Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s

Nature Methods volume 20, pages 432–441 (2023)Cite this article

Subjects

Abstract

Optogenetic tools for controlling protein–protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: BICYCL development and characterization.
Fig. 2: BICYCL-Green optogenetic systems in mammalian cells.
Fig. 3: BICYCL-Red optogenetic systems in mammalian cells.
Fig. 4: Genomically engineered BICYCL-Green gene expression cells.
Fig. 5: Bidirectional switching of BICYCLs for spatial optogenetic control.
Fig. 6: Multiplexing BICYCLs with other optogenetic tools in mammalian cells.

Similar content being viewed by others

Data availability

All data associated with this study are present in the article and the Supplementary Information. Source data are provided with this paper.

References

  1. Goglia, A. G. & Toettcher, J. E. A bright future: optogenetics to dissect the spatiotemporal control of cell behavior. Curr. Opin. Chem. Biol. 48, 106–113 (2019).

    Article  CAS  PubMed  Google Scholar 

  2. Kolar, K., Knobloch, C., Stork, H., Znidaric, M. & Weber, W. OptoBase: a web platform for molecular optogenetics. ACS Synth. Biol. 7, 1825–1828 (2018).

    Article  CAS  PubMed  Google Scholar 

  3. Redchuk, T. A., Omelina, E. S., Chernov, K. G. & Verkhusha, V. V. Near-infrared optogenetic pair for protein regulation and spectral multiplexing. Nat. Chem. Biol. 13, 633–639 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hallett, R. A., Zimmerman, S. P., Yumerefendi, H., Bear, J. E. & Kuhlman, B. Correlating in vitro and in vivo activities of light-inducible dimers: a cellular optogenetics guide. ACS Synth. Biol. 5, 53–64 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Toettcher, J. E., Gong, D., Lim, W. A. & Weiner, O. D. Light control of plasma membrane recruitment using the Phy-PIF system. Methods Enzymol. 497, 409–423 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Guntas, G. et al. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl Acad. Sci. USA 112, 112–117 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Wang, H. et al. LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat. Methods 13, 755–758 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhou, Y. et al. A small and highly sensitive red/far-red optogenetic switch for applications in mammals. Nat. Biotechnol. 40, 262–272 (2022).

    Article  CAS  PubMed  Google Scholar 

  9. Kainrath, S., Stadler, M., Reichhart, E., Distel, M. & Janovjak, H. Green-light-induced inactivation of receptor signaling using cobalamin-binding domains. Angew. Chem. Int. Ed. Engl. 56, 4608–4611 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chatelle, C. et al. A green-light-responsive system for the control of transgene expression in mammalian and plant cells. ACS Synth. Biol. 7, 1349–1358 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Fushimi, K., Ikeuchi, M. & Narikawa, R. The expanded red/green cyanobacteriochrome lineage: an evolutionary hot spot. Photochem. Photobiol. 93, 903–906 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. Rockwell, N. C., Martin, S. S. & Lagarias, J. C. Red/green cyanobacteriochromes: sensors of color and power. Biochemistry 51, 9667–9677 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Ikeuchi, M. & Ishizuka, T. Cyanobacteriochromes: a new superfamily of tetrapyrrole-binding photoreceptors in cyanobacteria. Photochem. Photobiol. Sci. 7, 1159–1167 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Ong, N. T. & Tabor, J. J. A miniaturized Escherichia coli green light sensor with high dynamic range. Chembiochem 19, 1255–1258 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Blain-Hartung, M. et al. Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells. J. Biol. Chem. 293, 8473–8483 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Losi, A., Gardner, K. H. & Moglich, A. Blue-light receptors for optogenetics. Chem. Rev. 118, 10659–10709 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fushimi, K. et al. Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin. Front. Microbiol. 7, 588 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Muller, K. et al. Synthesis of phycocyanobilin in mammalian cells. Chem. Commun. 49, 8970–8972 (2013).

    Article  Google Scholar 

  19. Reis, J. M. et al. Discovering selective binders for photoswitchable proteins using phage display. ACS Synth. Biol. 7, 2355–2364 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Fairbrother, W. J. et al. Novel peptides selected to bind vascular endothelial growth factor target the receptor-binding site. Biochemistry 37, 17754–17764 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Woloschuk, R. M., Reed, P. M. M., McDonald, S., Uppalapati, M. & Woolley, G. A. Yeast two-hybrid screening of photoswitchable protein–protein interaction libraries. J. Mol. Biol. 432, 3113–3126 (2020).

