Development of fluorogenic probes for quick No-Wash live-cell imaging of intracellular proteins

Article abstract of DOI:10.1021/ja405745v

We developed novel fluorogenic probes for no-wash live-cell imaging of proteins fused to PYP-tag, which is a small protein tag recently reported by our group. Through the design of a new PYP-tag ligand, specific intracellular protein labeling with rapid kinetics and fluorogenic response was accomplished. The probes crossed the cell membrane, and cytosolic and nuclear localizations of PYP-tagged proteins without cell washing were visualized within a 6-min reaction time. The fluorogenic response was due to the environmental effect of fluorophore upon binding to PYP-tag. Furthermore, the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization, movement, and function of cellular proteins.

Full text of DOI:10.1021/ja405745v

Article
pubs.acs.org/JACS
Development of Fluorogenic Probes for Quick No-Wash Live-Cell
Imaging of Intracellular Proteins
Yuichiro Hori,†,‡,§ Tomoya Norinobu,† Motoki Sato,† Kyohei Arita,∥ Masahiro Shirakawa,⊥
and Kazuya Kikuchi*,†,‡
‡
†Graduate School of Engineering, and Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan
§JST, PRESTO, Suita, Osaka 565-0871, Japan
∥Graduate School of Medical Life Science, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan
⊥Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
S
* Supporting Information
ABSTRACT: We developed novel fluorogenic probes for no-
wash live-cell imaging of proteins fused to PYP-tag, which is a
small protein tag recently reported by our group. Through the
design of a new PYP-tag ligand, specific intracellular protein
labeling with rapid kinetics and fluorogenic response was
accomplished. The probes crossed the cell membrane, and
cytosolic and nuclear localizations of PYP-tagged proteins
without cell washing were visualized within a 6-min reaction
time. The fluorogenic response was due to the environmental
effect of fluorophore upon binding to PYP-tag. Furthermore,
the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling
system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization,
movement, and function of cellular proteins.
Furthermore, because the removal of free fluorescence-
quenched probes is not necessary, prompt imaging is possible.
It is, however, still challenging to visualize intracellular proteins
by fluorogenic probes without a wash-out process. Successful
examples have been restricted to SNAP-tag,11−13 BL-tag,14 and
fluorogen-activating proteins (FAPs).15 However, most of these
reports showed some limitations. One of them is that special
treatment for probe introduction into cells was required.12
Another limitation is that long incubation caused the
nonspecific illumination of cellular components.15 Moreover,
in some of the reports, quick imaging of intracellular proteins
was not shown and labeling reactions were conducted with long
incubation time (2 h or more).11,14 Only silicon−rhodamine
(SiR) probes for SNAP-, CLIP-, and Halo-tags allowed protein
imaging with moderate incubation time (30 min).13 In this
study, by adopting a fluorogenic mechanism based on an
environmental-sensitive fluorophore, we created novel fluoro-
genic probes for specific rapid labeling of proteins inside living
cells with no-wash procedures to overcome these limitations.
We have previously reported a protein-labeling system using
Photoactive yellow protein (PYP) tag and its fluorogenic
probes, FCTP and FCANB.16,17 PYP-tag is a soluble protein
derived from purple bacteria.18 PYP-tag covalently binds to the
thioester derivative of cinnamic acid or coumarin through
INTRODUCTION
■
Protein labeling by synthetic fluorescence probes is a powerful
technique used to investigate protein function and localization
in living cells.1 Advances have been made in this field through
the development of specific pairs of fluorescence probes and
protein (peptide) tags, which allow live-cell imaging of proteins.
