A Rapid SNAP-Tag Fluorogenic Probe Based on an Environment

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A Rapid SNAP-tag Fluorogenic Probe Based on EnvironmentSensitive Fluorophore for No-Wash Live Cell Imaging Tao-Kai Liu, Pei-Ying Hsieh, Yu-De Zhuang, Chi-Yang Hsia, Chi-Ling Huang, Hsiu-Ping Lai, Hung-Sheung Lin, I-Chia Chen, Hsin-Yun Hsu, and Kui-Thong Tan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500502n • Publication Date (Web): 08 Aug 2014 Downloaded from http://pubs.acs.org on August 12, 2014

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ACS Chemical Biology

A Rapid SNAP-tag Fluorogenic Probe Based on EnvironmentSensitive Fluorophore for No-Wash Live Cell Imaging Tao-Kai Liu,†,§ Pei-Ying Hsieh,+,§ Yu-De Zhuang,† Chi-Yang Hsia,† Chi-Ling Huang,† Hsiu-Ping Lai,† Hung-Sheung Lin,† I-Chia Chen,†,‡ Hsin-Yun Hsu,*,#,+ Kui-Thong Tan*,†,‡

†

Department of Chemistry, National Tsing Hua University, 101 Sec. 2, Kuang-Fu Rd,

Hsinchu 30013, Taiwan (ROC) ‡

Frontier Research Center on Fundamental and Applied Sciences of Matters, National

Tsing Hua University, 101 Sec. 2, Kuang-Fu Rd, Hsinchu 30013, Taiwan (ROC) #

Department of Applied Chemistry and Institute of Molecular Science, National Chiao-

Tung University, No.1001 Ta-Hsueh Road, Hsinchu 30010, Taiwan (ROC) +

Center for Interdisciplinary Science (CIS), National Chiao-Tung University, No.1001 Ta-

Hsueh Road, Hsinchu 30010, Taiwan (ROC)

1 ACS Paragon Plus Environment


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Abstract

One major limitation of labeling proteins with synthetic fluorophores is the high fluorescence background which necessitates extensive washing steps to remove unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on environment-sensitive fluorophore for the labeling with SNAP-tag proteins. The probe exhibits dramatic fluorescence turn-on of 280-folds upon being labeled to SNAP-tag. The major advantages of our fluorogenic probe are the dramatic fluorescence turn-on, ease of synthesis, high selectivity and rapid labeling with SNAP-tag. No-wash labeling of both intracellular and cell surface proteins were successfully achieved in living cells and the localization of these proteins was specifically visualized.


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Introduction

Labeling proteins with synthetic fluorescent molecules is a powerful technique to study protein localization, trafficking and function in live cells. 1 ,

2

As compared with the

fluorescent proteins, protein labeling allows for the incorporation of a wide range of fluorophores with superior photophysical properties, coupled with the precise control of the location and timing of labeling by the appropriate regulation of probe delivery. 3, 4 However, to achieve high signal-to-noise ratio for visualization and quantification, extensive and time consuming washing of cells to remove unreacted fluorophore is required, rendering the continuous monitoring of the dynamics of protein pools virtually impossible.

Fluorogenic probes, which exhibit fluorescent enhancement upon labeling to the protein tag, are desirable in protein labeling as it helps to reduce labeling background and substantially enhances the signal-to-noise ratio. However, most of the fluorogenic probes reported to date have some limitations. For example, while the labeling of tetracysteine-tag with its biarsenical probes (FIAsH/ReAsH) shows a dramatic increase in fluorescence, live cell no-wash labeling is not possible due to the cytotoxicity of the labeling reagents.5, 6 To conduct no-wash protein labeling in live cells, many fluorogenic probes based on the principles of Förster resonance energy transfer (FRET) have been reported for the labeling of protein-tags such as SNAP-tag,7, 8, 9 BL-tag,10 TMP-tag,11 and PYP-tag. 12 , 13 However, most of these probes exhibit only moderate fluorescence enhancement upon labeling. Furthermore, FRET-based fluorogenic probes normally 3 ACS Paragon Plus Environment


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require lengthy synthetic steps to prepare and have large molecular sizes which often lead to low cell permeability and long incubation time for labeling intracellular proteins. Thus, new fluorogenic probes which can have the combination of high selectivity, fast labeling rate, high fluorescent turn-on ratio and cell-permeable is highly desirable for nowash labeling of intracellular proteins in live cells.

