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New codons for efficient production of unnatural proteins in a semisynthetic organism


Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with additional sense codons. Here, we report a systematic analysis of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded noncanonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to incorporate multiple, different ncAAs site specifically into a protein should now allow the development of proteins with novel activities, and possibly even SSOs with new forms and functions.

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Fig. 1: Protein production in nonclonal SSOs using unnatural codons and anticodons.
Fig. 2: Protein production and analyses of codon orthogonality in clonal SSOs.
Fig. 3: Simultaneous decoding of two unnatural codons.
Fig. 4: Simultaneous decoding of three unnatural codons.

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Data availability

Annotated plasmid sequences from this study are available via Genbank (accession numbers MN882182MN882190) as detailed in Supplementary Table 4. All data supporting the findings of this study are available within the paper and the supplementary information or from the corresponding author upon reasonable request.


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This work was supported by the National Institutes of Health (GM118178 to F.E.R., GM123735 to Y.Z. and GM128376 to R.J.K.). E.C.F. was supported by a Boehringer Ingelheim Fonds PhD Fellowship. K.H. was supported by a JSPS Overseas Research Fellowship. A.W.F. and M.P.L. were supported by a National Science Foundation Graduate Research Fellowship (NSF/DGE-1346837). R.K. was supported by NASA Exobiology (NNX14AP59G).

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Authors and Affiliations



F.E.R., Y.Z. and E.C.F. conceived the project. E.C.F. and F.E.R. designed experiments. E.C.F., K.H., A.W.F. and V.T.D. performed and analyzed experiments. R.J.K. and R.K. synthesized unnatural DNA oligonucleotides. A.W.F., M.P.L. and R.A. provided technical assistance. F.E.R. provided project leadership. E.C.F. and F.E.R. wrote the manuscript.

Corresponding author

Correspondence to Floyd E. Romesberg.

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Competing interests

The authors declare the following competing financial interests: a patent application has been filed based on the use of UBPs in SSOs (PCT/US2018/041509).

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Supplementary Figs. 1–11 and Tables 1–4.

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Fischer, E.C., Hashimoto, K., Zhang, Y. et al. New codons for efficient production of unnatural proteins in a semisynthetic organism. Nat Chem Biol 16, 570–576 (2020).

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