    Article  CAS  PubMed  Google Scholar 

  22. Muller, K. et al. Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Muller, K., Engesser, R., Timmer, J., Zurbriggen, M. D. & Weber, W. Orthogonal optogenetic triple-gene control in Mammalian cells. ACS Synth. Biol. 3, 796–801 (2014).

    Article  PubMed  Google Scholar 

  24. Moreno, M. V., Rockwell, N. C., Mora, M., Fisher, A. J. & Lagarias, J. C. A far-red cyanobacteriochrome lineage specific for verdins. Proc. Natl Acad. Sci. USA 117, 27962–27970 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fushimi, K. et al. Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin. Proc. Natl Acad. Sci. USA 116, 8301–8309 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tachibana, S. R. et al. An engineered biliverdin-compatible cyanobacteriochrome enables a unique ultrafast reversible photoswitching pathway. Int. J. Mol. Sci. 22, 5252 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dyson, M. R. Fundamentals of expression in mammalian cells. Adv. Exp. Med. Biol. 896, 217–224 (2016).

    Article  CAS  PubMed  Google Scholar 

  28. Guinn, M. T., Coraci, D., Guinn, L. & Balazsi, G. Reliably engineering and controlling stable optogenetic gene circuits in mammalian cells. J. Vis. Exp. https://doi.org/10.3791/62109 (2021).

  29. Zhu, D., Johnson, H. J., Chen, J. & Schaffer, D. V. Optogenetic application to investigating cell behavior and neurological disease. Front. Cell. Neurosci. 16, 811493 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schneider, N. et al. Liquid–liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice. Sci. Adv. 7, eabd3568 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Muller, K. et al. A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells. Nucleic Acids Res. 41, e77 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Levskaya, A., Weiner, O. D., Lim, W. A. & Voigt, C. A. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Niopek, D. et al. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat. Commun. 5, 4404 (2014).

    Article  CAS  PubMed  Google Scholar 

  34. Niopek, D., Wehler, P., Roensch, J., Eils, R. & Di Ventura, B. Optogenetic control of nuclear protein export. Nat. Commun. 7, 10624 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. van Bergeijk, P., Adrian, M., Hoogenraad, C. C. & Kapitein, L. C. Optogenetic control of organelle transport and positioning. Nature 518, 111–114 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Fellouse, F. A. & Sidhu, S. S. in Making and Using Antibodies: A Practical Handbook (eds Howard, G. C. & Kaser, M. R.) Ch. 8 (CRC Press, 2007).

  37. Kunkel, T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl Acad. Sci. USA 82, 488–492 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Chen, G. & Sidhu, S. S. Design and generation of synthetic antibody libraries for phage display. Methods Mol. Biol. 1131, 113–131 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Keller, S. et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066–5073 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Brautigam, C. A., Zhao, H., Vargas, C., Keller, S. & Schuck, P. Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions. Nat. Protoc. 11, 882–894 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhao, H., Piszczek, G. & Schuck, P. SEDPHAT: a platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 76, 137–148 (2015).

    Article  CAS  PubMed  Google Scholar 

  42. Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Beyer, H. M. et al. AQUA cloning: a versatile and simple enzyme-free cloning approach. PLoS ONE 10, e0137652 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Ruijter, J. M. et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 37, e45 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Mates, L. et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 41, 753–761 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Kowarz, E., Loscher, D. & Marschalek, R. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol. J. 10, 647–653 (2015).