Here, we described a novel fluorogenic probe BGSBD based on an environmentsensitive fluorophore for the rapid labeling of SNAP-tag proteins in different cellular compartments of living cells (Figure 1a). The SNAP-tag protein is one of the most prominent labeling techniques for the covalent reaction with O6-benzylguanine (BG) derivatives.14, 15 It is well-known for its rapid reaction rate with BG derivatives and nontoxicity in cells. Covalent labeling of SNAP-tag with an affinity ligand or optical probe has been used for protein interaction studies, 16 drug discovery, 17 super-resolution imaging applications, 18 and the construction of fluorescent sensors. 19 , 20 Most of the SNAP-tag fluorogenic probes were created based on the FRET strategy by introducing a fluorescent quencher either at the C-8 or N-9 position of guanine. However, chemical modifications at these positions have been shown to reduce the labeling rate dramatically and required long incubation time to label intracellular SNAP-tagged proteins. Although a rapid SNAP-tag fluorogenic probe was reported recently by using environment-sensitive Nile red fluorophore, this strategy can only work in plasma membrane.21 Our aim in this paper is to develop a new probe via shorter synthetic steps in high yield which not only can achieve high fluorescence turn-on ratios upon SNAPtag labeling (larger than 200-fold) but can also preserve the fast labeling rate to the


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SNAP-tag protein. Furthermore, the probe should be cell permeable to allow for rapid no-wash imaging of intracellular proteins.

Results and Discussion

Design and synthesis of fluorogenic substrates: In

our

strategy,

an

environment-sensitive

fluorophore,

4-sulfamonyl-7-

aminobenzoxadiazole (SBD), was attached on the benzyl amino moiety of BG to form the fluorogenic probe BGSBD for fluorescence turn-on labeling of SNAP-tag protein. The SBD fluorophore has been used in the applications such as protein detection and imaging of cellular temperature. 22, 23 Although NBD is a more common environment sensitive flurorophore with a similar benzoxadiazole chemical structure, we worked on the SBD fluorophore as it is more sensitive to changes in polarity and the hydrogen bonding ability of a solvent. In addition, different derivatives of BGSBD where the BG moiety is attached either on 4‐sulfamonyl or 7‐amino position of SBD can be synthesized for optimization (Figure 1b). Our underlying rationale for considering an environment-sensitive fluorophore to construct the fluorogenic probe for SNAP-tag is that the BG binding pocket of SNAP-tag is hydrophobic.24, 25 In the absence of SNAPtag, the fluorogenic probe would display weak fluorescence in aqueous buffer. Upon covalent reaction of SNAP-tag with BGSBD, the environment-sensitive SBD fluorophore would be located close to the BG hydrophobic pocket to cause the SBD fluorophore to emit stronger fluorescence.



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The synthesis toward BGSBD is rather straightforward which can be accomplished by coupling the benzyl amino moiety of BG with the fluorophore SBD-OH (Scheme 1). The precursor SBD-OH was prepared by first functionalization of commercially available compound 1 with sulfonyl chloride, followed by the sequential attachment of sarcosine and methylamine to the fluorophore. The final product BGSBD can be obtained in 5 steps with a total yield of 65 %. With this synthetic strategy, purification of any SBD-OH intermediate was not necessary and only the final product BGSBD was purified by reverse phase HPLC. As compared to the existing FRET-based fluorogenic probes, our approach has the advantage of having shorter synthetic steps with higher yield and does not require any purification of the fluorophore intermediates.

