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The HaloTag as a general scaffold for far-red tunable chemigenetic indicators

Nature Chemical Biology volume 17, pages 718–723 (2021)Cite this article

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Abstract

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for ‘chemigenetic’ fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

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Fig. 1: Strategy for engineering chemigenetic indicators from HaloTag.
Fig. 2: HaloCaMP engineering and in vitro characterization.
Fig. 3: Synthesis of Si-rhodamine derivatives and characterization of HaloCaMP in neuron cultures.
Fig. 4: Chemigenetic voltage indicators.

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Data availability

Plasmids have been deposited at Addgene (www.addgene.org) as follows: pAAV-synapsin-HaloCaMP1a (plasmid 138327), pAAV-synapsin-HaloCaMP1b (plasmid 138328), pCAG-HASAP1 (plasmid 138325) and pCAG-HArcLight1 (plasmid 138326). Crystal structures of HaloTag–TMR and HaloCaMP1b635 have been deposited at the Protein Data Bank (www.rcsb.org) with accession codes 6U32 and 6U2M, respectively. Source data from experiments in this study are available from the authors upon reasonable request. Source data are provided with this paper.

References

  1. Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).

    Article  CAS  Google Scholar 

  2. Lin, M. Z. & Schnitzer, M. J. Genetically encoded indicators of neuronal activity. Nat. Neurosci. 19, 1142–1153 (2016).

    Article  Google Scholar 

  3. Dana, H. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 16, 649–657 (2019).

    Article  CAS  Google Scholar 

  4. Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137–141 (2001).

    Article  CAS  Google Scholar 

  5. St-Pierre, F. et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nat. Neurosci. 17, 884–889 (2014).

    Article  CAS  Google Scholar 

  6. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).

    Article  Google Scholar 

  7. Qian, Y. et al. A genetically encoded near-infrared fluorescent calcium ion indicator. Nat. Methods 16, 171–174 (2019).

    Article  CAS  Google Scholar 

  8. Ng, D. N. & Fromherz, P. Genetic targeting of a voltage-sensitive dye by enzymatic activation of phosphonooxymethyl-ammonium derivative. ACS Chem. Biol. 6, 444–451 (2011).

    Article  CAS  Google Scholar 

  9. Ghitani, N., Bayguinov, P. O., Ma, Y. & Jackson, M. B. Single-trial imaging of spikes and synaptic potentials in single neurons in brain slices with genetically encoded hybrid voltage sensor. J. Neurophysiol. 113, 1249–1259 (2015).

    Article  Google Scholar 

  10. Liu, P., Grenier, V., Hong, W., Muller, V. R. & Miller, E. W. Fluorogenic targeting of voltage-sensitive dyes to neurons. J. Am. Chem. Soc. 139, 17334–17340 (2017).

    Article  CAS  Google Scholar 

  11. Deo, C., Sheu, S. H., Seo, J., Clapham, D. E. & Lavis, L. D. Isomeric tuning yields bright and targetable red Ca2+ indicators. J. Am. Chem. Soc. 141, 13734–13738 (2019).

    Article  CAS  Google Scholar 

  12. Grenier, V., Daws, B. R., Liu, P. & Miller, E. W. Spying on neuronal membrane potential with genetically targetable voltage indicators. J. Am. Chem. Soc. 141, 1349–1358 (2019).

    Article  CAS  Google Scholar 

  13. Deal, P. E. et al. Covalently tethered rhodamine voltage reporters for high speed functional imaging in brain tissue. J. Am. Chem. Soc. 142, 614–622 (2020).

    Article  CAS  Google Scholar 

  14. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  Google Scholar 

  15. Encell, L. P. et al. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr. Chem. Genomics 6, 55–71 (2012).

    Article  Google Scholar 

  16. Wombacher, R. & Cornish, V. W. Chemical tags: applications in live cell fluorescence imaging. J. Biophotonics 4, 391–402 (2011).

    Article  CAS  Google Scholar 

  17. Wang, A., Feng, J., Li, Y. & Zou, P. Beyond fluorescent proteins: hybrid and bioluminescent indicators for imaging neural activities. ACS Chem. Neurosci. 9, 639–650 (2018).

    Article  CAS  Google Scholar 

  18. England, C. G., Luo, H. & Cai, W. HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986 (2015).

    Article  CAS  Google Scholar 

  19. Abdelfattah, A. S. et al. Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science 365, 699–704 (2019).

    Article  CAS  Google Scholar 

  20. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    Article  CAS  Google Scholar 

  21. Grimm, J. B. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat. Methods 14, 987–994 (2017).

    Article  CAS  Google Scholar 

  22. Lavis, L. D. Teaching old dyes new tricks: biological probes built from fluoresceins and rhodamines. Annu. Rev. Biochem. 86, 825–843 (2017).