In this technique, objective proteins are fused with a protein
(peptide) tag and are labeled by a fluorescent probe through
the binding of the probe and tag. An early report of this
technique described the pairing of tetracystein tag and its
probes (FlAsH/ReAsH).2 Since then, protein-labeling systems
using various tags such as SNAP-tag,3 HaloTag,4 TMP-tag,5
LAP,6 Oligo-Asp tag,7 coiled-coil tag,8 and BL-tag9,10 have been
developed. This method has attracted attention as an
alternative to fluorescent proteins because it has the advantage
of various bright fluorophores that can be incorporated into
probes. In addition, the timing of the labeling can be easily
controlled, allowing for precise spatiotemporal analyses of
protein movement. On the other hand, unlabeled probes
remaining inside cells have the potential to cause an increase in
the background fluorescence and hinder the identification of
labeled proteins. Therefore, rigorous washing of cells is
required to remove free probes.
Recently, fluorogenic probes have been created as a solution
to this problem.2,10−16 These probes exhibit only low
background fluorescence in a free state and, as a result, enable
live-cell imaging of proteins with a high signal-to-noise ratio.
Received: June 8, 2013
© XXXX American Chemical Society
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transthioesterification with Cys69.16,17,19 The attractive feature
of this protein tag is its small size (125 amino acids). FCTP is
nonfluorescent because of the intramolecular association
between ligand and fluorophore moieties, and it becomes
fluorescent through the dissociation of the dyes upon protein
labeling. This probe requires more than 24 h for full PYP-tag
labeling. Owing to the slow labeling kinetics, a wash-out
process is necessary for protein imaging. FCANB was designed
to improve the labeling kinetics and enable the imaging of cell
surface proteins without a cell-washing procedure. Unfortu-
nately, wash-free imaging of intracellular proteins has not been
possible because the probe is not cell-permeable.
To solve this problem, we developed cell-permeable
fluorogenic probes that label PYP-tagged proteins expressed
inside living cells with a short incubation time. The probes
contain a novel PYP-tag ligand with a fluorogenic mechanism,
which is based on the environment sensitivity of a fluorophore.
The fluorescence was enhanced by the capture of the probes
into the low-polar pocket of PYP-tag. This mechanism allowed
us to design quencher-free fluorogenic probes with a simple
structure that can cross the cell membrane and rapidly label
intracellular proteins. In addition, this system was applied to the
imaging of localization of methyl-CpG-binding domain, MBD,
which is known to bind DNA containing 5-methylcytosine.20
Using this labeling method, DNA methylation in living cells
was successfully visualized.
was thought that a PYP-tag probe with this structure would
weakly fluoresce in aqueous buffer and emit strong fluorescence
when the probe binds to PYP-tag and, thereby, is brought into
protein interior, which is generally in a low-polar environment.
Based on this idea, we designed novel probes, TMBDMA and
CMBDMA, by incorporating trimethylamine or carboxylic acid
into the coumarin thioester derivative to increase water
solubility (Figure 1b). The ligand moiety, 7-dimethylamino-
coumarin-3-carboxylic acid, was synthesized from p-dimethyla-
minosalicylaldehyde in three steps and was then coupled with
2-(4-mercaptobenzamido)-N,N,N-trimethylethanaminium or 4-
(carboxymethyl)thiophenol to yield TMBDMA or CMBDMA,
respectively (Schemes S1, S2).
Labeling Reactions with Fluorogenic Response. First,
in vitro experiments were conducted to examine whether the
probes bind to PYP-tag. The labeling reactions of PYP-tag with
TMBDMA or CMBDMA were analyzed by SDS-PAGE. In
both cases, fluorescence was observed from the band of PYP-
tag (Figure 2a, b). This result demonstrates that these probes
RESULTS AND DISCUSSION
■
Probe Design. For no-wash imaging of intracellular
proteins, we explored a new PYP-tag ligand, which crosses
the cell membrane and shows a fluorogenic response upon
protein labeling. To this end, we focused on ligand structures,
which were known to be accommodated by PYP-tag. According
to previous reports, the protein binds to 4-dimethylaminocin-
namic acid thioester as well as 4-hydroxycinnamic acid and 7-
hydroxycoumarin thioesters.21 Considering the structural
similarity between cinnamic acid and coumarin structures, we
hypothesized that 7-dimethylaminocoumarin thioester deriva-
tives could bind to PYP-tag (Figure 1a). Importantly, 7-
dialkylaminiocoumarin derivatives are environment-sensitive
fluorophores, which are scarcely fluorescent in polar solvents
but become fluorescent in low-polar solvents.22 Therefore, it
Figure 2. Labeling reactions of PYP-tag with TMBDMA and
CMBDMA. PYP-tag (15 μM) was reacted with TMBDMA (25 μM)
(a, c) or with CMBDMA (25 μM) (b, d). The reactions were carried
out in the absence (a, b) or presence (c, d) of cell lysate. Images of
CBB-stained and fluorescence gel are shown on the left and right,
respectively.
