Syed et al., 2019 - Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine. International Journal of Molecular Sciences   20(10) Full text @ Int. J. Mol. Sci.

Figure 2

The site-specific incorporation of azido-lysine in the zebrafish embryos. (A) The Brightfield (left) or fluorescent (right) images of the zebrafish embryos injected with mRNA(s) for LifeAct-eGFP (top panel) or LifeAct-eGFP Y39* (codon for Y39 converted to stop codon UAG) with the orthogonal tRNAPyl CUA-mRNA aaRS for AzK/BCNK, with (middle panel) AzK but not without (bottom panel) AzK. Scale bar, 1 mm (B). The localization of LifeAct-eGFP or LifeAct-eGFP Y39AzK to cell–cell junctions and cortical speckles in the dechorionated embryos injected as described in (A). Scale bar represents 20 µm. (C) The mass-spectrometry identified AzK incorporation in LifeAct-eGFP Y39AzK. The lysates were prepared from ~1000 embryos injected with mRNA for LifeAct-eGFP Y39* with the orthogonal tRNAPyl-CUA -mRNA aaRS for AzK/BCNK with AzK. LifeAct-eGFP Y39AzK was immunoaffinity purified using GFP nanobodies and processed for mass spectrometry as described in the methods section. The MS/MS spectrum of precursor m/z = 791.3606 (M+2) corresponded to tryptic peptide FSVSGEGEGDATKGK with azido-lysine incorporated at the indicated position. The full y-ion fragment series is highlighted in blue. (D) An immunoprecipitation-Western blot from the zebrafish embryos injected with mRNA(s) for GST (left lane) or GST F52* (codon for F52 converted to stop codon UAG) with the orthogonal tRNAPyl-CUA -aaRS pair for AzK/BCNK, without (middle lane) or with (right lane) AzK. The immunoprecipitations and Western blots were performed with antibodies against GST.

Figure 3

The site-specific incorporation of bicylononyne-lysine and diazirine-lysine in the zebrafish embryos. (A) The Brightfield (left) or fluorescent (right) images of the zebrafish embryos injected with mRNA(s) for LifeAct-eGFP Y39* with the orthogonal tRNAPyl-CUA -mRNA aaRS for AzK/BCNK with (top panel) BCNK. Shown also are fields with multiple examples of each of the zebrafish embryos injected with mRNA(s) for LifeAct-eGFP Y39* with the orthogonal tRNAPyl-CUA -mRNA aaRS for AbK, with (middle panel) or without (bottom panel) AbK. (B) The localization of LifeAct-eGFP Y39BCNK (top panel) and LifeAct-eGFP Y39AbK to cell-cell junctions and cortical speckles in the dechorionized embryos injected as described in (A). (C) Shown in the top panel is an IP-Western blot from the zebrafish embryos injected with mRNA(s) for GST (left lane) or GFT F52* with the orthogonal tRNAPyl-CUA -aaRS pair for AzK/BCNK, without (middle lane) or with (right lane) BCNK. The bottom panel shows a similar experiment showing the incorporation of AbK into the zebrafish embryos using the appropriate amino-acid and the tRNAPyl-CUA –aaRS cocktail for AbK. The immunoprecipitations and Western blots were performed with antibodies against GST.

Acknowledgments:
ZFIN wishes to thank the journal International Journal of Molecular Sciences for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Int. J. Mol. Sci.