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Fluorogen-activating single-chain antibodies for imaging cell surface proteins

Nature Biotechnology volume 26, pages 235–240 (2008)Cite this article

A Corrigendum to this article was published on 01 April 2008

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Abstract

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores1. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

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Figure 1: Fluorogens and their use with yeast-displayed scFvs.
Figure 2: Fluorescence characterization of purified FAPs.
Figure 3: Fluorescence microscopy of FAPs displayed on live cells.

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GenBank/EMBL/DDBJ

Change history

  • 19 March 2008

    In the version of this article initially published online, an author’s middle initial was incorrectly given as “A” Brigitte A Schmidt should be Brigitte F Schmidt in the author list, and B.A.S should be B.F.S. in the authors’ contribution section. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  Google Scholar 

  2. Yao, J., Munson, K.M., Webb, W.W. & Lis, J.T. Dynamics of heat shock factor association with native gene loci in living cells. Nature 442, 1050–1053 (2006).

    Article  CAS  Google Scholar 

  3. Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  Google Scholar 

  4. Pertz, O., Hodgson, L., Klemke, R.L. & Hahn, K.M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006).

    Article  CAS  Google Scholar 

  5. Marks, K.M. & Nolan, G.P. Chemical labeling strategies for cell biology. Nat. Methods 3, 591–596 (2006).

    Article  CAS  Google Scholar 

  6. Adams, S.R. et al. 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 (2002).

    Article  CAS  Google Scholar 

  7. Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    Article  CAS  Google Scholar 

  8. Rozinov, M.N. & Nolan, G.P. Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores. Chem. Biol. 5, 713–728 (1998).

    Article  CAS  Google Scholar 

  9. Farinas, J. & Verkman, A.S. Receptor-mediated targeting of fluorescent probes in living cells. J. Biol. Chem. 274, 7603–7606 (1999).

    Article  CAS  Google Scholar 

  10. Hauser, C.T. & Tsien, R.Y. A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure. Proc. Natl. Acad. Sci. USA 104, 3693–3697 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Chen, I., Howarth, M., Lin, W. & Ting, A.Y. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2, 99–104 (2005).

    Article  CAS  Google Scholar 

  13. Reck-Peterson, S.L. et al. Single-molecule analysis of dynein processivity and stepping behavior. Cell 126, 335–348 (2006).

    Article  CAS  Google Scholar 

  14. Feldhaus, M.J. et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat. Biotechnol. 21, 163–170 (2003).

    Article  CAS  Google Scholar 

  15. Nygren, J., Svanvik, N. & Kubista, M. The interactions between the fluorescent dye thiazole orange and DNA. Biopolymers 46, 39–51 (1998).

    Article  CAS  Google Scholar 

  16. Babendure, J.R., Adams, S.R. & Tsien, R.Y. Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc. 125, 14716–14717 (2003).

    Article  CAS  Google Scholar 

  17. Iwaki, T., Torigoe, C., Noji, M. & Nakanishi, M. Antibodies for fluorescent molecular rotors. Biochemistry 32, 7589–7592 (1993).

    Article  CAS  Google Scholar 

  18. Siegel, R.W., Coleman, J.R., Miller, K.D. & Feldhaus, M.J. High efficiency recovery and epitope-specific sorting of an scFv yeast display library. J. Immunol. Methods 286, 141–153 (2004).

    Article  CAS  Google Scholar 

  19. Giudicelli, V. et al. IMGT/LIGM-DB, the IMGT comprehensive database of immunoglobulin and T cell receptor nucleotide sequences. Nucleic Acids Res. 34, D781–D784 (2006).

    Article  CAS  Google Scholar 

  20. Colby, D.W. et al. Engineering antibody affinity by yeast surface display. Methods Enzymol. 388, 348–358 (2004).

    Article  CAS  Google Scholar 

  21. Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

    Article  CAS  Google Scholar 

  22. Simeonov, A. et al. Blue-fluorescent antibodies. Science 290, 307–313 (2000).

    Article  CAS  Google Scholar 

  23. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  24. Huang, D. & Shusta, E.V. Secretion and surface display of green fluorescent protein using the yeast Saccharomyces cerevisiae. Biotechnol. Prog. 21, 349–357 (2005).

