Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes

Abstract

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

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Figure 1: Schematic representation of the artificial division of codon boxes.
Figure 2: Decoding by custom-made FIT systems that consisted of in vitro tRNA transcripts.
Figure 3: Artificial division of the Val GUN codon box.
Figure 4: Assignments of two different Naa in the Arg CGN codon box.
Figure 5: Simultaneous division of multiple codon boxes.
Figure 6: Artificial division of three codon boxes to express a peptide that contains a library of 23 amino acids and a non-standard N-methyl-macrocyclic peptide.

References

  1. 1

    Passioura, T., Katoh, T., Goto, Y. & Suga, H. Selection-based discovery of druglike macrocyclic peptides. Annu. Rev. Biochem. 83, 727–752 (2014).

    CAS  Article  Google Scholar 

  2. 2

    Craik, D. J., Fairlie, D. P., Liras, S. & Price, D. The future of peptide-based drugs. Chem. Biol. Drug Des. 81, 136–147 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Heinis, C. & Winter, G. Encoded libraries of chemically modified peptides. Curr. Opin. Chem. Biol. 26, 89–98 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Horswill, A. R. & Benkovic, S. J. Cyclic peptides, a chemical genetics tool for biologists. Cell Cycle 4, 552–555 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Yamagishi, Y. et al. Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem. Biol. 18, 1562–1570 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Hayashi, Y., Morimoto, J. & Suga, H. In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem. Biol. 7, 607–613 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Morimoto, J., Hayashi, Y. & Suga, H. Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew. Chem. Int. Ed. 51, 3423–3427 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Tanaka, Y. et al. Structural basis for the drug extrusion mechanism by a MATE multidrug transporter. Nature 496, 247–251 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Nemoto, N., Miyamoto-Sato, E., Husimi, Y. & Yanagawa, H. In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett. 414, 405–408 (1997).

    CAS  Article  Google Scholar 

  11. 11

    Roberts, R. W. & Szostak, J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl Acad. Sci. USA 94, 12297–12302 (1997).

    CAS  Article  Google Scholar 

  12. 12

    Murakami, H., Ohta, A., Ashigai, H. & Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nature Methods 3, 357–359 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Goto, Y. et al. Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol. 3, 120–129 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Goto, Y., Murakami, H. & Suga, H. Initiating translation with D-amino acids. RNA 14, 1390–1398 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Kawakami, T., Murakami, H. & Suga, H. Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem. Biol. 15, 32–42 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Xiao, H., Murakami, H., Suga, H. & Ferre-D'Amare, A. R. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454, 358–361 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem. Biol. 14, 1315–1322 (2007).

    CAS  Article  Google Scholar 

  18. 18

    Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nature Protocols 6, 779–790 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nature Biotechnol. 19, 751–755 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Forster, A. C. et al. Programming peptidomimetic syntheses by translating genetic codes designed de novo. Proc. Natl Acad. Sci. USA 100, 6353–6357 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Josephson, K., Hartman, M. C. & Szostak, J. W. Ribosomal synthesis of unnatural peptides. J. Am. Chem. Soc. 127, 11727–11735 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C. & Schultz, P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989).

    CAS  Article  Google Scholar 

  23. 23

    Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Mukai, T. et al. Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Res. 38, 8188–8195 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Mukai, T. et al. Genetic-code evolution for protein synthesis with non-natural amino acids. Biochem. Biophys. Res. Commun. 411, 757–761 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Johnson, D. B. F. et al. RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites. Nature Chem. Biol. 7, 779–786 (2011).

    CAS  Article  Google Scholar 

  27. 27

    Lajoie, M. J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).

    CAS  Article  Google Scholar 

  28. 28

    Hohsaka, T., Ashizuka, Y., Murakami, H. & Sisido, M. Incorporation of nonnatural amino acids into streptavidin through in vitro frame-shift suppression. J. Am. Chem. Soc. 118, 9778–9779 (1996).

    CAS  Article  Google Scholar 

  29. 29

    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).

    CAS  Article  Google Scholar 

  30. 30

    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).

    CAS  Article  Google Scholar 

  31. 31

    Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. & Chin, J. W. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441–444 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Lajoie, M. J. et al. Probing the limits of genetic recoding in essential genes. Science 342, 361–363 (2013).

    CAS  Article  Google Scholar 

  33. 33

    Komine, Y., Adachi, T., Inokuchi, H. & Ozeki, H. Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J. Mol. Biol. 212, 579–598 (1990).

    CAS  Article  Google Scholar 

  34. 34

    Murphy, F. V. IV & Ramakrishnan, V. Structure of a purine–purine wobble base pair in the decoding center of the ribosome. Nature Struct. Mol. Biol. 11, 1251–1252 (2004).

    CAS  Article  Google Scholar 

  35. 35

    Gustilo, E. M., Vendeix, F. A. & Agris, P. F. tRNA's modifications bring order to gene expression. Curr. Opin. Microbiol. 11, 134–140 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Crick, F. H. Codon–anticodon pairing: the wobble hypothesis. J. Mol. Biol. 19, 548–555 (1966).

    CAS  Article  Google Scholar 

  37. 37

    Vasil'eva, I. A. & Moor, N. A. Interaction of aminoacyl-tRNA synthetases with tRNA: general principles and distinguishing characteristics of the high-molecular-weight substrate recognition. Biochemistry. Biokhim. 72, 247–263 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Giege, R., Sissler, M. & Florentz, C. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26, 5017–5035 (1998).

    CAS  Article  Google Scholar 

  39. 39

    Tamura, K., Himeno, H., Asahara, H., Hasegawa, T. & Shimizu, M. In vitro study of E. coli tRNAArg and tRNALys identity elements. Nucleic Acids Res. 20, 2335–2339 (1992).

    CAS  Article  Google Scholar 

  40. 40

    Nureki, O. et al. Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli. J. Mol. Biol. 236, 710–724 (1994).

    CAS  Article  Google Scholar 

  41. 41

    Sylvers, L. A., Rogers, K. C., Shimizu, M., Ohtsuka, E. & Soll, D. A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry 32, 3836–3841 (1993).

    CAS  Article  Google Scholar 

  42. 42

    Cui, Z., Stein, V., Tnimov, Z., Mureev, S. & Alexandrov, K. Semisynthetic tRNA complement mediates in vitro protein synthesis. J. Am. Chem. Soc. 137, 4404–4413 (2015).

    CAS  Article  Google Scholar 

  43. 43

    Park, H. S. et al. Expanding the genetic code of Escherichia coli with phosphoserine. Science 333, 1151–1154 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Schlippe, Y. V., Hartman, M. C., Josephson, K. & Szostak, J. W. In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors. J. Am. Chem. Soc. 134, 10469–10477 (2012).

    Article  Google Scholar 

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Acknowledgements

We thank M. E. Harris and M. Ohuchi for a gift of plasmids that coded E. coli M1 RNA and C5 protein and their products. We thank S. Jongkees and J. Rogers for proofreading the manuscript. This research was supported by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) of Molecular Technologies to H.S., Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (B) to Y.G. (22750145) and Grants-in-Aid for JSPS Fellows to Y.I. (26-9576).

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Y.I., A.H., H.M., T.K., Y.G. and H.S. designed the experiments and analysed the data. Y.I. and A.H. performed the experiments. Y.I., T.K., Y.G. and H.S. wrote the paper.

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Correspondence to Hiroaki Suga.

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Iwane, Y., Hitomi, A., Murakami, H. et al. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nature Chem 8, 317–325 (2016). https://doi.org/10.1038/nchem.2446

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