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Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome

Abstract

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision1. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase–tRNACUA (MjTyrRS–tRNACUA)2,3 and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase–tRNACUA (MbPylRS–tRNACUA)4,5,6 orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli1,6,7. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome8,9 (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates8. By creating mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids10. Because the synthetase–tRNA pairs used have been evolved to incorporate numerous unnatural amino acids1,6,7, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.

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Figure 1: Selection and characterization of orthogonal quadruplet decoding ribosomes.
Figure 2: Enhanced incorporation of unnatural amino acids in response to amber and quadruplet codons with ribo-Q1.
Figure 3: Encoding an azide and an alkyne in a single protein by orthogonal translation.
Figure 4: Genetically directed cyclization of calmodulin by a Cu( i )-catalysed Huisgen’s [2+3]-cycloaddition.

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References

  1. Xie, J. & Schultz, P. G. A chemical toolkit for proteins–an expanded genetic code. Nature Rev. Mol. Cell Biol. 7, 775–782 (2006)

    Article  CAS  Google Scholar 

  2. Steer, B. A. & Schimmel, P. Major anticodon-binding region missing from an archaebacterial tRNA synthetase. J. Biol. Chem. 274, 35601–35606 (1999)

    Article  CAS  Google Scholar 

  3. Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 99, 11020–11024 (2002)

    Article  ADS  CAS  Google Scholar 

  4. Srinivasan, G., James, C. M. & Krzycki, J. A. Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 (2002)

    Article  ADS  CAS  Google Scholar 

  5. Polycarpo, C. et al. An aminoacyl-tRNA synthetase that specifically activates pyrrolysine. Proc. Natl Acad. Sci. USA 101, 12450–12454 (2004)

    Article  ADS  CAS  Google Scholar 

  6. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding Nε-acetyllysine in recombinant proteins. Nature Chem. Biol. 4, 232–234 (2008)

    Article  CAS  Google Scholar 

  7. Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair and click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009)

    Article  CAS  Google Scholar 

  8. Rackham, O. & Chin, J. W. A network of orthogonal ribosome·mRNA pairs. Nature Chem. Biol. 1, 159–166 (2005)

    Article  CAS  Google Scholar 

  9. Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nature Biotechnol. 25, 770–777 (2007)

    Article  Google Scholar 

  10. Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(i)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Edn Engl. 41, 2596–2599 (2002)

    Article  CAS  Google Scholar 

  11. Hohsaka, T. & Sisido, M. Incorporation of non-natural amino acids into proteins. Curr. Opin. Chem. Biol. 6, 809–815 (2002)

    Article  CAS  Google Scholar 

  12. Ohtsuki, T., Manabe, T. & Sisido, M. Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures. FEBS Lett. 579, 6769–6774 (2005)

    Article  CAS  Google Scholar 

  13. Murakami, H., Hohsaka, T., Ashizuka, Y. & Sisido, M. Site-directed incorporation of p-nitrophenylalanine into streptavidin and site-to-site photoinduced electron transfer from a pyrenyl group to a nitrophenyl group on the protein framework. J. Am. Chem. Soc. 120, 7520–7529 (1998)

    Article  CAS  Google Scholar 

  14. Rodriguez, E. A., Lester, H. A. & Dougherty, D. A. In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression. Proc. Natl Acad. Sci. USA 103, 8650–8655 (2006)

    Article  ADS  CAS  Google Scholar 

  15. Monahan, S. L., Lester, H. A. & Dougherty, D. A. Site-specific incorporation of unnatural amino acids into receptors expressed in mammalian cells. Chem. Biol. 10, 573–580 (2003)

    Article  CAS  Google Scholar 

  16. Anderson, J. C. et al. An expanded genetic code with a functional quadruplet codon. Proc. Natl Acad. Sci. USA 101, 7566–7571 (2004)

    Article  ADS  CAS  Google Scholar 

  17. Atkins, J. F. & Bjork, G. R. A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment. Microbiol. Mol. Biol. Rev. 73, 178–210 (2009)

    Article  CAS  Google Scholar 

  18. Stahl, G., McCarty, G. P. & Farabaugh, P. J. Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding. Trends Biochem. Sci. 27, 178–183 (2002)

    Article  CAS  Google Scholar 

  19. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006)

    Article  ADS  CAS  Google Scholar 

  20. Magliery, T. J., Anderson, J. C. & Schultz, P. G. Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of “shifty” four-base codons with a library approach in Escherichia coli. J. Mol. Biol. 307, 755–769 (2001)

    Article  CAS  Google Scholar 

  21. Khazaie, K., Buchanan, J. H. & Rosenberger, R. F. The accuracy of Qβ RNA translation. 1. Errors during the synthesis of Qβ proteins by intact Escherichia coli cells. Eur. J. Biochem. 144, 485–489 (1984)

    Article  CAS  Google Scholar 

  22. Laughrea, M., Latulippe, J., Filion, A. M. & Boulet, L. Mistranslation in twelve Escherichia coli ribosomal proteins. Cysteine misincorporation at neutral amino acid residues other than tryptophan. Eur. J. Biochem. 169, 59–64 (1987)

    Article  CAS  Google Scholar 

  23. Kramer, E. B. & Farabaugh, P. J. The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. RNA 13, 87–96 (2007)

    Article  CAS  Google Scholar 

  24. Chin, J. W. et al. Addition of p-azido-l-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 9026–9027 (2002)

    Article  CAS  Google Scholar 

  25. Mukai, T. et al. Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818–822 (2008)

    Article  CAS  Google Scholar 

  26. Camarero, J. A., Pavel, J. & Muir, T. W. Chemical synthesis of a circular protein domain: evidence for folding-assisted cyclization. Angew. Chem. Int. Ed. 37, 347–349 (1998)

    Article  CAS  Google Scholar 

  27. Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl Acad. Sci. USA 96, 13638–13643 (1999)

    Article  ADS  CAS  Google Scholar 

  28. Li, P. & Roller, P. P. Cyclization strategies in peptide derived drug design. Curr. Top. Med. Chem. 2, 325–341 (2002)

    Article  CAS  Google Scholar 

  29. Walensky, L. D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305, 1466–1470 (2004)

    Article  ADS  CAS  Google Scholar 

  30. Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A. & Walsh, C. T. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 407, 215–218 (2000)

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

We are grateful to P. B. Kapadnis for synthesizing CAK and to W. An for assistance with some experiments. J.W.C. is grateful to the ERC and the MRC for funding. K.W. is grateful to Trinity College, Cambridge for a fellowship.

Author Contributions K.W., H.N., L.D. and J.W.C. planned the experiments. K.W. selected and characterized ribo-Q, with help from L.D. K.W. and L.D. characterized amber and quadruplet incorporation by protein expression and mass spectrometry. L.D. and M.G.-A. performed protein expression experiments. H.N. demonstrated the mutual orthogonality of synthetase tRNA pairs, evolved synthetases, and characterized the double incorporation and protein cyclization, with help from M.G.-A. H.N., K.W., L.D. and J.W.C. analysed the data and wrote the paper.

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Correspondence to Jason W. Chin.

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This file contains Supplementary Methods, Supplementary Figures 1-13 with Legends, Supplementary Table 1 and Supplementary References. (PDF 5555 kb)

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Neumann, H., Wang, K., Davis, L. et al. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441–444 (2010). https://doi.org/10.1038/nature08817

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