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.

  • Compound

    Nε-(((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl)-L-lysine

  • Compound

    Nε-(tert-butyloxycarbonyl)-L-lysine

  • Compound

    Nε-((prop-2-yn-1-yloxy)carbonyl)-L-lysine

  • Compound

    Nε-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)-L-lysine

  • Compound

    3,3-dimethyl-2-((1E,3E)-5-((Z)-3-methyl-3-(6-oxo-6-((2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)hexyl)-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-5-sulfo-1-(3-sulfopropyl)-3H-indol-1-ium

  • Compound

    1-(6-((3-azidopropyl)amino)-6-oxohexyl)-2-((1E,3E)-5-((E)-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate

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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.

Author information

Author notes

    • Russell J Ernst
    •  & Toke P Krogager

    These authors contributed equally to this work.

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, England, UK.

    • Russell J Ernst
    • , Toke P Krogager
    • , Elizabeth S Maywood
    • , Roberto Zanchi
    • , Václav Beránek
    • , Thomas S Elliott
    • , Nicholas P Barry
    • , Michael H Hastings
    •  & Jason W Chin

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jason W Chin.

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    Supplementary Text and Figures

    Supplementary Results and Supplementary Figures 1–13.

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DOI

https://doi.org/10.1038/nchembio.2160

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