    Article  CAS  PubMed  Google Scholar 

  47. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  48. Peng, T. et al. A BaSiC tool for background and shading correction of optical microscopy images. Nat. Commun. 8, 14836 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Allan, C. et al. OMERO: flexible, model-driven data management for experimental biology. Nat. Methods 9, 245–253 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by Discovery grants from the Natural Sciences and Engineering Research Council of Canada (grant RGPIN-174255 to G.A.W.; grant RGPIN-06195 to M.U.) and the German Research Foundation (DFG) (grant ZU259/2-1 to M.D.Z., Collaborative Research Center 1208 project no. 267205415, NEXTplant GRK2466 and under Germany’s Excellence Strategy CEPLAS – EXC-2048/1 – Project ID 390686111 to M.D.Z.), Human Frontiers Scientific Program (HFSP RGY0063/2017 and HFSP RGP0067/2021to M.D.Z.) and the European Commission – Research Executive Agency (H2020 Future and Emerging Technologies (FET-Open) Project ID 801041 CyGenTiG to K.T., H.M.B. and M.D.Z.). H.M.B. was supported by the ‘Freigeist’ fellowship of the Volkswagen Foundation. We thank W. Houry (University of Toronto) for access to equipment, S. Kuschel (University of Düsseldorf) for technical assistance, T. Boissonnet for helping with image processing (University of Düsseldorf) and W. Weber (University of Freiburg) for the gift of IDR (intrinsically disordered region) plasmids. We also thank L. Koch, C. Diehl and U. Urquiza (University of Düsseldorf) and especially A. Jaikaran (University of Toronto) for careful reading and their suggestions to improve the paper.

Author information

Author notes

  1. These authors contributed equally: Jaewan Jang, Kun Tang.

Authors and Affiliations

  1. Department of Chemistry, University of Toronto, Toronto, Ontario, Canada

    Jaewan Jang, Jeffrey Youn & G. Andrew Woolley

  2. Institute of Synthetic Biology, Heinrich-Heine-Universität, Düsseldorf, Germany

    Kun Tang, Hannes M. Beyer & Matias D. Zurbriggen

  3. Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    Sherin McDonald & Maruti Uppalapati

  4. CEPLAS – Cluster of Excellence on Plant Science, Düsseldorf, Germany

    Matias D. Zurbriggen

Authors
  1. Jaewan Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeffrey Youn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sherin McDonald
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hannes M. Beyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matias D. Zurbriggen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maruti Uppalapati
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. G. Andrew Woolley
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.J., K.T., M.U., M.D.Z. and G.A.W. developed the concepts and designed the experiments. J.J., S.M. and M.U. designed and carried out the phage display experiments. J.J. carried out all protein expression, purification and in vitro analyses. J.J. designed, carried out and analyzed protein subcellular localization experiments. K.T. designed, carried out and analyzed all mammalian gene expression experiments. J.Y. purified the BAmGreen2.4 protein. H.M.B. and K.T. engineered stable cell lines, and designed and performed spatial expression patterning. G.A.W., M.U. and M.D.Z. supervised the project. J.J., K.T., M.U., M.D.Z. and G.A.W. wrote the paper. All authors contributed to the editing, and read the final version of the paper.

Corresponding authors

Correspondence to Matias D. Zurbriggen, Maruti Uppalapati or G. Andrew Woolley.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks J. Clark Lagarias, Matthieu Sainlos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

Additional information

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

Extended data

Extended Data Fig. 1 Performance of the BICYCL system with biliverdin (BV).

(a) Schematic showing mVenus-BAmRed anchored to the mitochondrial membrane via an NTOM20 tag. tagRFP-Amg2-BV is localized to the mitochondria in the dark and dispersed in the cytoplasm under far-red light. (b) mVenus fluorescence and tagRFP fluorescence confocal microscopy images of Amg2-tagRFP in HEK293T cells with 40 µM biliverdin added 24 h prior to imaging (direct fluorescence of BV could not be detected, as reported by Fushimi et al.17). Confocal images were obtained on cells co-transfected with pLL-tagRFP-Amg2 and NTOM20-mVenus-BAmRed1.4 (top) or BAmRed1.0 (bottom). (Scale bar: 20 µm). Cells were adapted to the dark state for 30 min prior to acquiring Pfr state images. The experiment was independently repeated four times with similar results. (c) Schematic of BICYCL-controlled gene expression with biliverdin. (d) Constructs tested for gene expression. CHO-K1 cells were transiently co-transfected with BICYCLs and the SEAP reporter. Cells were exposed to either 590 nm (20 μmol m−2 s−1), or 700 nm (20 μmol m−2 s−1) for 24 h. Data represent mean values ± SD; n = 3 independent samples. p values shown were calculated by 2-tailed unpaired t-test. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001; n.s., not significant. Source data are provided as a Source Data file. For plasmids and abbreviations, see Supplementary Table 2.

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–27 and Tables 1–3.

Reporting Summary

Peer Review File

Supplementary Data 1

Source data for Supplementary Information.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jang, J., Tang, K., Youn, J. et al. Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s. Nat Methods 20, 432–441 (2023). https://doi.org/10.1038/s41592-023-01764-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-023-01764-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing