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Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics

Nature Biotechnology volume 39, pages 686–690 (2021)Cite this article

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Abstract

Overcoming limitations of previous fluorescent light-up RNA aptamers for super-resolution imaging, we present RhoBAST, an aptamer that binds a fluorogenic rhodamine dye with fast association and dissociation kinetics. Its intermittent fluorescence emission enables single-molecule localization microscopy with a resolution not limited by photobleaching. We use RhoBAST to image subcellular structures of RNA in live and fixed cells with about 10-nm localization precision and a high signal-to-noise ratio.

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Fig. 1: Directed evolution and characterization of RhoBAST.
Fig. 2: Confocal, epifluorescence and SMLM with the RhoBAST:TMR-DN system.

Data availability

The data that support the findings of this study are available upon reasonable request from the corresponding authors. Aptamer sequences used for imaging ROIs are available in the Supplementary Information.

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Acknowledgements

M.S. and A.J. were supported by the Deutsche Forschungsgemeinschaft (DFG grant no. Ja794/11) and G.U.N. by the Helmholtz Association (Program Science and Technology of Nanosystems) and the DFG (GRK 2039). We thank the Nikon Imaging Center, Heidelberg for granting access to their facilities, U. Engel for technical advice in fluorescence microscopy and M. Mayer and L. Rohland for assistance with stopped-flow measurements. We gratefully acknowledge R. Ma and A. Kobitski for technical support with SMLM experiments and analysis. We thank BASF SE for kindly providing Lutensol AT50.

Author information

Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany

    Murat Sunbul, Annabell Martin, Daniel Englert, Franziska Grün & Andres Jäschke

  2. Institute of Applied Physics (APH), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

    Jens Lackner, Benjamin Hacene, Karin Nienhaus & G. Ulrich Nienhaus

  3. Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany

    G. Ulrich Nienhaus

  4. Institute of Biological and Chemical Systems (IBCS), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany

    G. Ulrich Nienhaus

  5. Department of Physics, University of Illinois at Urbana−Champaign, Urbana, IL, USA

    G. Ulrich Nienhaus

Authors
  1. Murat Sunbul
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  2. Jens Lackner
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  7. Karin Nienhaus
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  8. G. Ulrich Nienhaus
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Contributions

M.S., G.U.N. and A.J. designed the study. A.M. and M.S. evolved RhoBAST, and M.S., D.E. and F.G. characterized RhoBAST’s photophysical properties. M.S. created all plasmid constructs and strains and carried out confocal and SIM microscopy. J.L. carried out SMLM experiments and analyzed the data. J.L. and B.H. developed the assay for single-molecule binding kinetics and performed the experiments and analysis. M.S., K.N., G.U.N. and A.J. supervised the work. M.S. wrote the first draft, and all authors contributed to reviewing, editing and providing additional text for the manuscript.

Corresponding authors

Correspondence to Murat Sunbul, G. Ulrich Nienhaus or Andres Jäschke.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Don Lamb and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Supplementary Tables 1–5 and Supplementary Notes 1–3

Reporting Summary

Supplementary Video 1

Fluorescence decay of Pepper and RhoBAST

Supplementary Video 2

3D CGG repeat-containing FMR1-GFP mRNA aggregates

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Sunbul, M., Lackner, J., Martin, A. et al. Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics. Nat Biotechnol 39, 686–690 (2021). https://doi.org/10.1038/s41587-020-00794-3

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  • DOI: https://doi.org/10.1038/s41587-020-00794-3

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