    Article  CAS  Google Scholar 

  25. Baptista, M.S. & Indig, G.L. Effect of BSA binding on photophysical and photochemical properties of triarylmethane dyes. J. Phys. Chem. B 102, 4678–4688 (1998).

    Article  CAS  Google Scholar 

  26. Surrey, T. et al. Chromophore-assisted light inactivation and self-organization of microtubules and motors. Proc. Natl. Acad. Sci. USA 95, 4293–4298 (1998).

    Article  CAS  Google Scholar 

  27. Remington, S.J. Fluorescent proteins: maturation, photochemistry and photophysics. Curr. Opin. Struct. Biol. 16, 714–721 (2006).

    Article  CAS  Google Scholar 

  28. Colby, D.W. et al. Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display. J. Mol. Biol. 342, 901–912 (2004).

    Article  CAS  Google Scholar 

  29. Tanaka, T., Lobato, M.N. & Rabbitts, T.H. Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J. Mol. Biol. 331, 1109–1120 (2003).

    Article  CAS  Google Scholar 

  30. Proba, K., Worn, A., Honegger, A. & Pluckthun, A. Antibody scFv fragments without disulfide bonds made by molecular evolution. J. Mol. Biol. 275, 245–253 (1998).

    Article  CAS  Google Scholar 

  31. Motulsky, H. & Christopoulos, A. . Fitting Models to Biological Data Using Linear and Nonlinear Regression: A Practical Guide to Curve Fitting. (Oxford University Press, Oxford; New York, 2004).

  32. Kubin, R.F. & Fletcher, A.N. Fluorescence quantum yields of some rhodamine dyes. J. Lumin. 27, 455–462 (1982).

    Article  Google Scholar 

  33. Lacowicz, J. Principles of Fluorescence Spectroscopy edn 2. (Kluwer Academic/Plenum, London, 1999).

  34. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J. & Waggoner, A.S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4, 105–111 (1993).

    Article  CAS  Google Scholar 

  35. Sims, P.J., Waggoner, A.S., Wang, C.H. & Hoffman, J.F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13, 3315–3330 (1974).

    Article  CAS  Google Scholar 

  36. Mueller, W.H.I., Schuetz, H.J. & Meyer, G. Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis. Nucleic Acids Res. 95, 95–119 (1981).

    Article  Google Scholar 

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Acknowledgements

We thank Dane Wittrup for providing an aliquot of the yeast scFv surface display library, members of his laboratory for advice and Nicholas Bateman for work on scFv maturation. Supported by National Institutes of Health grant 7 U54 RR022241 and Pennsylvania Department of Health grant 4100020575.

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Authors and Affiliations

  1. Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, 15213, Pennsylvania, USA

    Christopher Szent-Gyorgyi, Brigitte F Schmidt, Yehuda Creeger, Gregory W Fisher, Kelly L Zakel, James A J Fitzpatrick, Marcel P Bruchez & Alan Waggoner

  2. Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, 15213, Pennsylvania, USA

    Sally Adler, Carol A Woolford, Qi Yan, Kalin V Vasilev, Peter B Berget & Jonathan W Jarvik

  3. Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, 15213, Pennsylvania, USA

    Marcel P Bruchez

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  1. Christopher Szent-Gyorgyi
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Contributions

B.F.S. synthesized fluorogens and derivatives; C.S.-G. and Y.C. cloned fluorogenic scFvs under the direction of C.S.-G.; K.L.Z. carried out cloning-related molecular biology and protein purification; under the direction of J.W.J., S.A. constructed mammalian expression vectors with contribution from K.V.V.; G.W.F. and Q.Y. created stable mammalian cell lines; G.W.F. and J.A.J.F. performed microscopy; C.A.W. implemented directed evolution mutagenesis with contribution from P.B.B.; C.S.-G. conducted spectroscopy and binding experiments and analysis; C.S.-G. drafted manuscript and figures; M.P.B., J.W.J., P.B.B. and A.W. contributed editorially to manuscript and to general design of research; A.W. conceived fluorogen strategy and oversaw entire project.

Corresponding authors

Correspondence to Christopher Szent-Gyorgyi or Alan Waggoner.

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Supplementary Tables 1, Figures 1–8, Methods (PDF 1556 kb)

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Szent-Gyorgyi, C., Schmidt, B., Creeger, Y. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol 26, 235–240 (2008). https://doi.org/10.1038/nbt1368

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