bind to PYP-tag, and 7-dimethylaminocoumarin is accommo-
dated as a ligand by PYP-tag. The binding mode was
considered to be covalent because the reaction mixture was
denatured by SDS and heat shock prior to analyses. To
investigate the binding specificity, labeling reactions were
carried out in cell lysate prepared from HEK293T. Although no
fluorescence was detected in the gel when the reaction mixtures
were incubated in cell lysate without PYP-tag, a clear single
fluorescent band appeared in a gel lane when the cell lysate
containing PYP-tag and each of the probes were loaded (Figure
2c, d). The band position was consistent with the molecular
weight of PYP-tag. Because glutathione is the most abundant in
living cells among thiol compounds, which may compete with
the labeling reactions, the effect of glutathione was also
examined. Even in the presence of physiological concentrations
Figure 1. (a) Principle of labeling system based on PYP-tag and
environment-sensitive fluorogenic probe. (b) Structures of new probes
for labeling PYP-tag.
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of glutathione (up to 10 mM),23 the probes were found to label
PYP-tag, although the fluorescence intensity of the labeled band
was slightly reduced (Figure S1). These results indicate that the
probes specifically bind to PYP-tag even in the presence of
various biomolecules.
126 M−1 s−1) was comparable to that of FCANB (Table 1,
Figure S5).
Despite the similar probe structures of TMBDMA and
CMBDMA, TMBDMA showed better kinetic feature than
CMBDMA. There are two possible reasons for this difference.
One is that electrostatic interaction occurs between PYP-tag
and the probes. PYP-tag is anionic under the physiological
conditions because the pI of the protein is 4.3.24 This finding
implies that the interaction of PYP-tag with cationic TMBDMA
is preferable to anionic CMBDMA. The other probable reason
is that TMBDMA contains a better leaving group than
CMBDMA. When Cys69 in PYP-tag attacks the thioester of
the probe, a thiophenyl compound is released from the probe.
Its leaving ability correlates with the labeling kinetics and is
dependent on the pKa of the thiol. The leaving group of
TMBDMA could have the thiol with a lower pKa than
CMBDMA because the leaving group of TMBDMA is
composed of thiophenol directly connected to an electron-
withdrawing carbonyl group, whereas the corresponding
thiophenol in CMBDMA is linked with a methylene group.
Accordingly, it is suggested that lowering of the pKa results in
increased kinetic constant of TMBDMA. The effect of charge
and pKa on labeling kinetics can also explain why TMBDMA
shows faster kinetics than FCANB, because FCANB is anionic
and contains a thiophenol leaving group linked with a
methylene group.
Next, spectral studies were conducted (Figure 3, Table 1,
Figure S2). Fluorescence spectra were recorded to clarify the
Figure 3. Fluorescence analyses for labeling reactions of PYP-tag with
its probes. (a) Fluorescence spectra of TMBDMA (red lines) and
CMBDMA (blue lines) in the absence (dotted lines) or presence
(solid lines) of PYP-tag. (b) Time course of fluorescence intensity of
TMBDMA (red circles) and CMBDMA (blue circles) at 487 nm in
the absence (disks) or presence (circles) of PYP-tag. The measure-
ments were conducted using samples in HEPES buffer (20 mM
HEPES, 150 mM NaCl (pH 7.4)) at 37 °C. The protein and probe
concentrations were 5 μM.