In vitro characterization of BGSBD labeled SNAP-tag protein: BGSBD shows extremely weak fluorescence (φ = 0.0008) in aqueous PBS buffer. However, after the SNAP-tag protein was labeled with the fluorophore, fluorescence was enhanced dramatically with a high turn-on ratio of 280-fold (φ = 0.1430, Figure 2a). Fluorescence enhancement was so significant that it can be observed easily under a handheld UV lamp (Figure 2a, inset). The reaction between BGSBD and SNAP-tag is highly selective and produced only one single product in the SNAP-tag containing cell lysate, as characterized by in-gel fluorescence analysis (Figure S1). Furthermore, fluorescence enhancement occurs only in the presence of the SNAP-tag protein, as no increase in fluorescence was observed in the cell lysate without SNAP-tag protein (Figure S2). The hydrophobic BG binding pocket is critical as the strong fluorescence of BGSBD labeled SNAP-tag was reduced dramatically in a protein denaturation buffer


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(Figure S3). As compared to the recently reported fluorescent turn-on labeling by using environment-sensitive fluorophores to label PYP-tag and TMP-tag where only 22-fold and 2-fold enhancement was obtained, our fluorogenic probe has achieved a much higher fluorescence turned-on ratio upon labeling to the SNAP-tag protein.26, 27

In bioimaging, one major concern for environment-sensitive probes is the potential background staining due to probe accumulation or nonspecific binding into lipid membranes which can also cause fluorescence increase. To our surprise, the BGSBD labeled SNAP-tag exhibits even stronger fluorescence, with higher quantum yield and greater blue-shifted emission than BGSBD in hydrophobic organic solvents (Figure 2b and Table S1). The maximum emission of BGSBD labeled SNAP-tag is about 37 nm blue-shifted as compared to BGSBD in dioxane and shows only partial overlap with the solvents. This feature can be of great merit for the no-wash labeling of BGSBD to SNAP-tag in bioimaging experiments, where the background fluorescence from the probe in the cell membrane or other hydrophobic environments can be effectively minimized by the choice of an appropriate optical filter. Derivatives of fluorogenic substrates for SNAP-tag protein labeling: A series of different derivatives of BGSBD were synthesized in our attempt to optimize the SNAP tag labeling performance (Figure 1b). The results are summarized in Table 1. We first investigated the possibility of other environment‐sensitive fluorophores to be used in this design for fluorescence turn‐on labeling of SNAP-tag protein. We attached BG to two well-known environment‐sensitive fluorophores, NBD-Cl and Dansyl-Cl, giving BGNBD and BGDansyl, respectively. Both BGNBD and BGDansyl showed only 7 ACS Paragon Plus Environment


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about 4-fold enhancement in fluorescence upon SNAP-tag labeling. This result suggests that the highly environment‐sensitive SBD fluorophore is a superior choice for SNAP‐tag labeling to achieve significant fluorescence enhancement. It has been reported that guanine can act as a fluorescence quencher when it was conjugated to a fluorophore.

28

To exclude the possibility of FRET-based fluorescence turn‐on

mechanism, fluorescent spectrum of SBD-OH (with no BG moiety) was measured in PBS buffer. SBD-OH exhibits very weak fluorescence and has similar fluorescence intensity and quantum yield as BGSBD in aqueous buffer (Table 1 and Figure S4). This result confirms that the BG moiety is not a fluorescent quencher for SBD fluorophore and the fluorescence enhancement of BGSBD is due to the position of SBD fluorophore in the BG hydrophobic binding pocket.

We also synthesized derivatives of BGSBD with different linker lengths to explore the optimal location of the SBD fluorophore in the BG hydrophobic pocket. By attaching a C6-linker in between the SBD fluorophore and BG, BG(C6)SBD gave fluorescence enhancement of only 5-fold. The low fluorescence turn on ratio is probably due to the longer linker chain which exposed the SBD fluorophore to the non‐hydrophobic protein surface. Surprisingly, when the linker was shortened either by directly conjugating BG and SBD via the sulfonyl moiety to form BG(S4)SBD or attaching BG to the 7‐amino position of SBD to give BGSBD(N7), fluorescence enhancement was reduced from 280‐fold to merely 13- and 2-fold, respectively. Therefore the proper positioning of the SBD fluorophore in the BG binding pocket is critical and BGSBD which consists of a sarcosine linker in between BG and the SBD fluorophore is our best fluorogenic probe,