    Article  CAS  Google Scholar 

  23. Baird, G. S., Zacharias, D. A. & Tsien, R. Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl Acad. Sci. USA 96, 11241–11246 (1999).

    Article  CAS  Google Scholar 

  24. Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  Google Scholar 

  25. Zheng, Q. et al. Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging. ACS Cent. Sci. 5, 1602–1613 (2019).

    Article  CAS  Google Scholar 

  26. Grimm, J. B. & Lavis, L. D. Synthesis of rhodamines from fluoresceins using Pd-catalyzed C–N cross-coupling. Org. Lett. 13, 6354–6357 (2011).

    Article  CAS  Google Scholar 

  27. Hansch, C., Leo, A. & Taft, R. W. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 91, 165–195 (1991).

    Article  CAS  Google Scholar 

  28. Wardill, T. J. et al. A neuron-based screening platform for optimizing genetically-encoded calcium indicators. PLoS ONE 8, e77728 (2013).

    Article  CAS  Google Scholar 

  29. Jin, L. et al. Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron 75, 779–785 (2012).

    Article  CAS  Google Scholar 

  30. Tsutsui, H., Jinno, Y., Tomita, A. & Okamura, Y. Optically detected structural change in the N-terminal region of the voltage-sensor domain. Biophys. J. 105, 108–115 (2013).

    Article  CAS  Google Scholar 

  31. Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S. & Tsien, R. Y. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349, 694–697 (1991).

    Article  CAS  Google Scholar 

  32. Grimm, J. B. et al. A general method to optimize and functionalize red-shifted rhodamine dyes. Nat. Methods 17, 815–821 (2020).

    Article  CAS  Google Scholar 

  33. Gottlieb, H. E., Kotlyar, V. & Nudelman, A. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512–7515 (1997).

    Article  CAS  Google Scholar 

  34. Suzuki, K. et al. Reevaluation of absolute luminescence quantum yields of standard solutions using a spectrometer with an integrating sphere and a back-thinned CCD detector. Phys. Chem. Chem. Phys. 11, 9850–9860 (2009).

    Article  CAS  Google Scholar 

  35. Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  Google Scholar 

  36. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  Google Scholar 

  37. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  38. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  39. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge the Molecular Biology, Cell Culture and Virus Production facilities at Janelia for assistance. This work was supported by the Howard Hughes Medical Institute. B.M. holds a postdoctoral fellowship from the Research Foundation–Flanders (FWO Vlaanderen). The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under contract no. DE-AC02-05CH11231.

Author information

Author notes

  1. Claire Deo

    Present address: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

  2. Hersh K. Bhargava

    Present address: Biophysics Graduate Program, University of California, San Francisco, San Francisco, CA, USA

  3. Natalie Falco

    Present address: Pharmacological Sciences Graduate Program, University of California, Irvine, Irvine, CA, USA

  4. Benjamien Moeyaert

    Present address: Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium

  5. These authors contributed equally: Claire Deo, Ahmed S. Abdelfattah.

  6. These authors jointly supervised this work: Luke D. Lavis, Eric R. Schreiter.

Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Claire Deo, Ahmed S. Abdelfattah, Hersh K. Bhargava, Adam J. Berro, Natalie Falco, Helen Farrants, Benjamien Moeyaert, Mariam Chupanova, Luke D. Lavis & Eric R. Schreiter

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Contributions

C.D., A.S.A., L.D.L. and E.R.S. conceived the project and wrote the manuscript. C.D. contributed organic synthesis, protein engineering, in vitro characterization of proteins, X-ray crystallography and neuron imaging. A.S.A. contributed protein engineering, neuron imaging and electrophysiology. H.K.B. contributed protein engineering, in vitro characterization and neuron imaging. A.J.B. contributed organic synthesis and X-ray crystallography. N.F. contributed organic synthesis. H.F. measured stopped-flow kinetics of purified HaloCaMP. B.M. and M.C. contributed protein engineering and neuron imaging. L.D.L. and E.R.S. directed the project.

Corresponding authors

Correspondence to Luke D. Lavis or Eric R. Schreiter.

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Competing interests

C.D., A.S.A., H.K.B., L.D.L. and E.R.S. have filed patent applications on chemigenetic indicators and azetidine-substituted fluorophores.

Additional information

Peer review information Nature Chemical Biology thanks Adam Cohen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Multiple conformations of tetramethylrhodamine (TMR) in the HaloTag-TMR structure (PDB 6U32).

