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Quenched Substrates for Live-Cell Labeling of SNAP-Tagged Fusion Proteins with Improved Fluorescent Background ¨ z,† Katharina Sto¨hr,† Daniel Siegberg,† Tanja Ehrhard,† Konstantinos Lymperopoulos,† Simin O ‡ § § § Sonja Schulmeister, Andrea C. Pfeifer, Julie Bachmann, Ursula Klingmu¨ller, Victor Sourjik,‡ and Dirk-Peter Herten*,† CellNetworks Cluster and Institute of Physical Chemistry, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany, and German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O6-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells. Current developments in fluorescence microscopy open new perspectives in biological research and molecular diagnostics due to improved sensitivity and resolution. In recent years, advanced fluorescence microscopy techniques emerged increasing the demand for fluorescent labels with specific properties. High * To whom correspondence should be addressed. E-mail: dirk.herten@ bioquant.uni-heidelberg.de. † CellNetworks Cluster and Institute of Physical Chemistry, Heidelberg University. ‡ Zentrum fu ¨ r Molekulare Biologie der Universita¨t Heidelberg. § German Cancer Research Centre (DKFZ).
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brightness, photostability, and constant emission rates are mandatory for most if not all methods, i.e., single-molecule fluorescence spectroscopy (SMFS) or stimulated emission depletion (STED) microscopy.1,2 More specialized photophysical properties, like photoactivation or photoswitching, are necessary for photoactivatable localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and related methods.3-5 Intended use in live cell microscopy further demands availability of functional groups for fast, specific, and efficient labeling, water solubility, nontoxicity, and reduced unspecific interactions with the complex cellular environment.6 Labeling of proteins based on genetic fusion with fluorescent proteins has become routine in cell biology to express fluorescently tagged proteins in living cells at controlled expression levels.7,8 Yet, their use with advanced microscopy techniques is still challenging. Not only have artifacts been reported on some of the fluorescent proteins, like delayed maturation or dimerization, but also limited applicability due to complex photophysics or rapid photobleaching.9-11 An alternative approach is the use of fluorescent semiconductor nanocrystals, i.e., quantum dots. Quantum dots feature not only high photostability owing to their semiconductor constitution but also a bright fluorescence and narrow emission spectra. Yet, their application in cell biology is (1) Moerner, W. E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12596–12602. (2) Klar, T. A.; Jakobs, S.; Hell, S. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8206–8210. (3) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Science 2006, 313, 1642–1645. (4) Rust, M. J.; Bates, M.; Zhuang, X. W. Nat. Methods 2006, 3, 793–795. (5) Heilemann, M.; van de Linde, S.; Schu ¨ ttpelz, M.; Kasper, R.; Seefeldt, B.; Mukherjee, A.; Tinnefeld, P.; Sauer, M. Angew. Chem., Int. Ed. 2008, 47, 6172–6176. (6) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217–224. (7) Shaner, N. C.; Steinbach, P. A.; Tsien, R. Y. Nat. Methods 2005, 2, 905– 909. (8) Shaner, N. C.; Campbell, R. E.; Steinbach, P. A.; Giepmans, B. N.; Palmer, A. E.; Tsien, R. Y. Nat. Biotechnol. 2004, 22, 1567–1572. (9) Seefeldt, B.; Kasper, R.; Seidel, T.; Tinnefeld, P.; Dietz, K. J.; Heilemann, M.; Sauer, M. J. Biophotonics 2008, 1, 74–82. (10) Hendrix, J.; Flors, C.; Dedecker, P.; Hofkens, J.; Engelborghs, Y. Biophys. J. 2008, 94, 4103–4113. (11) Jung, G.; Zumbusch, A. Microsc. Res. Tech. 2006, 69, 175–185. 10.1021/ac101521y 2010 American Chemical Society Published on Web 09/03/2010
still limited in view of specific labeling and probe delivery in living cells.12 In recent years, several methods for specific labeling of proteins in living cells have been developed that are based on defined conjugation chemistry established in the last decades.6,13 Fluorescent dyes have the advantage of low molecular weight and thus less influence on protein function aside of their defined biochemistry. Although their photostability is also limited they are usually more photostable and brighter than fluorescent proteins. Yet, specific labeling in living cells is limited to certain fluorescent dyes and mostly depends on the applied labeling method. Well known examples are the tetracystein-tag,14 the Histag,15 and the D4-tag to name the most common.16 More recently luminescent lanthanide-chelates have become available for specific labeling of proteins in living cells by introduction of the lanthanidebinding-tag (LBT).17 In addition to suchlike affinity tag based methods, covalent labeling of proteins has become possible by exploiting different enzymatic reactions.18,19 One example is the HaloTag, which is a mutant of a prokaryotic halogenase that can be genetically fused to proteins and labeled in a second step by fluorescently tagged chloralkenes.20 Another example is the labeling of Escherichia coli dihydrofolate reductase (eDHFR) tagged proteins with trimehoprim conjugates.21 More in the focus of our study, is the human O6-alkylguanosyltransferase (hAGT) whose function is DNA-repair by cleavage of alkyl-groups from guanines at their O6-position.22,23 Mutants of hAGT (SNAP-tag) can genetically be fused to the protein of interest and labeled subsequently