Live-Cell Imaging of PYP-Tagged Proteins on Cell
Surface. No-wash live-cell imaging of cell surface proteins on
cell membrane was carried out using TMBDMA and
CMBDMA. For the labeling of cell surface proteins, a PYP-
tag-encoding construct was prepared by fusing PYP-tag to the
epidermal growth factor receptor at the N-terminal extracellular
domain (PYP-EGFR). The protein expression on plasma
membrane of HEK293T cells was ascertained by immuno-
fluorescence staining as described in our previous report.17 The
probes were incubated with the cells for 30 min, and then
fluorescence images were taken with a confocal laser scanning
microscope without washing the cells. Fluorescence was
observed along the plasma membrane of the cells expressing
PYP-EGFR (Figure 4a). In contrast, nontransfected cells in the
image were not stained, and fluorescence was hardly detected in
the culture media. Additionally, nonspecific labeling was not
detected from cells expressing EGFR without PYP-tag. Similar
results were obtained with CMBDMA (Figure S6). When cells
were washed after the labeling reactions, the images displayed
Table 1. Spectral and Kinetic Properties of PYP-tag Probes
λabs
(nm)
λem
(nm)
ε
a
probe
(M−1 cm−1
)
Φf
k2 (M−1 s−1
)
TMBDMA
CMBDMA
451
449
450
501
499
487
34600
36400
27800
0.02
0.02
0.38
3950
126

b
PYP-probe
a
b
All data were obtained in triplicate experiments. Spectroscopic data
of PYP-tag-bound probe were obtained after the labeling reaction of
PYP-tag with CMBDMA was completed.
fluorescence properties of the probes. Both probes exhibited
only weak fluorescence in the absence of PYP-tag (TMBDMA,
Φf = 0.02; CMBDMA, Φf = 0.02) (Table 1). The binding of
the probes with PYP-tag induced remarkable fluorescence
enhancement (Figure 3a). Upon protein labeling, the
fluorescence intensity of both probes at 487 nm increased 22-
fold and 16-fold, respectively. There was no substantial change
of the fluorescence intensity of the probes in the absence of
PYP-tag during the 3-h incubation period (Figure S3). These
results demonstrate that both TMBDMA and CMBDMA are
fluorogenic probes for labeling PYP-tag. In addition, this
fluorescence response suggests that the dimethylaminocoumar-
in was captured into the low-polar binding pocket of PYP-tag.
Kinetic Analyses of Labeling Reactions. The kinetic
experiments of the protein labeling were conducted by
monitoring the fluorescence intensity of the probes. TMBDMA
labeled 50% of PYP-tag for 1.1 min (t1/2) under an
experimental condition, in which the protein and probe
concentrations were 5 μM (Figure 3b). On the other hand,
CMBDMA required a longer reaction time (t1/2 = 13 min)
under the same experimental conditions (Figure 3b, Figure S4).
For further detailed kinetic analyses, the second-order rate
constant for reactions between PYP-tag and TMBDMA/
CMBDMA was determined. TMBDMA showed a remarkable
value (k2 = 3950 M−1 s−1) that was 32-fold higher than FCANB
(k2 = 125 M−1 s−1).17 By contrast, the value of CMBDMA (k2 =
Figure 4. Live-cell imaging of PYP-EGFR by utilizing TMBDMA (1
μM) without (a) or with (b) a wash-out step. Images of PYP-EGFR-
and EGFR-expressing HEK293T cells are shown in upper and lower
panels, respectively. Fluorescence images and their overlays on phase
contrast images are displayed in the left and right of each panel,
respectively. The images were obtained with the excitation at 473 nm
by using a 490−590 emission filter. Scale bar: 10 μm.
C
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fluorescence localization, which were essentially similar to the
images acquired by the no-wash protocol (Figure 4b). These
results indicate that the probes specifically imaged PYP-tagged
proteins on the cell surface without washing.
