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A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells

Nature Chemical Biology volume 16, pages 69–76 (2020)Cite this article

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Abstract

Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.

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Fig. 1: Design, synthesis and fluorogenicity of Gemini-561.
Fig. 2: Isolation of Gemini-561 lighting-up aptamers by in vitro evolution.
Fig. 3: Characterization and engineering of the evolved molecule.
Fig. 4: Live-cell imaging of o-Coral expressed from Pol II and Pol III promoters.
Fig. 5: Comparative analysis of photostability by fluorescence microscopy and spectroscopy.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information file.

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Acknowledgements

We thank R. Leblay, A.-C. Helfer, E. Gutzwiller and G. Lieber for technical assistance as well as P. Romby and I. Caldelari for fruitful scientific discussion. We thank O. Kucherak for synthesis of intermediate 2, E. Real for scientific discussions and J. Valanciunaite and N. Humbert for the technical assistance. This work received financial support from Agence Nationale de la Recherche (BrightRiboProbes, grant no. ANR-16-CE11-0010-01/02 to M.R. and A.S.K.) and BacNet (grant no. ANR-10-BINF_02_02 to M.R.) and European Research Council ERC Consolidator grant BrightSens (no. 648528 to A.S.K.). This work has also been published under the framework of the LabEx (no. ANR-10-LABX-0036_NETRNA to M.R.) and benefits from a funding from the state managed by the French National Research Agency as part of the Investments for the Future Program. It was also supported by the Université de Strasbourg and the Centre National de la Recherche Scientifique.

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Author notes

  1. These authors contributed equally: Farah Bouhedda, Kyong Tkhe Fam.

Authors and Affiliations

  1. Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR 9002, Strasbourg, France

    Farah Bouhedda, Alexis Autour, Stefano Marzi & Michael Ryckelynck

  2. Université de Strasbourg, Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Illkirch, France

    Kyong Tkhe Fam, Mayeul Collot & Andrey Klymchenko

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Contributions

A.K., M.C. and M.R. proposed the concept of this study. M.C. synthesized Gemini-561. F.B. and K.T.F. performed main part of experimental work and data analysis with help and supervision of M.R., M.C. and A.K. F.B. and M.R. developed the aptamer o-Coral, including its variants and characterized their structure with the help of S.M. A.A. and F.B. characterized the contribution of the different selected mutations. K.T.F., M.C., F.B. and A.K. characterized Gemini-561–o-Coral fluorescence properties in solution. F.B. generated plasmids encoding o-Coral. K.T.F. realized all cellular studies and all fluorescence imaging of the cells, with help of A.K. and M.C.

Corresponding authors

Correspondence to Mayeul Collot, Andrey Klymchenko or Michael Ryckelynck.

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Competing interests

F.B., K.T.F., M.C., A.K., M.R., the University of Strasbourg and the CNRS have filed a patent application covering the technology presented in this manuscript.

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Bouhedda, F., Fam, K.T., Collot, M. et al. A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells. Nat Chem Biol 16, 69–76 (2020). https://doi.org/10.1038/s41589-019-0381-8

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