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achieving the largest fluorescence enhancement. Finally, we studied the effect of substituents on the 7-position of SBD fluorophore by replacing methyl amine with benzyl amine and glycine, to give probe BGSBD(Bn) and BGSBD(Gly). Both probes gave strong fluorescence increase upon SNAP‐tag labeling with fluorescence turn‐on ratio of 260- and 100-fold, respectively. This result suggests that our fluorogenic probe design can tolerate different substituents on 7-amino position of SBD. Based on this finding, our fluorogenic probe design not only can be applied to protein labeling, but also has the potential to be extended to other applications, such as characterization and analysis of ligand‐protein interactions. Currently, we are working toward developing this fluorogenic probe design as a new analytical tool for protein detection.

Kinetic analysis of BGSBD reaction with SNAP-tag: A kinetic analysis of the BGSBD labeling reaction with SNAP-tag was carried out by monitoring the increase of fluorescence intensity at protein and probe concentrations of 5 µM each. The time required to achieve 50% labeling of protein was calculated to be about 24 s (t1/2) and full labeling can be achieved within 3 minutes (Figure 3a). The second order rate constant (k2) for reaction between BGSBD and SNAP-tag was determined to be 7200 ± 1600 M-1s-1 (Figure 3b). With this design, the probe has successfully preserved the high labeling rate of SNAP-tag and the k2 value is comparable to those native BG substrates. As compared to all the FRET-based SNAPtag fluorogenic probes reported to date, the labeling rate of BGSBD on the SNAP-tag is about 10-times faster than the fluorescent quencher attached at the C-8 position7, 9 and around 1000-times faster than at the N-9 position of BG.8 Furthermore, the labeling rate



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of our probe is also faster than most of the fluorogenic labeling techniques reported so far.

No-wash live-cell imaging of SNAP-tagged proteins with BGSBD: To demonstrate the feasibility of applying BGSBD for no-wash bioimaging in different subcellular compartments, HCT-116 cells expressing SNAP-tag fusion proteins on the cell surface, in cytosol and in the nucleus were investigated. We first investigated the permeability of BGSBD to label intracellular SNAP-tag protein. For this goal, gene encoding SNAP-tag protein only was constructed on mammalian cells expression vector. The SNAP-tag expression HCT-116 cells and control experiment (cells without exogeneous SNAP-tag expression) were incubated with 5 µM BGSBD for 30 minutes and fluorescence images were taken without washing the cells. In the nontransfected cells, fluorescence was hardly detected in the presence of BGSBD (Figure 4, control). In contrast, a rapid and substantial rise in fluorescence was observed in cells expressing SNAP-tag which showed that BGSBD is a membrane permeable probe to label intracellular proteins. Toxicity test showed that the probe is non-toxic to the cells (Figure S5). To further establish the utility of the probe for live cell imaging, we applied the probe to label and visualize SNAP-tag expressed on cell surface and in nucleus. For the labeling of cell surface proteins, a SNAP-tag encoding construct was prepared by fusing SNAP-tag to the N-terminal of transmembrane anchoring domain of plateletderived growth factor receptor, named SNAP-PDGFR. For the labeling of proteins in nucleus, gene encoding SNAP-tag fused with nuclear localized human histone H2B (SNAP-H2B) was created. The fluorescence images of SNAP-PDGFR expression cells


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labeled with BGSBD revealed clear fluorescence along the plasma membrane. Furthermore, specific labeling of SNAP-H2B fusion proteins in nucleus was also observed. Co-staining of the cells with Hoechst 33342 and BGSBD validated that the probe visualized the nuclear localization of SNAP-H2B. Thus, these results show that our probe can be applied to visualize a wide range of target proteins at a specific location, either inside or outside of the cells, with no post-incubation washing of the cells.