with O6-benzylguanine (BG) derivatives carrying a specific label at the benzyl-group.22,24,25 The demand for suchlike improved labeling methods is exemplified by recent publications on multicolor labeling, on stimulated emission depletion (STED) nanoscopy in living cells, and even on conjugates that can be activated or converted by light using custom and commercially available labels.26-28 (12) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (13) Soh, N.; Makihara, K.; Ariyoshi, T.; Seto, D.; Maki, T.; Nakajima, H.; Nakano, K.; Imato, T. Anal. Sci. 2008, 24, 293–296. (14) 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. (15) Gaietta, G.; Deerinck, T. J.; Adams, S. R.; Bouwer, J.; Tour, O.; Laird, D. W.; Sosinsky, G. E.; Tsien, R. Y.; Ellisman, M. H. Science 2002, 296, 503–507. (16) Ojida, A.; Honda, K.; Shinmi, D.; Kiyonaka, S.; Mori, Y.; Hamachi, I. J. Am. Chem. Soc. 2006, 128, 10452–10459. (17) Franz, K. J.; Nitz, M.; Imperiali, B. ChemBioChem 2003, 4, 265–271. (18) Miller, L. W.; Cornish, V. W. Curr. Opin. Chem. Biol. 2005, 9, 56–61. (19) Chen, I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35–40. (20) Los, G.; Learish, R.; Karassina, N.; Zimprich, C.; McDougall, M. G.; Encell, L. P.; Friedman-Ohana, R.; Wood, M.; Vidugiris, G.; Zimmerman, K.; Otto, P.; Berstock, S.; Klaubert, D.; Wood, K. V. Cell Notes 2006, 14, 10–14. (21) Miller, L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W. Nat. Methods 2005, 2, 255–257. (22) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86–89. (23) Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9955–9959. (24) Ciocco, G. M.; Moschel, R. C.; Chae, M. Y.; McLaughlin, P. J.; Zagon, I. S.; Pegg, A. E. Cancer Res. 1995, 55, 4085–4091. (25) Damoiseaux, R.; Keppler, A.; Johnsson, K. ChemBioChem 2001, 2, 285– 287. (26) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R., Jr.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128–136. (27) Hein, B.; Willig, K. I.; Wurm, C. A.; Westphal, V.; Jakobs, S.; Hell, S. W. Biophys. J. , 98, 158–163.
We became interested in this labeling approach because guanine is well-known to specifically quench the fluorescence of certain dyes, like the oxazine dye MR 121, by photoinduced electron transfer (PET).29 Sauer et al. were the first to use intramolecular quenching of guanine for DNA probes that show a strong increase in their fluorescence emission by exploiting conformational changes in the DNA hairpin structures upon specific hybridization to a complementary sequence.30 Recently, we designed a set of suchlike Smart Probes to specifically identify the cDNA of different mycobacteria based on fundamental design principles.31 On the basis of our previous work, we explored the potential of intramolecular fluorescence quenching by guanine in BG-dye conjugates to improve the specificity of fluorescence emission of SNAP-tagged proteins (Figure 1). Here, we present the spectroscopic characterization of BG-dye conjugates with 21 different fluorescent dyes in the visible spectral range. We found four BG-dye conjugates that are not only significantly quenched but also show a strong (>10-fold) increase in their fluorescence emission upon covalent labeling of the SNAP-tag. Using a subset of nine BG-dye conjugates, we investigated their potential for live cell imaging. In E. coli expressing SNAP-tag, we found that quenched conjugates show less fluorescent background and hence a higher specificity. We will also shortly discuss our experience with labeling a SNAP-tag fusion protein in living mammalian cells. RESULTS In Vitro Assay. For spectroscopic studies of intramolecular quenching effects on the fluorescence emission, 21 differently labeled BG-dye conjugates were synthesized by labeling BG-NH2 with the respective dye activated as NHS-ester. According to the reaction scheme in Figure 1A, BG-dye conjugates react with the thiol-group in the active site of SNAPtag by nucleophilic substitution of the O6-guanine moiety thereby covalently labeling the protein with the derivatized benzylgroup. In live cell experiments, labeled product cannot be purified. The labeling yield of the reaction is therefore linked to the contribution of excess BG-dye conjugate to the total fluorescence signal. In contrast, BG-dye conjugates with reduced fluorescence emission could be used in higher excess and thus yield a higher labeling efficiency (Figure 1B). On the background of specific intramolecular quenching of fluorescence of oxazine dyes by guanine,29,30,32 we were particularly interested in the fluorescence intensities of the different BG-dye conjugates as well as the labeled protein after reaction with the SNAP-tag in comparison to the free dye. Following the reaction scheme in Figure 1B, the fluorescence intensities of quenched BG-dye conjugates like BG-MR 121 (Figure 1C) should be considerably lower compared to the free dye due to the close proximity of the quenching guanine moiety and rise significantly after reaction with SNAP-tag as the guanine is cleaved off and therefore no longer (28) Maurel, D.; Banala, S.; Laroche, T.; Johnsson, K. ACS Chem. Biol. 2010, 5, 507–516. (29) Nord, S.; Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Lieberwirth, U.; Seeger, S.; Wolfrum, J. J. Fluoresc. 1997, 7, 15S–18S. (30) Knemeyer, J. P.; Marme, N.; Sauer, M. Anal. Chem. 2000, 72, 3717–3724. (31) Sto ¨hr, K.; Hafner, B.; Nolte, O.; Wolfrum, J.; Sauer, M.; Herten, D. P. Anal. Chem. 2005, 77, 7195–7203. (32) Buschmann, V.; Weston, K. D.; Sauer, M. Bioconjugate Chem. 2003, 14, 195–204.
