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Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA

Abstract

Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

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Fig. 1: A bright green FR Okra–ACE (Okra505).
Fig. 2: Okra–ACE complex exhibits excellent photostability in live cells.
Fig. 3: Visualization of mRNA distributions in live bacterial and mammalian cells.
Fig. 4: Time-lapse imaging of SG dynamics in live cells.
Fig. 5: Dual-color SIM imaging of SGs in live cells.

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The data supporting the findings of this study are available in the paper and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

This research was supported by the National Key Research and Development Program of China (grant nos. 2022YFC3400100 to Y.Y. and X.C., 2019YFA0110500 to L.Z. and 2019YFA0904800 to Y.Y. and L.Z.), NSFC (grant nos. 32121005, 92357308, 32150028 and 21937004 to Y.Y.; 21907029 and 21877037 to L.Z.; 32250009 to X.C. and 32001051 to N.S.), STI2030-Major Projects (grant nos. 2021ZD0202200 and 2021ZD0202203 to X.C.), the Shanghai Municipal Education Commission (grant no. 2021 Sci & Tech 03-28 to Y.Y. and X.C.), the Shanghai Science and Technology Commission (23J21900400 to X.C.), the State Key Laboratory of Bioreactor Engineering (to Y.Y. and X.C.) and the Fundamental Research Funds for the Central Universities (to Y.Y. and X.C.).

Author information

Authors and Affiliations

Authors

Contributions

Concepts were conceived by Y.Y., L.Z. and X.C. Y.Y., L.Z., X.C., F.Z., N.S. and L.J. designed the experiments and analyzed the data. L.J. and B.B. synthesized the dyes. F.Z. performed SELEX experiment. F.Z., Y. Zhang, L.W., H.Y. and R.L. characterized the aptamer in vitro and constructed plasmids. F.Z. and N.S. performed live-cell imaging experiments. Y.S., X.H., Q.Z., Q.L., Z.C., Y. Zhuang and Y. Zhao gave technical support and conceptual advice. Y.Y., L.Z., X.C. and F.Z. wrote the manuscript.

Corresponding authors

Correspondence to Xianjun Chen, Linyong Zhu or Yi Yang.

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Competing interests

Y.Y., F.Z. and X.C. are named inventors of patent application number CN202110817172.8. The other authors declare no competing interests.

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Nature Chemical Biology thanks Murat Sunbul and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Viscosity responsiveness analysis of ACE.

(a) Schematic diagram of ACE and its fluorescence activation after fixation by RNA aptamer. (b) Viscosity coefficient measurement of ACE. 5 μM ACE were prepared in ethylene/glycerol solutions with different mixing ratios at room temperature. Fluorescence intensity was acquired using PerkinElmer FL6500/8500 fluorescence spectrophotometer. Viscosity was measured using NDJ-8S rotor viscometer. The value of viscosity sensitivity (x) was determined by logarithm plot of emission intensity as a function of viscosity based on the Förster-Hoffmann equation log10 (Intensity)=x·log10 (Viscosity)+A. (c) Excitation and emission spectra of ACE. 5 μM ACE was prepared in a series of ethylene glycol/glycerol solutions in which glycerin ratios were 0, 20%, 40%, 60%, 80%, 100% at room temperature, respectively. (d) Fluorescence measurement of ACE in different viscosity solutions. Data in (c) and (d) were normalized to the maximum fluorescence and presented as the mean ± s.d. from three biological replicates.

Source data

Extended Data Fig. 2 Mutational optimization of the aptamer.

(a) Mutation sites (green) in the loop region of A2-T2. (b) Fluorescence activation of ACE by different A2-T2 variants. The vertical coordinate represents the mutation sites and the horizontal coordinate represents the nucleotide types of the indicated site shown in (a). Data were normalized to the fluorescence of A2-T2 aptamer and presented as the mean of data from two independent experiments. (c), (e) and (g), fluorescence activation of ACE by different A2-T2 variants containing single mutation (c), double mutations (e), and multiple mutations (g). 2.5 μM ACE was incubated with 0.5 μM RNA aptamers. Data were normalized to the fluorescence of A2-T2. (d), (f) and (h), the affinity of ACE with different A2-T2 variants containing single mutation (d), double mutations (f), and multiple mutations (h). 2 nM RNA aptamer was incubated with increasing concentrations of ACE.

