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Genetic code expansion in the mouse brain

Abstract

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNAPylCUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.

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Figure 1: AAV-mediated incorporation of non-natural amino acids into proteins in dissociated neurons and bioorthogonal labeling.
Figure 2: Incorporation of non-natural amino acids into proteins in SCN slices and live mice.

References

  1. 1

    Davis, L. & Chin, J.W. Nat. Rev. Mol. Cell Biol. 13, 168–182 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Chin, J.W. Annu. Rev. Biochem. 83, 379–408 (2014).

    CAS  Article  Google Scholar 

  3. 3

    Xue, L., Karpenko, I.A., Hiblot, J. & Johnsson, K. Nat. Chem. Biol. 11, 917–923 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Neumann, H., Peak-Chew, S.Y. & Chin, J.W. Nat. Chem. Biol. 4, 232–234 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Lin, S. et al. J. Am. Chem. Soc. 133, 20581–20587 (2011).

    CAS  Article  Google Scholar 

  6. 6

    Hancock, S.M., Uprety, R., Deiters, A. & Chin, J.W. J. Am. Chem. Soc. 132, 14819–14824 (2010).

    CAS  Article  Google Scholar 

  7. 7

    Mukai, T. et al. Biochem. Biophys. Res. Commun. 371, 818–822 (2008).

    CAS  Article  Google Scholar 

  8. 8

    Gautier, A. et al. J. Am. Chem. Soc. 132, 4086–4088 (2010).

    CAS  Article  Google Scholar 

  9. 9

    Greiss, S. & Chin, J.W. J. Am. Chem. Soc. 133, 14196–14199 (2011).

    CAS  Article  Google Scholar 

  10. 10

    Bianco, A., Townsley, F.M., Greiss, S., Lang, K. & Chin, J.W. Nat. Chem. Biol. 8, 748–750 (2012).

    CAS  Article  Google Scholar 

  11. 11

    Li, F. et al. Angew. Chem. Int. Ed. Engl. 52, 9700–9704 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Rosenthal, N. & Brown, S. Nat. Cell Biol. 9, 993–999 (2007).

    CAS  Article  Google Scholar 

  13. 13

    Fossella, J.A. & Casey, B.J. Cogn. Affect. Behav. Neurosci. 6, 1–8 (2006).

    Article  Google Scholar 

  14. 14

    Buvoli, M., Buvoli, A. & Leinwand, L.A. Mol. Cell. Biol. 20, 3116–3124 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Kang, J.-Y. et al. Neuron 80, 358–370 (2013).

    CAS  Article  Google Scholar 

  16. 16

    Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J.E. Mol. Ther. 16, 1073–1080 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Brancaccio, M., Maywood, E.S., Chesham, J.E., Loudon, A.S.I. & Hastings, M.H.A. Neuron 78, 714–728 (2013).

    CAS  Article  Google Scholar 

  18. 18

    Schmied, W.H., Elsässer, S.J., Uttamapinant, C. & Chin, J.W. J. Am. Chem. Soc. 136, 15577–15583 (2014).

    CAS  Article  Google Scholar 

  19. 19

    Kim, J.H. et al. PLoS One 6, e18556 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Lang, K. & Chin, J.W. Chem. Rev. 114, 4764–4806 (2014).

    CAS  Article  Google Scholar 

  21. 21

    Elliott, T.S. et al. Nat. Biotechnol. 32, 465–472 (2014).

    CAS  Article  Google Scholar 

  22. 22

    Reppert, S.M. & Weaver, D.R. Nature 418, 935–941 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Hastings, M.H., Reddy, A.B., McMahon, D.G. & Maywood, E.S. Methods Enzymol. 393, 579–592 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Yoo, S.H. et al. Proc. Natl. Acad. Sci. USA 101, 5339–5346 (2004).

    CAS  Article  Google Scholar 

  25. 25

    Colwell, C.S. Nat. Rev. Neurosci. 12, 553–569 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Nguyen, D.P., Garcia Alai, M.M., Kapadnis, P.B., Neumann, H. & Chin, J.W. J. Am. Chem. Soc. 131, 14194–14195 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Kaech, S. & Banker, G. Nat. Protoc. 1, 2406–2415 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Lang, K. et al. J. Am. Chem. Soc. 134, 10317–10320 (2012).

    CAS  Article  Google Scholar 

  29. 29

    Beal, S.L. J. Pharmacokinet. Pharmacodyn. 28, 481–504 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Jaki, T. & Wolfsegger, M.J. Pharm. Stat. 10, 284–288 (2011).

    Article  Google Scholar 

  31. 31

    Uttamapinant, C., Sanchez, M.I., Liu, D.S., Yao, J.Z. & Ting, A.Y. Nat. Protoc. 8, 1620–1634 (2013).

    Article  Google Scholar 

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Acknowledgements

This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008, to J.W.C., MC_U105170643 to M.H.H.). V.B. is supported by an MRC case studentship (Nikon). We thank the MRC biomedical facility staff at ARES for their help.

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Authors

Contributions

J.W.C. defined the direction of research. R.J.E. designed the AAV vectors. V.B. and R.J.E. performed the experiments in rat cortical neurons. R.Z., E.S.M. and R.J.E. defined the amino acid delivery conditions in live mice. R.J.E. and T.P.K. performed the SCN slice experiments under the direction of M.H.H. R.Z. performed the labeling of brain sections containing 3 with 6 and performed microscopy. E.S.M. performed the experiments in live mice. T.S.E. provided amino acid 1. R.Z. provided amino acid 3, designed pharmacokinetic experiments, determined the plasma concentrations of 3 and analyzed pharmacokinetic data. N.P.B. performed quantitative microscopy and assisted imaging experiments. J.W.C. and R.J.E. wrote the paper with input from all authors.

Corresponding author

Correspondence to Jason W Chin.

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The authors declare no competing financial interests.

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Supplementary Results and Supplementary Figures 1–13. (PDF 4466 kb)

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Ernst, R., Krogager, T., Maywood, E. et al. Genetic code expansion in the mouse brain. Nat Chem Biol 12, 776–778 (2016). https://doi.org/10.1038/nchembio.2160

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