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

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

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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.

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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.

Data availability

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

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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

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  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

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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.

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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.

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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

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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

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