Source data

Extended Data Fig. 3 Validation of A2-T2 variants in live mammalian cells.

(a) Schematic representation of fluorescence measurement by flow cytometry. (b) Flow cytometry histograms of the fluorescence distributions for different A2-T2 variants. HEK293T cells transfected with plasmids expressing different A2-T2 variants embedded in an F30 scaffold were digested and suspended in 4% FBS/1× PBS solution containing 0.5 μM ACE 36 h after transfection. The fluorescence of the cells was analyzed using a flow cytometry with an FITC channel. Untransfected cells or cells transfected with the mock plasmid were used as the controls. (c) Quantification of the fluorescence and positive population of the cells in (b).

Source data

Extended Data Fig. 4 In vitro characterization of Okra-ACE.

(a) Absorption spectra of ACE (gray) and Okra-ACE complex (green). 1 μM ACE was incubated without or with 10 μM RNA aptamers. (b) Schematic view of the ON kinetics by a Sepharose bead, which coated by streptavidin protein and labeled by RNA coupled with biotin at the 5’ end and then added buffer containing corresponding ligands. (c) The ON kinetics of the association of ACE to Okra. Fluorescence images were taken immediately after the beads were incubated with 10 nM ACE. Scale bar, 10 μm. (d) Quantitative analysis of the Okra-ACE complex formation. The data were fitted to the formula of exponential rise to maximum y = y0 + a·(1-e-bx) (Methods for details). Data represent the mean fluorescence of three Sepharose beads. (e) The OFF kinetics of the disassociation of ACE to Okra. Okra-coated Sepharose beads were placed in buffer without ACE fluorophore and consecutive imaging was performed. Scale bar, 10 μm. (f) Quantitative analysis of the fluorescence of Okra-ACE complex. The data were fitted to the formula of exponential decay (y = y0 + a·e-bx) (Methods for details). Data represent the mean fluorescence of three Sepharose beads. (g) Potassium independence of Okra-ACE fluorescence. (h) Effect of pH on the fluorescence of Okra-ACE, Pepper-HBC530 or Broccoli-BI. (g-h) The concentration of RNA was 1 μM and fluorophore was 10 μM. Data were normalized to the maximum fluorescence and presented as the mean ± s.d. from three biological replicates.

Source data

Extended Data Fig. 5 Okra-ACE exhibited reduced photoisomerization in vitro.

In vitro photostability of Okra bound to ACE (0.1 μM) and Broccoli bound to BI (0.1 μM) or TBI (0.1 μM) was assessed by continuous imaging of Okra- and Broccoli-coated Sepharose beads, respectively. 1 μM free aptamer was added to bind the free fluorophore surrounding the beads. Shown is the initial light-induced rate of fluorescence loss in vitro. Before spectral correction, ACE exhibited markedly reduced photoisomerization compared to BI and TBI (a, b). After spectral correction, Okra505 exhibited a comparable photoisomerization rate with Broccoli-TBI, but still had a much reduced photoisomerization rate compared to Broccoli-BI (c, d). Data represent the mean fluorescence of 10 Sepharose beads.

Source data

Extended Data Fig. 6 Visualization of mRNA distributions in live mammalian cells.

(a) Schematic representation of constructs expressing ACTB mRNA tagged with different copies of dOkra, dPepper or dBroccoli from a CMV promoter. (b) Confocal imaging of live COS-7 cells expressing ACTB mRNA tagged with different fluorescent RNAs. COS-7 cells transfected with different FR were incubated with 0.5 μM ACE (Okra), 1 μM HBC530 (Pepper) or 10 μM BI (Broccoli) and imaged. COS-7 cells transfected with construct expressing ACTB alone were used as the controls. Scale bars, 20 μm. (c) Quantitative analysis of the fluorescence of Okra-ACE, Pepper-HBC530 and Broccoli-BI fluorescence in individual cells. Statistical comparison was performed by a two-tailed t test. ***P < 0.001. Data represent the mean ±s.d. (N = 151, 170, 157, 151, 151, 152, 155, 150 and 152 cells from left to right).

