Abstract
Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.
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Acknowledgements
We thank M. Papiz (Liverpool University, UK), E. Giraud (Institute for Research and Development, France), W. Weber (University of Freiburg, Germany) and F. Zhang (Broad Institute of MIT and Harvard, USA) for plasmids, and A. Leopold (University of Helsinki), A. Kaberniuk and A. Shemetov (both from Albert Einstein College of Medicine) for useful suggestions. We thank Biomedicum Imaging Unit, AAV Gene Transfer and Cell Therapy and Biomedicum Flow Cytometry core facilities staff (University of Helsinki) for the technical assistance. This work was supported by grants GM105997, GM108579 and NS099573 from the US National Institutes of Health, ERC-2013-ADG-340233 from the EU FP7 program, and 263371 and 266992 from the Academy of Finland.
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T.A.R. and K.G.C. characterized the proteins in vitro, and T.A.R., E.S.O. and K.G.C. analyzed the optogenetic pair in mammalian cells. V.V.V. planned and directed the project and together with T.A.R., E.S.O. and K.G.C. designed the experiments, analyzed the data and wrote the manuscript.
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Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–18 (PDF 2816 kb)
Supplementary Video 1
Sequential light-induced relocalization of the iRIS tool from a cytoplasm to the nucleus and to the plasma membrane in a single cell after the illumination periods (either 5 min with 460 nm of 1 mW cm−2 or 5 min with 740 nm of 1 mW cm−2 light) followed by the dark relaxation periods. (AVI 33424 kb)
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Redchuk, T., Omelina, E., Chernov, K. et al. Near-infrared optogenetic pair for protein regulation and spectral multiplexing. Nat Chem Biol 13, 633–639 (2017). https://doi.org/10.1038/nchembio.2343
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DOI: https://doi.org/10.1038/nchembio.2343
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