Journal of the American Chemical Society
Communication
this conjugate and Tz1-fluorescein diacetate. Following the
optimized protocol shown in Figure 3A, we observed labeling
signal specific to the nuclei of transfected cells, despite the
presence of LplA in both the cytosol and the nucleus. Negative
controls with TCO2 omitted, wild-type LplA, or an inactive
LAP mutant abolished labeling signals (Figure 3B). We also
examined the labeling specificity by lysing cells after Tz1-
fluorescein diacetate treatment and imaging the fluorescence of
the lysate after gel separation. Supporting Figure 8 shows that a
single protein corresponding to the size of LAP-BFP was
selectively labeled over the endogenous proteome.
ACKNOWLEDGMENTS
■
Funding was provided by the National Institutes of Health
(R01 GM072670 and P20 RR017716), the American Chemical
Society, the Dreyfus Foundation, and MIT. Spectra were
obtained with instrumentation supported by NSF grants CHE-
0840401 and CHE-0541775. We thank Chayasith Uttamapi-
nant and Jennifer Yao (MIT) for plasmids, reagents, and
helpful discussions, as well as Carolyn Kwa (MIT) for
assistance with neuron cultures. Masahito Yamagata (Harvard)
provided the neuroligin-1 plasmid. A.T. was funded by a
Novartis undergraduate fellowship.
We were unable to achieve fluorogenic labeling inside cells
because high fluorescence signal was observed inside
untransfected cells as well as cells free of TCO2 treatment,
immediately upon loading of both Tz1-fluorophore conjugates
(data not shown). We were, however, able to wash away off-
target dyes after 2 h. In COS-7 cells, where the required dye
washout time was shorter, we successfully labeled actin
filaments (LAP-β-actin) and intermediate filaments (vimen-
tin-LAP) with high specificity (Figure 3C). Supporting Figure 9
shows that actin and vimentin filaments labeled by Tz1-TMR
colocalized perfectly with filaments detected by immunofluor-
escence staining in the same cells, providing another indication
of labeling specificity.
In summary, we have found that the tetrazine−trans-
cyclooctene Diels−Alder cycloaddition is highly efficient for
fluorescence labeling of cell surface proteins and sufficiently
bioorthogonal for labeling of intracellular proteins. We utilized
this fast chemistry for the extension of PRIME to a panel of
useful fluorophores, including tetramethylrhodamine and Alexa
647, while retaining a level of specificity comparable to that
obtained with direct fluorophore ligation by PRIME.1 This
method is generally applicable to different proteins in various
cell types.
On the cell surface, we achieved fluorogenic labeling using
tetrazine−fluorescein but failed to accomplish fluorogenic
labeling with Alexa 647 because its red-shifted fluorescence
emission was not significantly quenched by Tz1 (Supporting
Figure 4B). Inside the cell, we observed a trade-off between the
reactivity and stability of two different tetrazine structures. We
surmise that, while monoaryl-substituted Tz1 is significantly
more reactive than diaryl Tz2 toward trans-cyclooctene, the
former is also more prone to cross-reactivity with endogenous
nucleophiles or dienophiles. Our study therefore illustrates the
need for next-generation tetrazines that are less kinetically
hindered by protective substitutions and more able to quench
the fluorescence of red dyes.
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ASSOCIATED CONTENT
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* Supporting Information
Synthesis and characterization of trans-cyclooctenes and
tetrazine−fluorophore conjugates; validation of trans-cyclo-
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cell imaging experiments and experimental details; and
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AUTHOR INFORMATION
■
Corresponding Author
Author Contributions
‡These authors contributed equally.
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dx.doi.org/10.1021/ja209325n | J. Am. Chem.Soc. 2012, 134, 792−795