Quick Imaging of Intracellular Proteins without
Wash-Out Process. We investigated whether the probes
pass through the cell membrane and label intracellular proteins
without washing cells. Genes encoding PYP-tag fused to
maltose-binding protein (MBP-PYP), and nuclear localization
signals (PYP-NLS) were created and expressed in HEK293T
cells. From the immunofluorescence images, MBP-PYP was
expressed mainly in the cytosol (Figure S7a), and PYP-NLS
was localized to the nuclei, as expected (Figure S7b). Live-cell
images were obtained with a no-wash protocol immediately
after TMBDMA was incubated with cells expressing MBP-PYP
for 30 min. As a result, fluorescence was detected mostly in the
cytosol of the cells (Figure 5a). This localization pattern
Figure 6. Time-lapse live-cell imaging of PYP-NLS with TMBDMA.
(a) Measurement was made every 1 min after the addition of the
probe (1 μM). PYP-NLS was expressed in HEK293T cells. The images
were obtained with the excitation at 473 nm by using a 490−590
emission filter. (b) Plots of average fluorescence intensity of
TMBDMA against incubation time (N = 3). Scale bar: 5 μm.
Figure 5. No-wash live-cell imaging of intracellular proteins with
TMBDMA. PYP-tagged MBP or NLS-fused PYP-tag was localized to
cytosol (a) or nuclei (b) of HEK293T cells and was labeled with
TMBDMA (1 μM). Fluorescence images and their overlays on phase
contrast images are displayed in the left and right of each panel,
respectively. The images were obtained with the excitation at 473 nm
by using a 490−590 emission filter. Scale bar: 10 μm.
to that of previously reported fluorogenic protein-labeling
probes, SiR derivatives.13
Imaging Analyses of DNA Methylation in Nuclei. DNA
methylation is an important epigenetic reaction, which controls
gene expression.25 Methylation occurs in the 5-position of
cytosine and is typically observed in CpG sequences. DNA
sequences rich in 5-methylcytosine (5-mC) generally form
heterochromatin, in which gene expression is suppressed.
Methyl-CpG-binding domain 1 (MBD1) specifically binds to 5-
mC20 and is involved with heterochromatin formation. In
mammals, this epigenetic modification plays a significant role in
the homeostasis of somatic cells, the differentiation of
embryonic and induced-pluripotent stem cells (ES and iPS
cells), and carcinogenesis.25 Hence, the live-cell data of the
DNA methylation state offers valuable epigenetic information.
We applied the probes to the imaging of 5-mC by labeling the
fusion protein of PYP-tag and MBD1 (1-112) (PYP-MBD).
After NIH3T3 cells were transfected with the gene of PYP-
MBD, incubation with TMBDMA or CMBDMA was carried
out for 10 or 15 min, respectively. Fluorescence signals were
clearly detected as characteristic puncta in the nuclei (Figure
7a). The localization of the fluorescence overlapped with
Hoechst33342. Since Hoechst33342 mainly stains heterochro-
matin,26 which is rich in 5-mC, it is suggested that PYP-MBD
was predominantly localized to heterochromatin in these cells.
This result is consistent with a previous report.27
coincided with the results of immunofluorescence staining. No
cell expressing MBP without PYP-tag exhibited fluorescence.
Specific labeling of PYP-NLS by TMBDMA was also observed
(Figure 5b). Co-staining of the cells with Hoechst 33342 and
TMBDMA confirmed that the probe visualized the nuclear
localization of PYP-NLS (Figure S8). CMBDMA also imaged
the specific localization of MBP-PYP and PYP-NLS, as with the
images obtained with TMBDMA (Figure S9). This is surprising
for us because an anionic probe does not efficiently permeate
the cell membrane in general. All of the no-wash imaging data
were indistinguishable from those obtained in labeling
experiments with a washing step (Figure S10). These results
clearly show that both of the probes are cell-permeable and
specifically label intracellular PYP-tag fusion protein in targeted
subcellular regions without the requirement of washing.
