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A general strategy to red-shift green fluorescent protein-based biosensors

Nature Chemical Biology volume 16, pages 1434–1439 (2020)Cite this article

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Abstract

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.

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Fig. 1: Green-to-red conversion of sfGFP by aY.
Fig. 2: Fluorescence imaging of glucose-induced metabolic dynamics in pancreatic MIN6 β-cells.

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Data availability

The gene sequences for MjaYRS and EcaYRS have been deposited to GenBank under the accession numbers MT002434 and MT002433, respectively. The plasmids for pEvol-MjaYRS (Plasmid no. 153557) and pMAH-EcaYRS (Plasmid no. 153558) have been deposited to Addgene. Materials, associated protocols and other supporting data are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Greenwald, E. C., Mehta, S. & Zhang, J. Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of signaling networks. Chem. Rev. 118, 11707–11794 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mishin, A. S. et al. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Biochemistry 47, 4666–4673 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Sawin, K. E. & Nurse, P. Photoactivation of green fluorescent protein. Curr. Biol. 7, R606–R607 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Elowitz, M. B., Surette, M. G., Wolf, P. E., Stock, J. & Leibler, S. Photoactivation turns green fluorescent protein red. Curr. Biol. 7, 809–812 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Sattarzadeh, A., Saberianfar, R., Zipfel, W. R., Menassa, R. & Hanson, M. R. Green to red photoconversion of GFP for protein tracking in vivo. Sci. Rep. 5, 11771 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Ai, M. et al. Green-to-red photoconversion of GCaMP. PLoS ONE 10, e0138127 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Bogdanov, A. M. et al. Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol. 5, 459–461 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Saha, R. et al. Light driven ultrafast electron transfer in oxidative redding of green fluorescent proteins. Sci. Rep. 3, 1580 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Bae, J. H. et al. Expansion of the genetic code enables design of a novel ‘gold’ class of green fluorescent proteins. J. Mol. Biol. 328, 1071–1081 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, L., Xie, J., Deniz, A. A. & Schultz, P. G. Unnatural amino acid mutagenesis of green fluorescent protein. J. Org. Chem. 68, 174–176 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Wang, L. et al. Significant expansion and red-shifting of fluorescent protein chromophore determined through computational design and genetic code expansion. Biophys. Rep. 4, 273–285 (2018).

    Article  CAS  PubMed  Google Scholar 

  14. Villa, J. K. et al. Fluorescence modulation of green fluorescent protein using fluorinated unnatural amino acids. Molecules 22, 1194 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Fu, C. et al. Genetically encoding quinoline reverses chromophore charge and enables fluorescent protein brightening in acidic vesicles. J. Am. Chem. Soc. 140, 11058–11066 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Groff, D., Wang, F., Jockusch, S., Turro, N. J. & Schultz, P. G. A new strategy to photoactivate green fluorescent protein. Angew. Chem. Int. Ed. Engl. 49, 7677–7679 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Reddington, S. C. et al. Different photochemical events of a genetically encoded phenyl azide define and modulate GFP fluorescence. Angew. Chem. Int. Ed. Engl. 52, 5974–5977 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Reddington, S. C. et al. Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence. Chem. Sci. 6, 1159–1166 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Wang, F., Niu, W., Guo, J. & Schultz, P. G. Unnatural amino acid mutagenesis of fluorescent proteins. Angew. Chem. Int. Ed. Engl. 51, 10132–10135 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Liu, X. et al. Significant expansion of the fluorescent protein chromophore through the genetic incorporation of a metal-chelating unnatural amino acid. Angew. Chem. Int. Ed. Engl. 52, 4805–4809 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Ayyadurai, N. et al. Development of a selective, sensitive, and reversible biosensor by the genetic incorporation of a metal-binding site into green fluorescent protein. Angew. Chem. Int. Ed. Engl. 50, 6534–6537 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Chen, Z. J., Ren, W., Wright, Q. E. & Ai, H. W. Genetically encoded fluorescent probe for the selective detection of peroxynitrite. J. Am. Chem. Soc. 135, 14940–14943 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Chen, S., Chen, Z. J., Ren, W. & Ai, H. W. Reaction-based genetically encoded fluorescent hydrogen sulfide sensors. J. Am. Chem. Soc. 134, 9589–9592 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Seyedsayamdost, M. R., Xie, J., Chan, C. T., Schultz, P. G. & Stubbe, J. Site-specific insertion of 3-aminotyrosine into subunit ɑ2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation. J. Am. Chem. Soc. 129, 15060–15071 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Sakamoto, K. et al. Genetic encoding of 3-iodo-l-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography. Structure 17, 335–344 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Ren, W., Truong, T. M. & Ai, H. W. Study of the binding energies between unnatural amino acids and engineered orthogonal tyrosyl-tRNA synthetases. Sci. Rep. 5, 12632 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Takimoto, J. K., Adams, K. L., Xiang, Z. & Wang, L. Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells. Mol. BioSyst. 5, 931–934 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Oki, K., Sakamoto, K., Kobayashi, T., Sasaki, H. M. & Yokoyama, S. Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog. Proc. Natl Acad. Sci. USA 105, 13298–13303 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K. & Tsien, R. Y. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl Acad. Sci. USA 97, 11990–11995 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ai, H. W., Henderson, J. N., Remington, S. J. & Campbell, R. E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. & Tsien, R. Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Chen, Z. J. & Ai, H. W. A highly responsive and selective fluorescent probe for imaging physiological hydrogen sulfide. Biochemistry 53, 5966–5974 (2014).