Flow cytometric analysis of BGSBD labeled cytosolic SNAP‐‐tag protein: The applications of flow cytometric techniques for the analysis of cell populations enables scientists to evaluate cells based on both intrinsic and extrinsic properties. Multiple characteristics of thousands of cells can be investigated within a few minutes by using appropriate fluorescent probes such as dyes, quantum dots, or fluorescent proteins. 29 ,

30

, 31 However, as for live-cell imaging, flow cytometric analysis of

intracellular proteins using current labeling methods also requires extensive washing to remove the unreacted fluorophores. Due to the distinctive fluorescence emission of BGSBD upon labeled to SNAP-tag and its membrane permeability, we anticipate its potential application in flow cytometry to label intracellular proteins. To demonstrate the feasibility of no-wash labeling in flow cytometric applications, 5 µM BGSBD was employed to label HCT-116 cells expressing SNAP-tag in the cytosol for 30 minutes. The labeled cells were then submitted to flow cytometric analysis without washout steps. Figure 5a-c shows dot plots for HCT-116 cells only (blank control), nontransfected cells incubated with BGSBD (negative control) and SNAP-tag



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expressing cells labeled with BGSBD, respectively.

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As shown in Figure 4b,

nontransfected HCT-116 cells were hardly labeled by BGSBD and displayed minimal fluorescence background. On the other hand, robust fluorescence labeling was observed for cells expressing cytosolic SNAP-tags which mostly appeared in the fluorescence-gated window (Figure 5c). The total counts of the cells that produced fluorescence intensity above the threshold were 39, 321 and 2884 (5000 cells were counted in each condition) for blank control, negative control and BGSBD labeled HCT116 cell expressing cytosolic SNAP-tag proteins, respectively (Figure 5d).

Conclusions

In conclusion, we have created a novel fluorogenic probe BGSBD based on the environment-sensitive fluorophore SBD for rapid labeling of SNAP-tag to overcome the washout steps during protein labeling. Due to the large fluorescence enhancement and unique emission spectra, no-wash labeling of intracellular proteins as well as cell surface proteins was successfully achieved, and the localization of the proteins was specifically visualized. This no-wash protein labeling technique can also be applied in the flow cytometric analysis. The major advantages of our fluorogenic probes are the dramatic fluorescence turn-on, the ease of synthesis, high selectivity, cell-permeable and rapid reaction with SNAP-tag. With this design, the probe BGSBD has successfully preserved the high labeling rate of SNAP-tag with its BG substrates. While fluorescent proteins will continue to be an important tool in bioimaging, the development of a nowash chemical labeling technique may provide an alternative avenue for researchers to 12 ACS Paragon Plus Environment

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have precise control of the location and time of labeling which is crucial for real-time monitoring of high dynamic processes in living cells.



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ASSOCIATED CONTENT Supporting Information For complete experimental methods and figures see Supplementary Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]; [email protected] Author Contributions §

T.-K. L. and P.-Y. H. contributed equally to this work.

Notes The authors declare no competing financial interests.

Acknowledgements We are grateful to the Ministry of Science and Technology (Grant No.: 102-2113-M007004-MY2 and 101-2113-M009-006-MY2) and Ministry of Education ("Aim for the Top University Plan"; Grant No.: 102N2011E1), Taiwan (ROC) for financial support.


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(a)

(b)

BGSBD: R1 = NHMe, R2 = Sarcosine-BG R1

BG(C6)SBD: R1 = NHMe, R2 = Sarcosine-C6linker-BG

N O N O S O R2

BG(S4)SBD: R1 = NHMe, R2 = NH-BG BGSBD(N7): R1 = NH-BG, R2 = N(Me)2 BGSBD(Bn): R1 = NH-Bn, R2 = Sarcosine-BG BGSBD(Gly): R1 = Gly-OEt, R2 = Sarcosine-BG

Figure 1. (a) Design of fluorescence turn-on probe for SNAP-tag protein labeling. Upon reaction with SNAP-tag protein, the environment-sensitive fluorophore SBD is located in the hydrophobic BG binding pocket, whereby the surrounding hydrophobic environment can cause the fluorophore to exhibit stronger fluorescence. In the absence of SNAP-tag protein, the fluorogenic probe has low fluorescence. (b) Chemical structures of BGSBD and its derivatives.