We performed time-lapse imaging experiments to determine
the incubation time necessary for fluorescence detection of
PYP-tag inside cells (Figure 6, Figure S11). After addition of
TMBDMA to cells expressing PYP-NLS, fluorescence images
were periodically recorded. Fluorescence started to appear in
nuclei just 2 min after addition of the probe. The labeling
reaction was almost completed within 6 min. In the case of
CMBDMA, the fluorescence intensity was saturated 30 min
after the addition of the probe. Consistent with in vitro kinetic
analyses, it took longer to finish the labeling reaction in
comparison with TMBDMA. Nevertheless, the incubation time
of CMBDMA for labeling intracellular proteins was comparable
DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-
azadC), was used to examine whether the localization pattern
of the fusion protein was altered. This inhibitor is mis-
incorporated into DNA instead of dCTP, and it inactivates
DNA methyltransferases by trapping the enzyme.28 Thus, the
incubation of the inhibitor during cell division leads to the
suppression of DNA methylation. In the presence of 5-azadC,
fluorescence derived from the complex between the probes and
D
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high signal-to-noise ratio. This protein-labeling system offers a
practical method for the analyses of protein expression and
localization, and it will be an attractive tool for verifying
versatile biological events, including epigenetic phenomena.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and supplemental results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
kkikuchi@mls.eng.osaka-u.ac.jp
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JST, PRESTO, by MEXT of Japan
(Grants 20675004, 24108724, 25220207, and 25620133 to
K.K. and 22685016, 25560403 to Y.H.), by the Funding
Program for World-Leading Innovative R&D on Science and
Technology from JSPS, by CREST from JST, and by Asahi
Glass Foundation.
Figure 7. Live-cell imaging of localization of PYP-MBD in the absence
(a) or presence (b) of 5-azadeoxycytidine. PYP-MBD was expressed in
NIH3T3 cells. Images of cells treated with TMBDMA and CMBDMA
are shown in upper and lower panels, respectively. Fluorescence
derived from PYP-MBD and Hoechst33342 is displayed as green and
red colors, respectively. In their overlay images, orange colors indicate
the overlap of the localizations of PYP-MBD and Hoechst33342. The
images were obtained with the excitation at 473 nm by using a 490−
590 emission filter. Scale bar: 10 μm.
REFERENCES
■
(1) (a) Jing, C.; Cornish, V. W. Acc. Chem. Res. 2011, 44, 784−792.
(b) Yano, Y.; Matsuzaki, K. Biochim. Biophys. Acta 2009, 1788, 2124−
2131.
(2) (a) Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.;
Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien, R. Y. J. Am. Chem. Soc. 2002,
124, 6063−6076. (b) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science
1998, 281, 269−272.
(3) (a) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R., Jr.;
Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128−
136. (b) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel,
H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86−89.
PYP-MBD was detected in some spots, which were not stained
with Hoechst33342 (Figure 7b). These results clearly indicate
that PYP-MBD was bound to 5-mC-containing DNA in the
absence of 5-azadC and was released from the DNA by the
addition of the inhibitor. In other words, this observation
indicates that DNA methylation was imaged by monitoring the
localization of PYP-MBD. Importantly, the probe precisely
visualized PYP-MBD in living cells. The precision of the
imaging analyses was accomplished by the simple probe design
that solved the problem of cell-permeability, labeling specificity,
labeling kinetics, and fluorogenic property for protein-labeling
probes.
(4) (a) Los, G. V.; Wood, K. Methods Mol. Biol. 2007, 356, 195−208.
(b) Lee, H. L.; Lord, S. J.; Iwanaga, S.; Zhan, K.; Xie, H.; Williams, J.
C.; Wang, H.; Bowman, G. R.; Goley, E. D.; Shapiro, L.; Twieg, R. J.;
Rao, J.; Moerner, W. E. J. Am. Chem. Soc. 2010, 132, 15099−15101.
(5) (a) Chen, Z.; Jing, C.; Gallagher, S. S.; Sheetz, M. P.; Cornish, V.
W. J. Am. Chem. Soc. 2012, 134, 13692−13699. (b) Miller, L. W.; Cai,
Y.; Sheetz, M. P.; Cornish, V. W. Nat. Methods 2005, 2, 255−257.