    Article  CAS  PubMed  Google Scholar 

  34. Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chen, Z. & Ai, H. W. Single fluorescent protein-based indicators for zinc ion (Zn2+). Anal. Chem. 88, 9029–9036 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Marvin, J. S. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat. Methods 10, 162–170 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Marvin, J. S. et al. A genetically encoded fluorescent sensor for in vivo imaging of GABA. Nat. Methods 16, 763–770 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Patriarchi, T. et al. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360, eaat4422 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Zhao, Y. et al. SoNar, a highly responsive NAD+/NADH sensor, allows high-throughput metabolic screening of anti-tumor agents. Cell Metab. 21, 777–789 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tao, R. et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism. Nat. Methods 14, 720–728 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tantama, M., Martinez-Francois, J. R., Mongeon, R. & Yellen, G. Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio. Nat. Commun. 4, 2550 (2013).

    Article  PubMed  Google Scholar 

  42. Lobas, M. A. et al. A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP. Nat. Commun. 10, 711 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fridlyand, L. E. & Philipson, L. H. Glucose sensing in the pancreatic beta cell: a computational systems analysis. Theor. Biol. Med. Model 7, 15 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Brown, W., Liu, J. & Deiters, A. Genetic code expansion in animals. ACS Chem. Biol. 13, 2375–2386 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Ernst, R. J. et al. Genetic code expansion in the mouse brain. Nat. Chem. Biol. 12, 776–778 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, L. Engineering the genetic code in cells and animals: biological considerations and impacts. Acc. Chem. Res. 50, 2767–2775 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Korosak, D. & Slak Rupnik, M. Collective sensing of β-cells generates the metabolic code. Front. Physiol. 9, 31 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Gray, J. P., Alavian, K. N., Jonas, E. A. & Heart, E. A. NAD kinase regulates the size of the NADPH pool and insulin secretion in pancreatic β-cells. Am. J. Physiol. Endocrinol. Metab. 303, E191–E199 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Plecitá-Hlavatá, L. et al. Glucose-stimulated insulin secretion fundamentally requires H2O2 signaling by NADPH oxidase 4. Diabetes 69, 1341–1354 (2020).

    Article  PubMed  Google Scholar 

  50. Dan Dunn, J., Alvarez, L. A., Zhang, X. & Soldati, T. Reactive oxygen species and mitochondria: a nexus of cellular homeostasis. Redox Biol. 6, 472–485 (2015).

    Article  Google Scholar 

  51. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Young, T. S., Ahmad, I., Yin, J. A. & Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361–374 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Zhang, Z. & Marshall, A. G. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J. Am. Soc. Mass Spectrom. 9, 225–233 (1998).