Stereo view of the region of the bound dye ligand with the HaloTag protein backbone represented as grey cartoon, Asp106 and the bound HaloTag ligand are shown as sticks with grey carbon atoms, and the TMR dye is modeled in two conformations, represented as sticks with black or white carbon atoms. The TMR conformation with black carbon atoms is the one represented in Fig. 1e,f. The 2fo-fc electron density map for the bound dye ligand is shown as purple mesh, contoured at 0.8σ. Although the electron density is strong and continuous for the HaloTag ligand, density for the dye moiety is weaker and broken, suggesting conformational heterogeneity.

Extended Data Fig. 2 Exploration of circular permutation sites within HaloTag for sensor design.

a, Crystal structure of HaloTag (grey cartoon ribbons) bound to TMR–HaloTag ligand (sticks), illustrating circular permutation sites tested as green spheres labeled with the amino acid number. b, Schematic of the sensor context for testing HaloTag circular permutations, with cpHaloTag inserted between helices 3 and 4 of a VSD (top), the field stimulus pattern for stimulation of neurons expressing voltage sensors with cpHaloTag variants (middle), and representative fluorescence traces of each voltage sensor variant in stimulated neurons (bottom).

Source data

Extended Data Fig. 3 Synthesis of Si-rhodamine derivatives.

Synthesis routes and yields of azetidine-substituted Si-rhodamine derivatives (a) and their corresponding HaloTag ligands (b).

Extended Data Fig. 4 Spectroscopy of azetidine-substituted Si-rhodamine derivatives.

a, Absorption, (b) normalized excitation and (c) normalized fluorescence emission spectra of novel azetidine-substituted Si-rhodamines. d, Correlation of experimental λex versus inductive Hammett constants (σΙ) for Si-rhodamines. e, Normalized absorption (bold line) and fluorescence (dashed line) spectra of Si-rhodamine HaloTag ligands, in the presence (colored line) or absence (black line) of HaloTag protein. Values were normalized to the HaloTag-bound spectra. All spectra were measured at C = 5 µM in 10 mM HEPES pH = 7.4. In the case of the HaloTag ligands, 0.1 mg.mL-1 CHAPS was added to the buffer.

Source data

Extended Data Fig. 5 Calcium titrations.

Solution calcium titration curves for purified HaloCaMP1a (a) and HaloCaMP1b (b) labeled with Si-rhodamine ligands.

Source data

Extended Data Fig. 6 Labeling of HaloCaMP in neuron cultures.

Cultured hippocampal neurons expressing HaloCaMP1a-GFP (a) or HaloCaMP1b-GFP labeled with with JF635-HTL (1 μM, 30 min). GFP channel (left panel), JF635 channel (middle panel) and merge (right panel); scale bars: 50 μm.

Extended Data Fig. 7 Comparison of HaloCaMP with NIR-GECO1.

a, Comparison of the relative fluorescence brightness (product of quantum yield and extinction coefficient) measured in purified protein solutions. b-d, Measurements in primary neuron cultures with field stimulation-driven action potentials of (b) maximum ΔF/F0 (c) fluorescence rise time, and (d) fluorescence decay time.

Source data

Extended Data Fig. 8 Fluorescence response in neuron culture of the initial variant of HASAP loaded with JF635-HTL.

Black triangles denote the timing of electrical stimuli from the field electrode. Compare with Fig. 4i after additions of the R467G mutation.

Source data

Extended Data Fig. 9 Fluorescence vs. Voltage for HASAP1635.

Fluorescence traces of HASAP1635 in response to a series of voltage steps (from -110 mV to +50 mV in 20 mV increments). Image acquisition rate = 400 Hz. Representative traces from one cell of N = 8 cells for HASAP1635.

Source data

Extended Data Fig. 10 Fluorescence vs. Voltage for HArcLight1635.

Fluorescence traces of HArcLight1635 in response to a series of voltage steps (from -110 mV to +50 mV in 20 mV increments). Image acquisition rate = 400 Hz. Representative traces from one cell of N = 9 cells.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Tables 1–6 and Supplementary Notes.

Reporting Summary

Supplementary Video 1

Raw fluorescence video, captured at 400 Hz and displayed in real time, of a rat hippocampal neuron in culture expressing HASAP1 and labeled with JF635–HaloTag ligand. Voltage is clamped at −70 mV and stepped using a patch electrode to +30 mV for 1 s.

Source data

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Deo, C., Abdelfattah, A.S., Bhargava, H.K. et al. The HaloTag as a general scaffold for far-red tunable chemigenetic indicators. Nat Chem Biol 17, 718–723 (2021). https://doi.org/10.1038/s41589-021-00775-w

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