(6) (a) Yao, J. Z.; Uttamapinant, C.; Poloukhtine, A.; Baskin, J. M.;
Codelli, J. A.; Sletten, E. M; Bertozzi, C. R.; Popik, V. V.; Ting, A. Y. J.
Am. Chem. Soc. 2012, 134, 3720−3728. (b) Uttamapinant, C.; White,
CONCLUSION
■
́ ́
K. A.; Baruah, H.; Thompson, S.; Fernandez-Suarez, M.; Puthenveetil,
We created novel PYP-tag-probes with a fluorogenic ligand, 7-
dimethylaminocoumarin. The labeling rate of TMBDMA
dramatically accelerated in comparison with a previous probe,
FCANB. Furthermore, no-wash labeling of intracellular
proteins as well as cell surface proteins was successfully
achieved, and the localization of the proteins was specifically
visualized. Importantly, clear live-cell images were acquired in a
short incubation time (<6 min). Moreover, the probes were
utilized for the imaging of MBD localization, and DNA
methylation was visualized by combining the labeling method
with inhibitor analyses. As far as we know, TMBDMA is the
most rapid imaging probe for intracellular proteins among
reported fluorogenic probes that covalently label protein tags
inside living cells.
S.; Ting, A. Y. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10914−10919.
(7) Nonaka, H.; Fujishima, S. H.; Uchinomiya, S. H.; Ojida, A.;
Hamachi, I. J. Am. Chem. Soc. 2010, 132, 9301−9309.
(8) (a) Ono, S.; Yano, Y.; Matsuzaki, K. Biopolymers 2012, 98, 234−
238. (b) Hori, Y.; Egashira, Y.; Kamiura, R.; Kikuchi, K. Chembiochem
2010, 11, 646−668. (c) Tsutsumi, H.; Nomura, W.; Abe, S.; Mino, T.;
Masuda, A.; Ohashi, N.; Tanaka, T.; Ohba, K.; Yamamoto, N.;
Akiyoshi, K.; Tamamura, H. Angew. Chem., Int. Ed. 2009, 48, 9164−
9166.
(9) (a) Mizukami, S.; Yamamoto, T.; Yoshimura, A.; Watanabe, S.;
Kikuchi, K. Angew. Chem., Int. Ed. 2011, 50, 8750−8752. (b) Watanabe,
S.; Mizukami, S.; Akimoto, Y.; Hori, Y.; Kikuchi, K. Chem.Eur. J.
2011, 17, 8342−8349.
(10) (a) Watanabe, S.; Mizukami, S.; Hori, Y.; Kikuchi, K.
Bioconjugate Chem. 2010, 21, 2320−2326. (b) Mizukami, S.;
Watanabe, S.; Hori, Y.; Kikuchi, K. J. Am. Chem. Soc. 2009, 131,
5016−5017.
The pair of PYP-tag and the fluorogenic probes contains the
advantage of a small tag size, rapid in-cell-labeling kinetics, and
E
dx.doi.org/10.1021/ja405745v | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(11) (a) Komatsu, T.; Johnsson, K.; Okuno, H.; Bito, H.; Inoue, T.;
Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2011, 133, 6745−6751.
(b) Zhang, C. J.; Li, L.; Chen, G. Y.; Xu, Q. H.; Yao, S. Q. Org. Lett.
2011, 13, 4160−4163.
(12) Sun, X.; Zhang, A.; Baker, B.; Sun, L.; Howard, A.; Buswell, J.;
Maurel, D.; Masharina, A.; Johnsson, K.; Noren, C. J.; Xu, M. Q.;
Correa
(13) Lukinavici
Yang, G.; Plass, T.; Mueller, V.; Reymond, L.; Correa
̂
, I. R., Jr. Chembiochem 2011, 12, 2217−2226.
us, G.; Umezawa, K.; Olivier, N.; Honigmann, A.;
, I. R., Jr.; Luo, Z.