    Article  CAS  PubMed  Google Scholar 

  54. Kobayashi, T. et al. Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion. Proc. Natl Acad. Sci. USA 102, 1366–1371 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chatterjee, A., Xiao, H., Bollong, M., Ai, H. W. & Schultz, P. G. Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells. Proc. Natl Acad. Sci. USA 110, 11803–11808 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kellogg, R. E. & Bennett, R. G. Radiationless intermolecular energy transfer. III. Determination of phosphorescence efficiencies. J. Chem. Phys. 41, 3042–3045 (1964).

    Article  CAS  Google Scholar 

  57. Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank R. Campbell, L. Looger, B. Khakh, G. Yellen and P. Schultz for plasmids; S. Chen for MIN6 cells; and other members of the Ai laboratory for discussion and assistance with experiments. We thank W. Ren and A. Ji for early exploration of this project. Research reported in this publication was supported in part by the University of Virginia and the National Institutes of Health under awards nos. R01GM118675, R01GM129291, U01CA230817 and R01DK122253.

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Authors and Affiliations

  1. Center for Membrane and Cell Physiology, Department of Molecular Physiology and Biological Physics, Department of Chemistry, and the UVA Cancer Center, University of Virginia, Charlottesville, VA, USA

    Shen Zhang & Hui-wang Ai

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  1. Shen Zhang
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Contributions

H.-w.A. conceived and supervised the project. S.Z. performed all experiments. H.-w.A. and S.Z. analyzed the data and prepared the manuscript.

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Correspondence to Hui-wang Ai.

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Extended data

Extended Data Fig. 1 Fluorescence excitation and emission of aY-modified fluorescence proteins.

Fluorescence excitation (black) and emission (red) profiles of aY-modified cpsGFP a, mTFP1 b, cpYFP c, and Citrine d.

Extended Data Fig. 2 Characterization of aY-modified G-GECO1 (Ca2+ sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for G-GECO1 after addition of 1 mM EGTA (cyan) or 100 μM Ca2+ (dark cyan), and aY-G-GECO1 after addition of 1 mM EGTA (magenta) or 100 μM Ca2+ (red). b, Representative images of HeLa cells expressing aY-G-GECO1 in response to sequential addition of 5 μM histamine, 1 mM CaCl2 with 10 μM ionomycin, and 2 mM EGTA with 5 μM ionomycin. Scale bar: 30 µm. c, Quantitative traces for randomly selected five cells in panel b. Intensities are normalized to the values at t = 0 s. The time points for addition of chemicals are shown as arrows. Ca2+ oscillations in response to histamine were observed as expected. These experiments were repeated three times with similar results using independent biological samples.

Extended Data Fig. 3 Characterization of aY-modified ZnGreen1 (Zn2+ sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for ZnGreen1 after addition of 1 mM EDTA (cyan) or 100 μM Zn2+ (dark cyan), and aY- ZnGreen1 after addition of 1 mM EDTA (magenta) or 100 μM Zn2+ (red). b, Representative images of HEK 293 T cells expressing aY-ZnGreen1 in response to sequential addition of 50 μM ZnCl2 with 5 μM pyrithione, and 200 μM TPEN. Scale bar: 20 µm. c, Quantitative traces for randomly selected six single cells (gray dots) and their average (red line). Intensities are normalized to the values at t = 0 s. The time points for addition of chemicals are shown as arrows. These experiments were repeated three times with similar results using independent biological samples.

Extended Data Fig. 4 Characterization of aY-modified iGluSnFR (glutamate sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for iGluSnFR before (cyan) and after (dark cyan) addition of 100 μM L-glutamate, and aY-iGluSnFR before (magenta) and after (red) addition of 100 μM L-glutamate. b, Representative images of HEK 293 T cells expressing cell-surface-localized aY-iGluSnFR in response to addition of 100 μM L-glutamate. c, Cultured mouse hippocampal neurons in response to 90 mM KCl (excitatory Tyrode’s saline buffer). Neurons were co-transfected to express cell-surface-localized aY-iGluSnFR (red fluorescence) and a Ca2+ indicator, G-GECO1 (green fluorescence). Quantitative traces for four different regions are presented. Intensities are normalized to the values at t = 0 s. These experiments were repeated three times with similar results using independent biological samples. Scale bar: 20 µm.