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Figure 1. (A) Sketch of the labeling reaction with a standard organic fluorophore and (B) sketch of the labeling of the SNAP protein with a quenched BG-dye conjugate. In the labeling reaction, guanine acts as a leaving group and is substituted by a thiol group of a cysteine residue in the binding pocket of SNAP. The dye shows an increase in fluorescence emission upon conjugation with SNAP. (C) Structure of the benzylguanine-MR 121 conjugate. (D) Fluorescence spectra of the ATTO 655 NHS-Ester (dashed line), the BG-ATTO 655 conjugate (dotted line), and the SNAP-ATTO 655 conjugate (solid line) after 3 h reaction time at 25 °C with a 1000-fold excess of purified SNAP-protein.
available for intramolecular electron transfer. To test this hypothesis, we measured the emission spectra of ATTO 655 as NHS ester (10 nM) (Figure 1D, dashed line), as BG-conjugated (Figure 1D, dotted line) and after reaction of the BG-ATTO 655 with the purified SNAP-protein (1000-fold molar excess) (Figure 1D, solid line) at 25 °C. The decreased relative fluorescence intensity of BG-ATTO 655 (dotted line) with respect to the free dye (dashed line) and the increase upon labeling of SNAP-tag confirmed our expectations. To find more suitable dyes which feature the desired characteristics, we conjugated many of the commercial available dyes into BG-conjugates and studied their properties. Most importantly, the fluorescence intensities of the free dyes and the BG-dye conjugates were measured first to identify dyes which are quenched significantly in the presence of the BG-moiety. In a second step, the change in fluorescence intensity upon reaction of the BG-dye conjugate with the purified SNAP-tag was monitored by adding a 1000-fold molar excess of purified SNAPtag to 10 nM solution of the respective BG-dye conjugates. The reaction time was chosen to be 5 h at a reaction temperature of 37 °C. Comparison of the relative fluorescence intensities of the BG-dye conjugates IBG in Table 1 shows that most dyes are influenced by the presence of the BG-moiety. MR 121, ATTO 655, and ATTO 488 maintain less than 10% of their original fluorescence emission as BG-dye conjugates being the strongest quenched fluorophores. In contrast, two rhodamines, Alexa 568 and Alexa 594, as well as the carbocyanine Cy5 even show an increase in fluorescence upon conjugation with BG. In contrast to the behavior of the BG-dye conjugates, the changes in fluorescence emission of the respective conjugates with the SNAP-tag seems to be less correlated with their structural properties. Most of them are strongly influenced in their fluorescence emission upon conjugation to the SNAP-tag and most of them show an enhanced brightness. The strongest increase in 8188
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fluorescence intensity compared to the dye-NHS-ester by at least a factor of 4 was observed for ATTO 495 and ATTO 647N, respectively. Only ATTO 488 (14%) and ATTO 620 (34%) are quenched upon conjugation with SNAP-tag. From these data, we are inclined to favor the hypothesis that intramolecular quenching in BG-dye conjugates is more related to the molecular properties of the fluorescent dyes than the influence of the protein which is governed by a multitude of possible interactions with its different amino acids. This notion is reflected in the respective fluorescence lifetime data (see Supporting Information, Table S1) where strong quenching correlates with shortened lifetimes of BG-dye conjugates to 35-70% in comparison to the free dye while the change in lifetime is much smaller (