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Scheme 1. (a) Design of fluorogenic probe BGSBD for the fast labeling of SNAP-tag protein. Upon reaction with SNAP-tag, the SBD fluorophore is located in the hydrophobic BG binding pocket, whereby the surrounding hydrophobic environment can cause the fluorophore to exhibit stronger fluorescence. (b) Reaction conditions for the synthesis of BGSBD: (i) ClSO3H, neat, 0°C (1.5hr) then 150°C (6hr), (80%); (ii) Sarcosine t-butyl ester, TEA, DCM, RT, 10 mins, (90%); (iii) NH2Me, MeOH, RT, 1hr, (quantitative); (iv) TFA, DCM, RT, 1hr, (quantitative); (v) BG-NH2, EDC.HCl, HOBt, TEA, DMF, RT, overnight (90%).


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(a)

(b)

Figure 2. Fluorescence analyses for the labeling reactions of SNAP-tag with fluorogenic probe BGSBD. (a) Fluorescence spectra of BGSBD in the absence (red line) or presence (black line) of SNAP-tag protein. λex = 435 nm. The inset shows the images of the solution in a cuvette before (left) and after labeling (right) under excitation with a UV lamp (365 nm). The fluorescence turn-on ratio was calculated from the relative fluorescence intensity of BGSBD at λem = 516 nm. Labeling condition: 5 µM BGSBD and 10 µM SNAP-tag protein in PBS buffer (1 % DMSO) at 37°C for 30 minutes. (b) Comparison of fluorescence response of BGSBD in various organic solvents and upon labeled with SNAP-tag.



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Table 1. Spectral properties of fluorogenic probes at free and SNAP-tag bound states. Probe

λabs

λemfree

λembound

ε

Фfree

Фbound

RFI

BGSBD

435

584

516

13000

0.0008

0.1426

280

BGNBD

477

539

530

6300

0.0230

0.0750

4

BGDansyl

331

527

530

8600

0.0292

0.0948

4

SBD-OH

434

596

NA

8800

0.0012

NA

NA

BG(C6)SBD

437

591

557

8400

0.0017

0.0076

5

BG(S4)SBD

438

607

556

4800

0.0013

0.0179

13

BGSBD(N7)

430

570

557

10000

0.0095

0.0125

2

BGSBD(Bn)

432

585

516

6400

0.0050

0.3380

260

BGSBD(Gly)

428

572

509

4500

0.0031

0.0488

100

(a)

NBDNHMe in ACN was used as a quantum yield reference with the value of

0.38.32 (b) The RFIs were calculated from the ratio of fluorescence intensity between the free and SNAP-tag bound probe by using maximum emission wavelength of the SNAPtag bound probe.


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(a)

(b)

Figure 3. Kinetic study of SNAP-tag labeling with BGSBD. (a) Time course of fluorescence increase for the reaction of BGSBD with SNAP-tag. The fluorescence intensity was recorded at 518 nm with excitation at 435 nm. (b) Fluorescence increase of BGSBD (1 – 5 µM) in labeling of SNAP-tag (100 nM). The inset shows the linear relationship plot of probe concentration versus calculated kobs (R2 = 0.99).



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Figure 4. No-wash live-cell imaging of SNAP-tagged proteins labeled with BGSBD. HCT-116 cells expressing SNAP-tag fusion proteins at various subcellular locations. (i) Cells without exogeneous SNAP-tag expression, (ii) SNAP-tag for both cytosol and nucleus, (iii) SNAP-PDGFR for cell surface and (iv) SNAP-H2B for nucleus. SNAPtagged proteins labeled with BGSBD are shown in green, nucleus labeled with Hoechst 33342 are shown in blue. The fluorescence images were obtained with excitation at 442 nm and emission at 520±20 nm.