̌
̂
G.; Schultz, C.; Lemke, E. A.; Heppenstall, P.; Eggeling, C.; Manley, S.;
Johnsson, K. Nat. Chem. 2013, 5, 132−139.
(14) Mizukami, S.; Watanabe, S.; Akimoto, Y.; Kikuchi, K. J. Am.
Chem. Soc. 2012, 134, 1623−1629.
(15) Szent-Gyorgyi, C.; Schmidt, B. F.; Creeger, Y.; Fisher, G. W.;
Zakel, K. L.; Adler, S.; Fitzpatrick, J. A.; Woolford, C. A.; Yan, Q.;
Vasilev, K. V.; Berget, P. B.; Bruchez, M. P.; Jarvik, J. W.; Waggoner, A.
Nat. Biotechnol. 2008, 26, 235−240.
(16) Hori, Y.; Ueno, H.; Mizukami, S.; Kikuchi, K. J. Am. Chem. Soc.
2009, 131, 16610−16611.
(17) Hori, Y.; Nakaki, K.; Sato, M.; Mizukami, S.; Kikuchi, K. Angew.
Chem., Int. Ed. 2012, 51, 5611−5614.
(18) (a) Kumauchi, M.; Hara, M. T.; Stalcup, P.; Xie, A.; Hoff, W. D.
Photochem. Photobiol. 2008, 84, 956−969. (b) Meyer, T. E. Biochim.
Biophys. Acta 1985, 806, 175−183.
(19) (a) Cordfunke, R.; Kort, R.; Pierik, A.; Gobets, B.; Koomen, G.
J.; Verhoeven, J. W.; Hellingwerf, K. J. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 7396−7401. (b) Hoff, W. D.; Devreese, B.; Fokkens, R.;
Nugteren-Roodzant, I. M.; Van Beeumen, J.; Nibbering, N.;
Hellingwerf, K. J. Biochemistry 1996, 35, 1274−1281. (c) Imamoto,
Y.; Ito, T.; Kataoka, M.; Tokunaga, F. FEBS Lett. 1995, 374, 157−160.
(20) Clouaire, T.; Stancheva, I. Cell. Mol. Life Sci. 2008, 65, 1509−
1522.
(21) Kroon, A. R.; Hoff, W. D.; Fennema, H. P.; Gijzen, J.; Koomen,
G. J.; Verhoeven, J. W.; Crielaard, W.; Hellingwerf, K. J. J. Biol. Chem.
1996, 271, 31949−31956.
(22) (a) Nad, S.; Kumbhakar, M.; Pal, H. J. Phys. Chem. A 2003, 107,
4808−4816. (b) Jones, G.; Jackson, W. R.; Kanoktanaporn, S.;
Halpern, A. M. Opt. Commun. 1980, 33, 315−320.
(23) Wu, G.; Fang, Y. Z.; Yang, S.; Lupton, J. R.; Turner, N. D. J.
Nutr. 2004, 134, 489−492.
(24) McRee, D. E.; Meyer, T. E.; Cusanovich, M. A.; Parge, H. E.;
Getzoff, E. D. J. Biol. Chem. 1986, 261, 13850−13851.
(25) (a) Cedar, H.; Bergman, Y. Nat. Rev. Genet. 2009, 10, 295−304.
(b) Delcuve, G. P.; Rastegar, M.; Davie, J. R. J. Cell. Physiol. 2009, 219,
243−250.
(26) (a) Amirand, C.; Viari, A.; Ballini, J. P.; Rezaei, H.; Beaujean, N.;
Jullien, D.; Kas, E.; Debey, P. J. Cell Sci. 1998, 111, 3551−3561.
̈
(b) Zhang, W. H.; Srihari, R.; Day, R. N.; Schaufele, F. J. Biol. Chem.
2001, 276, 40373−40376.
(27) Kobayakawa, S.; Miike, K.; Nakao, M.; Abe, K. Genes Cells 2007,
12, 447−460.
(28) Juttermann, R.; Li, E.; Jaenisch, R. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 11797−11801.
F
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