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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.

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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.

References

  1. Sigal, Y. M., Zhou, R. & Zhuang, X. Science 361, 880–887 (2018).

    Article  CAS  Google Scholar 

  2. Schmidt, A., Gao, G., Little, S. R., Jalihal, A. P. & Walter, N. G. Wiley Interdiscip. Rev. RNA 11, e1587 (2020).

  3. Su, Y. & Hammond, M. C. Curr. Opin. Biotechnol. 63, 157–166 (2020).

    Article  CAS  Google Scholar 

  4. Wirth, R., Gao, P., Nienhaus, G. U., Sunbul, M. & Jäschke, A. J. Am. Chem. Soc. 141, 7562 (2019).

    Article  CAS  Google Scholar 

  5. Chen, X. et al. Nat. Biotechnol. 37, 1287–1293 (2019).

    Article  CAS  Google Scholar 

  6. Sunbul, M. & Jäschke, A. Angew. Chem. Int. Ed. 52, 13401–13404 (2013).

    Article  CAS  Google Scholar 

  7. Arora, A., Sunbul, M. & Jäschke, A. Nucleic Acids Res. 43, e144 (2015).

    PubMed  PubMed Central  Google Scholar 

  8. Braselmann, E. et al. Nat. Chem. Biol. 14, 964–971 (2018).

    Article  CAS  Google Scholar 

  9. Sunbul, M. & Jäschke, A. Nucleic Acids Res. 46, e110 (2018).

    Article  Google Scholar 

  10. Li, Y., Ishitsuka, Y., Hedde, P. N. & Nienhaus, G. U. ACS Nano 7, 5207–5214 (2013).

    Article  CAS  Google Scholar 

  11. Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. J. Am. Chem. Soc. 136, 16299–16308 (2014).

    Article  CAS  Google Scholar 

  12. Song, W. et al. Nat. Chem. Biol. 13, 1187–1194 (2017).

    Article  CAS  Google Scholar 

  13. Autour, A. et al. Nat. Commun. 9, 656 (2018).

    Article  Google Scholar 

  14. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Nat. Protoc. 12, 1198–1228 (2017).

    Article  CAS  Google Scholar 

  15. Litke, J. L. & Jaffrey, S. R. Nat. Biotechnol. 37, 667–675 (2019).

    Article  CAS  Google Scholar 

  16. Kim, H. & Jaffrey, S. R. Cell Chem. Biol. 26, 1725–1731 e1726 (2019).

    Article  CAS  Google Scholar 

  17. Hagerman, R. J. & Hagerman, P. Nat. Rev. Neurol. 12, 403–412 (2016).

    Article  CAS  Google Scholar 

  18. Fox, A. H., Nakagawa, S., Hirose, T. & Bond, C. S. Trends Biochem. Sci. 43, 124–135 (2018).

    Article  CAS  Google Scholar 

  19. Steinberg, R., Knupffer, L., Origi, A., Asti, R. & Koch, H. G. FEMS Microbiol. Lett. 365 (2018).

  20. Wu, Y. & Shroff, H. Nat. Methods 15, 1011–1019 (2018).

    Article  CAS  Google Scholar 

  21. Strack, R. L., Disney, M. D. & Jaffrey, S. R. Nat. Methods 10, 1219–1224 (2013).

    Article  CAS  Google Scholar 

  22. Grimm, J. B. et al. Nat. Methods 14, 987–994 (2017).

    Article  CAS  Google Scholar 

  23. Bajar, B. T. et al. Sci. Rep. 6, 20889 (2016).

    Article  CAS  Google Scholar 

  24. Schindelin, J. et al. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  25. Manz, C. et al. Nat. Chem. Biol. 13, 1172–1178 (2017).

    Article  CAS  Google Scholar 

  26. Li, Y., Shang, L. & Nienhaus, G. U. Nanoscale 8, 7423–7429 (2016).

    Article  CAS  Google Scholar 

  27. Ober, R. J., Ram, S. & Ward, E. S. Biophys. J. 86, 1185–1200 (2004).

    Article  CAS  Google Scholar 

  28. Deschout, H. et al. Nat. Methods 11, 253–266 (2014).

    Article  CAS  Google Scholar 

  29. Guizar-Sicairos, M., Thurman, S. T. & Fienup, J. R. Opt. Lett. 33, 156–158 (2008).

    Article  Google Scholar 

  30. Fedorov, A. et al. Magn. Reson. Imaging 30, 1323–1341 (2012).

    Article  Google Scholar 

Download references

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.

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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

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  1. Murat Sunbul
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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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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.

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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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