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Figure 5. No-wash flow cytometric analysis of HCT-116 cells expressing cytosolic SNAP-tag labeled with BGSBD. Dot plots for (a) HCT-116 cells only (blank control), (b) nontransfected cells incubated with BGSBD (negative control), (c) SNAP-tag expressing cells labeled with BGSBD. Solid lines on the dot plots indicate the fluorescence threshold. (d) The total counts of the cells that produced fluorescence intensity above the threshold.



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References:

1

Johnsson, N., and Johnsson, K. (2007) Chemical tools for biomolecular imaging. ACS

Chem. Biol. 2, 31-38. 2

Jing, C., and Cornish, V. W. (2011) Chemical tags for labeling proteins inside living

cells. Acc. Chem. Res. 44, 784-792. 3

Haugland, R. P. (2005) The Handbook: A Guide to Fluorescent Probes and Labeling

Technologies, 10th ed.; Invitrogen: Carlsbad, CA. 4

Lavis, L. D., and Raines, R. T. (2008) Bright ideas for chemical biology. ACS Chem.

Biol. 3, 142-155. 5

Adams, S. R., Campbell, R. E., Gross, L. A., Martin, B. R., Walkup, G. K., Yao, Y.,

Llopis, J., and Tsien, R. Y. (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063-6076. 6

Hoffmann, C., Gaietta, G., Zürn, A., Adams, S. R., Terrillon, S., Ellisman, M. H., Tsien,

R. Y., and Lohse, M. J. (2010) Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat. Protoc. 5, 1666-1677. 7

Komatsu, T., Johnsson, K., Okuno, H., Bito, H., Inoue, T., Nagano, T., and Urano, Y.

(2011) Real-time measurements of protein dynamics using fluorescence activationcoupled protein labeling method. J. Am. Chem. Soc. 133, 6745-6751. 8

Zhang, C. J., Li, L., Chen, G. Y., Xu, Q. H., and Yao, S. Q. (2011) One- and two-

photon live cell imaging using a mutant SNAP-Tag protein and its FRET substrate pairs. Org. Lett. 13, 4160-4163.


Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9

Sun, X., Zhang, A., Baker, B., Sun, L., Howard, A., Buswell, J., Maurel, D., Masharina,

A., Johnsson, K., Noren, C. J., Xu, M. Q., and Corrêa, I. R. Jr. (2011) Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. Chembiochem 12, 2217-2226. 10

Mizukami, S., Watanabe, S., Akimoto, Y., and Kikuchi, K. (2012) No-wash protein

labeling with designed fluorogenic probes and application to real-time pulse-chase analysis. J. Am. Chem. Soc. 134, 1623-1629. 11

Jing, C., and Cornish, V. W. (2013) A fluorogenic TMP-tag for high signal-to-

background intracellular live cell imaging. ACS Chem. Biol. 8, 1704-1712. 12

Hori, Y., Ueno, H., Mizukami, S., and Kikuchi, K. (2009) Photoactive yellow protein-

based protein labeling system with turn-on fluorescence intensity. J. Am. Chem. Soc. 131, 16610-16611. 13

Hori, Y., Nakaki, K., Sato, M., Mizukami, S., and Kikuchi, K. (2012) Development of

protein-labeling probes with a redesigned fluorogenic switch based on intramolecular association for no-wash live-cell imaging. Angew. Chem. Int. Ed. 51, 5611-5614. 14

Keppler, A., Pick, H., Arrivoli, C., Vogel, H., and Johnsson, K. (2004) Labeling of

fusion proteins with synthetic fluorophores in live cells. Proc. Natl. Acad. Sci. USA 101, 9955-9959. 15

Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K.