Extended Data Fig. 5 Characterization of aY-modified iGABASnFR (a,b) and dLight1.2 (c,d) as biosensors for GABA and dopamine, respectively.

a, c, Fluorescence excitation (dash line) and emission (solid line) profiles for iGABASnFR or dLight1.2 before (cyan) and after (dark cyan) addition of 1 mM GABA or 100 µM dopamine, and aY-iGABASnFR or aY-dLight1.2 before (magenta) and after (red) addition of 1 mM GABA or 100 µM dopamine. b, d, Representative images of HEK 293 T cells expressing surface-localized aY-iGABASnFR (b) or aY-dLight1.2 (d) in response to GABA or dopamine. These experiments were repeated three times with similar results using independent biological samples. Scale bar: 20 µm.

Extended Data Fig. 6 Characterization of aY-modified SoNar (NAD+/NADH sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for SoNar after addition of 20 µM NAD+ (cyan) or NADH (dark cyan), and aY-SoNar after addition of 20 µM NAD+ (magenta) or NADH (red). b, Representative images of HEK 293 T cells expressing aY-SoNar in response to addition of 1 mM pyruvate. Scale bar: 20 µm. c, Quantitative traces for randomly selected four single cells in panel b. Intensities are normalized to the values at t = 0 s. The time points for addition of chemicals are shown as arrows. These experiments were repeated three times with similar results using independent biological samples.

Extended Data Fig. 7 Characterization of aY-modified iNap1 (NADPH sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for iNap1 before (cyan) and after (dark cyan) addition of 100 µM NADPH, and aY-iNap1 before (magenta) and after (red) addition of 100 µM NADPH. b, Representative images of HEK 293 T cells expressing aY- iNap1 in response to addition of 1 mM diamide. Scale bar: 20 µm. c, Quantitative traces for randomly selected five single cells (gray dots) and their average (red line). Intensities are normalized to the values at t = 0 s. The time points for addition of chemicals are shown as arrows. These experiments were repeated three times with similar results using independent biological samples.

Extended Data Fig. 8 Characterization of aY-modified PercevalHR (ATP sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for PercevalHR before (cyan) and after (dark cyan) addition of 1 mM ATP, and aY-PercevalHR before (magenta) and after (red) addition of 1 mM ATP. b, Representative images of HeLa cells expressing aY- PercevalHR in response to 10 mM 2-deoxy-D-glucose (2-DG). Scale bar: 20 µm. c, Quantitative traces for randomly selected five single cells (gray dots) and their average (red line). Intensities are normalized to the values at t = 0 s. The time points for addition of chemicals are shown as arrows. These experiments were repeated three times with similar results using independent biological samples.

Extended Data Fig. 9 Characterization of aY-modified iATPSnFR1.1 (ATP sensor).

a, Fluorescence excitation (dash line) and emission (solid line) profiles for iATPSnFR1.1 before (cyan) and after (dark cyan) addition of 1 mM ATP, and aY-iATPSnFR1.1 before (magenta) and after (red) addition of 1 mM ATP. b, Representative images of HeLa cells intracellular expressing aY-iATPSnFR1.1 in response to 10 mM 2-deoxy-D-glucose (2-DG). Scale bar: 20 µm. c, Quantitative traces for randomly selected eight single cells (gray dots) and their average (red line). Intensities are normalized to the values at t = 0 s. The time points for addition of chemicals are shown as arrows. These experiments were repeated three times with similar results using independent biological samples.

Extended Data Fig. 10 Representative dual-color images of MIN6 cells co-expressing SoNar and mitochondrial aY-SoNar.

MIN6 β-cells were sequentially imaged using GFP and RFP channels. Scale bar: 20 µm. This experiment was repeated three times independently with similar results.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Video 1

aY-G-GECO1 in HeLa cells in response to histamine, Ca2+ and EGTA sequentially.

Supplementary Video 2

aY-iGluSnFR and G-GECO1 in a mouse hippocampal neuron in response to high K+ depolarization.

Supplementary Video 3

SoNar and Mito-aY-SoNar in MIN6 cells in response to high glucose.

Source data

Source Data Fig. 1

Source data for Fig. 1d

Source Data Fig. 2

Statistical source data for Fig. 2h

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Zhang, S., Ai, Hw. A general strategy to red-shift green fluorescent protein-based biosensors. Nat Chem Biol 16, 1434–1439 (2020). https://doi.org/10.1038/s41589-020-0641-7

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