Source data

Extended Data Fig. 7 The effects of ATP depletion at different time points after NaAsO2 induction.

HeLa cells expressing ACTB-4×dOkra were incubated with 2DG and CCCP at different time points after NaAsO2 induction. Cells were co-transfected with a plasmid expressing G3BP1-BFP protein, which is a marker for stress granules. The images were taken 60 min after NaAsO2 induction. To validate the results, the non-transfected HeLa cells with the same treatment were fixed 60 min after NaAsO2 induction, and ACTB mRNA and G3BP1 protein were labeled using the Cy3-conjugated-FISH probe and Alexa Fluor 647-conjugated antibody, respectively. Scale bars, 10 μm. At least three independent experiments were carried out with similar results.

Extended Data Fig. 8 SIM imaging using Okra505.

(a) Schematic representation of the protein-RNA tethered reporting system based on the interaction of tdMCP and the MS2 binding hairpin RNA. (b) Raw images of Okra-ACE for SIM imaging. HeLa cells expressing Okra-MS2 and tdMCP-TOMM20 were imaged by structured illumination microscopy (SIM) imaging in the presence of 1 μM ACE. Scale bar, 10 μm. (c) Wide field and SIM imaging of Okra tethered to the outer mitochondrial membrane by TOMM20 protein. The imaged were shown in maximum-intensity projection along the z dimension through the cell volume. Two areas indicated as the white rectangle were enlarged. Scale bars, 5 μm (left), 1 μm (right). (d) Line profiles of the fluorescence from Okra-ACE of the dashed white lines in (c). For b and c, at least two independent experiments were carried out with similar results.

Source data

Extended Data Fig. 9 Bioorthogonality of Okra505 with Pepper620.

(a) Predicted secondary structure of Okra and Pepper and the structures of their ligands. (b) The excitation and emission spectra of Okra505 and Pepper620. (c) The activation fluorescence of Okra505 with Pepper620 in vitro. Data were normalized with Okra-ACE or Pepper-HBC620 and presented as the mean ± s.d. from three biological replicates. (d) Dual-color imaging of Okra505 and Pepper620. HEK293T cells expressing F30-Okra and circular-Pepper or each alone were incubated with 0.5 μM ACE and 1 μM HBC620 and imaged. The nucleus was stained with Hoechst 33342 (blue). Scale bars, 10 μm.

Source data

Extended Data Fig. 10 Dual-color SIM imaging using Okra505 with Pepper620.

(a) Schematic representation of the protein‒RNA tethered reporting system based on the interactions of boxB-λN and MS2-MCP. Okra and Pepper are fused with boxB and MS2 to obtain Okra-boxB and Pepper-MS2, respectively. SNAP-tag-labeled λN and BFP-labeled tdMCP are fused to LifeAct and TOMM20, which enables the fusion proteins to be localized to filamentous actin and the outer mitochondrial membrane, respectively. (b) Dual-color SIM imaging using Okra505 and Pepper620 in live mammalian cells. HEK293T cells expressing the components in (a) were incubated with 1 μM ACE and 1 μM HBC620 and studied by 3D-SIM imaging. Scale bars, 10 μm. At least two independent experiments were carried out with similar results.

Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Table 1–5 and Notes 1 and 2.

Reporting Summary

Supplementary Video 1

The time course of SG formation after NaAsO2 induction.

Supplementary Video 2

The reversible and dynamic nature of SGs after withdrawal of stress.

Supplementary Video 3

Blocking of SG assembly by 2DG and CCCP.

Supplementary Video 4

ATP-dependent SG disassembly after withdrawal of NaAsO2.

Supplementary Video 5

The dynamics of SGs in cells after NaAsO2 induction.

Supplementary Video 6

The dynamics of SGs in ATP-depleted cells after NaAsO2 induction.

Supplementary Data 1

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Zuo, F., Jiang, L., Su, N. et al. Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01629-x

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