(2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86-89.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16

Page 24 of 27

Gautier, A., Nakata, E., Lukinavicius, G., Tan, K.-T., and Johnsson, K. (2009)

Selective cross-linking of interacting proteins using self-labeling tags. J. Am. Chem. Soc. 131, 17954-17962. 17

Chidley, C., Haruki, H., Pedersen, M. G., Muller, E., and Johnsson, K. (2011) A yeast-

based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis. Nat. Chem. Biol. 7, 375-383. 18

Lukinavičius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T.,

Mueller, V., Reymond, L., Corrêa, I. R. Jr., Luo, Z. G., Schultz, C., Lemke, E. A., Heppenstall, P., Eggeling, C., Manley, S., and Johnsson, K. (2013) A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132-139. 19

Brun, M. A., Tan, K.-T., Nakata, E., Hinner, M. J., and Johnsson, K. (2009)

Semisynthetic fluorescent sensor proteins based on self-labeling protein tags. J. Am. Chem. Soc. 131, 5873-5884. 20

Shih, P.-M., Liu, T.-K., and Tan, K.-T. (2013) Fluorescence amplified detection of

proteases by the catalytic activation of a semisynthetic sensor. Chem. Comm. 49, 62126214. 21

Prifti, E., Reymond, L., Umebayashi, M., Hovius, R., Riezman, H., and Johnsson, K.

(2014) A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red. ACS Chem. Biol. 9, 606-612. 22

Zhuang, Y.-D., Chiang, P.-Y., Wang, C.-W., and Tan, K.-T. (2013) Environment-

sensitive fluorescent turn-on probes targeting hydrophobic ligand-binding domains for selective protein detection. Angew. Chem. Int. Ed. 52, 8124-8128. 24 ACS Paragon Plus Environment

Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


23

Okabe, K., Inada, N., Gota, C., Harada, Y., Funatsu, T., and Uchiyama, S. (2012)

Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. Nat. Commun. 3, 705. 24

Mollwitz, B., Brunk, E., Schmitt, S., Pojer, F., Bannwarth, M., Schiltz, M.,

Rothlisberger, U., and Johnsson, K. (2012) Directed evolution of the suicide protein O⁶alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability. Biochemistry, 51, 986-994. 25

Wibley, J. E., Pegg, A. E., and Moody, P. C. (2000) Crystal structure of the human

O(6)-alkylguanine-DNA alkyltransferase. Nucleic Acids Res. 28, 393-401. 26

Hori, Y., Norinobu, T., Sato, M., Arita, K., Shirakawa, M., and Kikuchi, K. (2013)

Development of fluorogenic probes for quick no-wash live-cell imaging of intracellular proteins. J. Am. Chem. Soc. 135, 12360-12365. 27

Liu, W., Li, F., Chen, X., Hou, J., Yi, L., and Wu, Y.-W. (2014) A rapid and fluorogenic

TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells. J. Am. Chem. Soc. 136, 4468-4471. 28

Stöhr, K., Siegberg, D., Ehrhard, T., Lymperopoulos, K., Öz, S., Schulmeister, S.,

Pfeifer, A. C., Bachmann, J., Klingmüller, U., Sourjik, V., and Herten, D.-P. (2010) Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background. Anal. Chem. 82, 8186-8193. 29

Brown, M., and Wittwer, C. (2000) Flow cytometry: principles and clinical applications

in hematology. Clin. Chem. 46, 1221-1229.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Page 26 of 27

Wu, L., Huang, T., Yang, L., Pan, J., Zhu, S., and Yan, X. (2011) Sensitive and

selective bacterial detection using tetracysteine-tagged phages in conjunction with biarsenical dye. Angew. Chem. Int. Ed. 50, 5873-5877. 31

Hangauer, M. J., and Bertozzi, C. R. (2008) A FRET-based fluorogenic phosphine for

live-cell imaging with the Staudinger ligation. Angew. Chem. Int. Ed. 47, 2394-2397. 32

Uchiyama, S., Santa, T., and Imai, K. (1999) Fluorescence characteristics of six 4,7-

disubstituted benzofurazan compounds: an experimental and semi-empirical MO study. J. Chem. Soc., Perkin Trans. 2, 2525-2532.


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BG

POI

SBD

POI

SNAP‐tag No‐wash Labeling

Before Labeling

After Labeling

ACS Paragon Plus Environment

SNAP‐tag SBD

A Rapid SNAP-Tag Fluorogenic Probe Based on an Environment

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