designer are available at


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This work was supported by the UK Medical Research Council (MRC; MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR (all to J.W.C.). D.L.D. and S.B.O. were supported by the Boehringer Ingelheim Fonds. We thank M. Skehel at the MRC-LMB mass spectrometry facility for performing mass spectrometry.

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Authors and Affiliations



D.L.D., S.B.O. and J.W.C. conceived the study. D.L.D. performed all wet-lab experiments and managed data. S.B.O. developed the automated orthogonal mRNA design, with input from D.L.D. D.L.D. developed the aaRS operons. A.T.B. developed the tRNA operon generator and analysed the tandem mass spectrometry data. D.L.D., S.B.O. and J.W.C. wrote the paper with input from A.T.B.

Corresponding author

Correspondence to Jason W. Chin.

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The authors declare no competing interests.

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Peer review information Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Tables 1–5.

Reporting Summary

Supplementary Table 1

O-mRNA calculations / experimental data

Supplementary Table 2

GFP yields

Supplementary Table 3

5′UTRS aaRS operons

Supplementary Table 4

Plasmid list

Supplementary Table 5

Source Data SI

Source data

Source Data Fig. 2

Source data for Fig. 2

Source Data Fig. 3

Source data for Fig. 3

Source Data Fig. 4

Source data for Fig. 4

Source Data Fig. 5

Source data for Fig. 5

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Dunkelmann, D.L., Oehm, S.B., Beattie, A.T. et al. A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat. Chem. 13, 1110–1117 (2021).

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