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Live-cell imaging of RNA dynamics using bright and stable fluorescent RNAs

Nature Protocols (2026)Cite this article

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Abstract

RNAs exhibit complex dynamics in cells, including expression, splicing, localization, translation and degradation, and these processes are highly coordinated and tightly regulated both spatially and temporally. To better understand the biological function of diverse RNAs, approaches that allow monitoring of RNA with high spatiotemporal resolution are essential. Fluorescent RNAs (FRs), fluorescent protein–like entities consisting of RNA aptamers and their cognate fluorogenic dyes, have emerged as a promising approach for imaging RNA dynamics in live cells. We recently reported the development of several high-performance FRs, named Pepper, Clivia and Okra, that show advantageous properties, including high cellular brightness and photostability, low ion dependence and/or large Stokes shifts, and have been used to image diverse RNA species in live cells. In this protocol, we provide easy, efficient and generalizable strategies for using FRs to visualize different RNA species in bacteria and mammalian cells by expressing the RNA of interest tagged with one or more copies of the aptamer. We also provide a detailed procedure for multiplexed RNA imaging using orthogonal FRs and the steps to perform super-resolution live imaging of RNAs. The protocol typically takes 5–7 d, including cloning, transfection of mammalian cells or transformation of bacteria, live imaging and results analysis. This protocol is applicable to the real-time monitoring of the localization and dynamics of RNAs of interest in live cells.

Key points

  • Single or tandem copies of the RNA aptamers Pepper, Okra or Clivia are fused to a target RNA expressed exogenously in bacteria or mammalian cells. The aptamer binds its cognate fluorogenic dye, rendering the RNA fluorescent for live imaging.

  • Because of their small size and high brightness and stability, fluorescent RNA tags permit live imaging of diverse RNAs, including small noncoding and messenger RNAs, while the orthogonality of the tags allows them to be multiplexed.

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Fig. 1: Overview of different approaches for RNA labeling.
Fig. 2: Pepper, Clivia and Okra fluorescent RNAs.
Fig. 3: Overview of the strategy of engineering target RNA for RNA labeling.
Fig. 4: Overview of the procedure for FR-based RNA imaging.
Fig. 5: Imaging FR-tagged mRNA in live E. coli cells.
Fig. 6: Imaging of FR-tagged ncRNA and mRNA in live mammalian cells.
Fig. 7: Multiplexed RNA imaging in live mammalian cells.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers9,11,13. The raw datasets are too large to be publicly shared but are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (2022YFC3400100 to Y.Y. and X.C.; 2024YFA180006 to N.S.), NSFC (32121005, 92581203, 22437001 and 92357308 to Y.Y.; 32250009 to X.C.; 32501340 to F.Z.; 32401077 to X.X.), STI2030-Major Projects (2021ZD0202200 and 2021ZD0202203 to X.C.), the Shanghai Municipal Education Commission (2021 Sci & Tech 03-28), the Shanghai Science and Technology Commission (23J21900400 to X.C.), a grant from the Tianfu Jincheng Laboratory (TFJCPI20250016 to Y.Y.), the Shanghai Sailing Program (24YF2709300 to F.Z.), the Shanghai Municipal Health Commission (20254Y0019 to F.Z.), Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (JYB2025XDXM404), 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

Author notes

  1. These authors contributed equally: Fangting Zuo, Ni Su, Xin Xie.

Authors and Affiliations

  1. Yangpu Hospital, School of Medicine, Tongji University, Shanghai, China

    Fangting Zuo

  2. Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, School of Pharmacy, East China University of Science and Technology, Shanghai, China

    Fangting Zuo, Xin Xie, Mengyue Fang, Yuzheng Zhao, Xianjun Chen & Yi Yang

  3. Frontiers Medical Center, Tianfu Jincheng Laboratory, Chengdu, China

    Ni Su & Yi Yang

  4. School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China

    Li Jiang & Linyong Zhu

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Contributions

Concepts were conceived by Y.Y. and X.C. Y.Y., X.C., F.Z., N.S. and X.X. designed the experiments and analyzed the data. F.Z. and X.X. performed plasmid construction. F.Z. and N.S. performed live-cell imaging and FISH experiments. M.F., L.J, Y.Z. and L.Z. gave technical support and conceptual advice. Y.Y., X.C., F.Z., N.S. and X.X. wrote the manuscript.

Corresponding authors

Correspondence to Xianjun Chen or Yi Yang.

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Nature Protocols thanks Michael Ryckelynck and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references

Chen, X. et al. Nat. Biotechnol. 37, 1287–1293 (2019): https://doi.org/10.1038/s41587-019-0249-1

Jiang, L. et al. Nat. Methods 20, 1563–1572 (2023): https://doi.org/10.1038/s41592-023-01997-7

Zuo, F. et al. Nat. Chem. Biol. 20, 1272–1281 (2024): https://doi.org/10.1038/s41589-024-01629-x

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Zuo, F., Su, N., Xie, X. et al. Live-cell imaging of RNA dynamics using bright and stable fluorescent RNAs. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01343-z

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