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

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.

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.

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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.

Author information

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Authors

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.

Corresponding author

Correspondence to Hui-wang Ai.

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The authors declare no competing interests.

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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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