Angewandte
Chemie
for 4 h with the first UAA (TCO*) which was chased with 4 h
of incubation with the second UAA (BCN). Living cells
expressing IRTAG were labeled by incubation with first azide-
Cy5 (10 min) and then with Methyl-Tet-Cy3 (Me-Tet-Cy3)
(10 min). As can be seen in Figure 3b (first panel), confocal
plasma membrane for both channels. SCO selectively reacts
with H-Tet but not Me-Tet on the timescale of our experi-
ments and thus this reaction is orthogonal to the SPIEDAC
between TCO* and Me-Tet. We dub this subreaction type
selectivity-enhanced SPIEDAC (seSPIEDAC). Note that as
TCO* is highly reactive with both Me-Tet and H-Tet,
experimental conditions must be chosen to ensure that all
TCO* is consumed before proceeding to the second labeling
step (see Figure 4(SI) for details). Furthermore, we show in
Figure 2d and Figure 4(SI) that increasing the speed of
seSPIEDAC by using the highly ring-strained BCN instead of
SCO is not possible due to the reactivity of BCN towards Me-
Tet (Figure 1(SI) and Note 2(SI); see also Figure 1).
Since Cy5 and Atto532 are commonly used for local-
ization-based SRM, we performed dual-color SRM measure-
ments.[11] The confocal (Figure 3) and widefield images
showed overlapping plasma membrane staining of IR in
both colors after dual-color labeling the TCO* and SCO.
However, SRM revealed a heterogeneous distribution of IR
clusters at the membrane (Figure 4a). Notably, clustering of
other growth factor receptors has also been observed in
previous SRM studies.[12]
To demonstrate the generality of our approach, we
labeled virus-like particles (VLPs) assembled by the co-
expression of influenza virus proteins hemagglutinin (HA)
and matrix protein 1 (M1) (for a review see Ref. [13]). Viral
genomes are compact and often contain overlapping genes,
which makes inserting genetically encoded tags into viral
proteins a particular challenge. We generated a TAG mutant
Figure 3. a) Outline of the expression and labeling scheme employed
for dual-color labeling of IRTAG. b) Confocal images of dual-color
labeling of IR with different combinations of UAAs and dyes. The left
panels show a combination of SPIEDAC (TCO*/Me-Tet-Cy3 and
SPAAC labeling (BCN/azide-Cy5). The right panels show a combination
of SPIEDAC (TCO*/Me-Tet-Cy5) and seSPIEDAC (SCO/H-Tet-Atto532).
c) Virus-like particles (VLPs) visualized by dual-color labeling achieved
by SPIEDAC (TCO*/Me-Tet-Cy5; top) and seSPIEDAC (SCO/H-Tet-
Atto532; bottom). Scale bars are 20 mm.
of HA and expressed it together with M1 and the tRNAPyl
/
PylRSAF in HEK293T cells. We repeated the pulse–chase
protocol using TCO*, SCO as UAAs and Me-Tet-Cy5 and H-
Tet-Atto532 as fluorescence labels. As shown in Figure 3c,
Atto532- and Cy5-stained filamentous protrusions, corre-
sponding to assembled VLPs, became visible. The enhanced
resolution of SRM makes it possible to visualize individual
filaments (Figure 4b). Notably, there is significant spatial
overlap between the two colors, suggesting that proteins
translated at different times are incorporated into the same
assembling VLPs.
imaging was used to visualize the membrane staining of
IRTAG !TCO* with Me-Tet-Cy3 from the SPIEDAC reaction.
The short incubation of IRTAG !BCN with azide-Cy5 gave no
results. As detailed in Figure 3(SI), this could be explained by
the speed of the SPAAC reaction, which is three to four
orders of magnitudes slower than the SPIEDAC reaction. We
therefore set out to find an alternative fast orthogonal click
reaction.
We observed previously that tetrazines can react with
strained alkene and alkyne UAAs (Figure 1).[3a,c] Due to the
markedly different reaction properties of strained alkynes
and alkenes,[4a] we reasoned that it might be possible to
identify tetrazine-UAA combinations that permit orthogonal
labeling. The previously developed cyclooctynyl-lysine deriv-
ative (SCO) is accepted by the same tRNAPyl/PylRSAF pair
and in similar yields as TCO* (Figure 2a,b).[3b] While TCO*
reacts with H-Tet and Me-Tet with reactions rates exceeding
1000mꢀ1 sꢀ1 in in vitro kinetic assays and labeling experiments,
SCO shows no substantial reactivity with Me-Tet under the
tested conditions (see Figure 1(SI) for reaction kinetics;
Figure 2d and Figure 1). However, the SPIEDAC reaction of
SCO with H-Tet is still about two orders of magnitude faster
than the SPAAC reaction of BCN with azide.[3a,10] We
repeated the pulse–chase experiment with TCO* and SCO
(Figure 3a), followed by labeling with Me-Tet-Cy5, and then
H-Tet-Atto532 for 10 min. As shown in Figure 3b, this
combination resulted in the bright labeling of the IR in the
In summary, we have tuned the genetically encoded
SPIEDAC reaction into two mutually orthogonal SPIEDAC
reactions which can be used to perform rapid labeling of
proteins in living cells. In this sense, this expands the existing
repertoire of biocompatible click labeling methods using an
expanded genetic code from SPIEDAC and SPAAC to
SPIEDAC, seSPIEDAC, and SPAAC. The exact origin of
the orthogonality achieved between seSPIEDAC and SPIE-
DAC is still subject to further investigations, such as
theoretical calculations.[4a] The two rapid SPIEDAC reactions
allowed SRM-compatible dual-color-labeling experiments in
mammalian cells, while the slow reactivity of SPAAC seemed
insufficient for rapid high-contrast labeling of live cells.
TCO* offers higher biostability and incorporation effi-
ciency than the original TCO. TCO* reacts rapidly with both
tested tetrazines (Me-Tet, H-Tet), while BCN is much less
reactive with Me-Tet. Under the experimental conditions,
SCO reacted with H-Tet only and not with Me-Tet. Dual-
color labeling was achieved using a promiscuous tRNA/RS
Angew. Chem. Int. Ed. 2014, 53, 2245 –2249
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2247
Nikic, Ivana
