Journal of the American Chemical Society
Communication
To investigate whether the rapid BCN−tetrazine ligation
would provide advantages for site-specific labeling of proteins
on mammalian cells, we expressed an epidermal growth factor
receptor (EGFR)−GFP fusion bearing an amber codon at
position 128 (EGFR(128TAG)GFP) in HEK-293 cells
containing the BCNRS/tRNACUA pair cultured in the presence
of 1 (0.5 mM). Full-length EGFR-1-GFP was produced in the
presence of 1, resulting in bright green fluorescence at the cell
membrane. To label 1 with tetrazine−fluorophore conjugates,
we incubated cells with 11 (400 nM), changed the medium,
and imaged the red fluorescence arising from TAMRA labeling.
The TAMRA fluorescence colocalized nicely with the cell-
surface EGFR-GFP fluorescence. Clear labeling of cells bearing
EGFR-1-GFP was observed within 2 min, the first time point
we could measure; additional time points demonstrated that
the labeling was saturated within 2 min (Figure 5b and Figures
S11−S14); similar results were obtained with 12. Incorporation
of 2 into the EGFR−GFP fusion led to similarly rapid and
efficient labeling with 11 (Figure 5b and Figures S15 and S16).
In contrast, it took 2 h before we observed any specific labeling
of cells bearing EGFR-4-GFP under identical conditions
(Figure S14).7 In control experiments we observed neither
labeling of cells bearing EGFR-5-GFP nor nonspecific labeling
of cells that did not express EGFR-GFP. We observed weak but
measurable labeling of EGFR-GFP expressed in HEK-293 cells
from EGFR(128TAG)GFP in the presence of the BCNRS/
tRNACUA pair and 3 (Figure S17). These observations are
consistent with the isomerization of a fraction of 3 in
mammalian cells and with our observations in E. coli.
To demonstrate the rapid labeling of an intracellular protein
in mammalian cells, we expressed a transcription factor, jun,
with a C-terminal mCherry fusion from a gene bearing an
amber codon in the linker between JunB (jun) and mCherry. In
the presence of amino acid 1 and the BCNRS/tRNACUA pair,
the jun-1-mCherry protein was produced in HEK cells and, as
expected, localized in the nuclei of cells (Figure 5c and Figure
S18). Labeling with cell-permeable conjugate 17 (200 nM)
resulted in green fluorescence that colocalized nicely with the
mCherry signal at the first time point analyzed (after 15 min of
labeling and 90 min of washing). No specific labeling was
observed in nontransfected cells in the same sample or in
control cells expressing jun-5-mCherry, further confirming the
specificity of intracellular labeling.
that it may be possible to extend the labeling approach
described here to site-specific protein labeling in animals.
ASSOCIATED CONTENT
* Supporting Information
■
S
Complete references and supplementary results and methods.
This material is available free of charge via the Internet at
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the MRC (Grants U105181009
and UD99999908) and the NIH (Grant P20 RR017716 from
the NCRR COBRE Program). We thank Han Zhang, Fiona
Townsley, and Trevor Rutherford for experimental assistance.
REFERENCES
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In conclusion, we have reported the exceptionally rapid
fluorogenic reaction of BCN with a range of tetrazines under
aqueous conditions at room temperature. The reaction between
a strained alkene (e.g., a norbornene or trans-cyclooctene) and
a tetrazine may lead to a mixture of diastereomers and
regioisomers as well as isomers from dihydropyridazine
isomerization.3,4 In contrast, the BCN−tetrazine reaction
leads to the formation of a single product. This will be an
advantage in applications where homogeneity in the orientation
of probe attachment may be important, including single-
molecule spectroscopy, super-resolution microscopy, and
FRET approaches.
We have demonstrated the efficient site-specific incorpo-
ration of 1 and 2 into proteins in E. coli and mammalian cells
and their efficient, specific, fluorogenic, and rapid labeling with
tetrazine fluorophore conjugates. While we have demonstrated
the advantages of this approach in vitro, in E. coli, and in live
mammalian cells, the ability to incorporate unnatural amino
acids in C. elegans using the PylRS/tRNACUA pair29 suggests
10320
dx.doi.org/10.1021/ja302832g | J. Am. Chem. Soc. 2012, 134, 10317−10320