Near-infrared (NIR, 740–780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1–QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1–QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1–QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3–5 d. Use of NIR light–controlled optogenetic systems should advance basic and biomedical research.
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This work was supported by grants GM122567 and NS103573 from the US National Institutes of Health, ERC-2013-ADG-340233 from the EU 7th Framework Programme (FP7), and grants 263371 and 266992 from the Academy of Finland.
The authors declare no competing financial interests.
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(a) Light penetration depth at 480 nm, 560 nm, 670 nm and 720 nm wavelengths in a muscle tissue. Adapted with permission from suppl. ref. 31, Nature Publishing Group (b) Molar extinction coefficient of oxygenated hemoglobin (HbO2) and deoxygenated hemoglobin (Hb). NIR tissue transparency window is shown as a red box. a adapted from Rumyantsev, K.A., Turoverov, K.K. & Verkhusha, V.V. Near-infrared bioluminescent proteins for two-color multimodal imaging. Sci. Rep. 6, 636588 (2016), Macmillan Publishers. b adapted with permission from Shcherbakova, D.M., Shemetov, A.A., Kaberniuk, A.A. & Verkhusha, V.V. Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools. Annu. Rev. Biochem., 84, 519–550, © 2015 by Annual Reviews, http://www.annualreviews.org.
(a) Mode of function of the light-inducible transcription activation system. NIR light converts BphP1 into the Pr state and induces its heterodimerization with PpsR2. The NLS fused to PpsR2 facilitates translocation of the heterodimer to the nucleus where BphP1 fusions interact with tetO DNA repeats via its fused TetR. VP16 fused to PpsR2 recruits the transcription initiation complex and triggers transcription of a reporter gene. (b) Kinetics of the light-to-dark ratio of the SEAP signal detected in the culture media of a HeLa cell line stably expressing BphP1-mCherry-TetR and co-transfected with NLS-PpsR2-VP16-producing plasmid and pTRE-Tight-SEAP (7× tetO) reporter plasmid. Samples were illuminated by 740 nm pulsing light (30 s ON, 180 s OFF) of 1 mW cm−2. Adapted from ref. 4, Nature Publishing Group.
(a) Rluc8 bioluminescence detected in mice with subcutaneously injected HeLa cells stably expressing BphP1-mCherry-TetR and co-transfected with the NLS-PpsR2-VP16-producing plasmid and pTRE–Tight–Rluc8 reporter plasmid kept either in the dark (top) or illuminated with 740/25 nm light of 1 mW cm−2 (bottom) for 48 h. (b) Rluc8 signals detected in dark-treated animals and in illuminated animals shown in (a) (n = 3; error bars are s.e.m.). All animal experiments in this Protocol were performed in an AAALAC approved facility according to the permission #20160313 approved by the Albert Einstein College of Medicine Animal Usage Committee. a adapted from ref. 4, Nature Publishing Group.
(a) iRIS relocalization to the nucleus in HeLa cells in response to 460 nm illumination. Scale bar, 10 μm. (b) Intensity profile of mCherry fluorescence of iRIS in the cell shown in (a) marked with a dashed line before (black line) and after (red line) 10 min of 460 nm illumination. (c) iRIS relocalization to the plasma membrane in HeLa cells under 740 nm illumination. Scale bar, 10 μm. (d) Intensity profile of mCherry fluorescence of iRIS in the cell shown in (c) marked with a dashed line before (black line) and after (red line) 10 min of 740 nm illumination. Adapted from ref. 5, Nature Publishing Group.
iRIS relocalization to the nucleus (a) and to the plasma membrane (b) in HeLa cells in response to 460 nm and 740 nm illumination, respectively. Scale bar, 10 μm. Good and poor quality results are shown, white arrows indicate non-specific signals (mCherry fluorescence on the plasma membrane under 460 nm light (a) and in the nucleus under 740 nm light (b)).
Non-transfected HeLa cells (a) are used to create a gate for FACS sorting of mCherry positive cells (b).
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Redchuk, T., Kaberniuk, A. & Verkhusha, V. Near-infrared light–controlled systems for gene transcription regulation, protein targeting and spectral multiplexing. Nat Protoc 13, 1121–1136 (2018). https://doi.org/10.1038/nprot.2018.022
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