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

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

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.

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. 1.

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

    CAS 
    Article 

    Google Scholar
     

  2. 2.

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

  3. 3.

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

    CAS 
    Article 

    Google Scholar
     

  4. 4.

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

    CAS 
    Article 

    Google Scholar
     

  5. 5.

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

    CAS 
    Article 

    Google Scholar
     

  6. 6.

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

    CAS 
    Article 

    Google Scholar
     

  7. 7.

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

    PubMed 
    PubMed Central 

    Google Scholar
     

  8. 8.

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

    CAS 
    Article 

    Google Scholar
     

  9. 9.

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

    Article 

    Google Scholar
     

  10. 10.

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

    CAS 
    Article 

    Google Scholar
     

  11. 11.

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

    CAS 
    Article 

    Google Scholar
     

  12. 12.

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

    CAS 
    Article 

    Google Scholar
     

  13. 13.

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

    Article 

    Google Scholar
     

  14. 14.

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

    CAS 
    Article 

    Google Scholar
     

  15. 15.

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

    CAS 
    Article 

    Google Scholar
     

  16. 16.

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

    CAS 
    Article 

    Google Scholar
     

  17. 17.

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

    CAS 
    Article 

    Google Scholar
     

  18. 18.

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

    CAS 
    Article 

    Google Scholar
     

  19. 19.

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

  20. 20.

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

    CAS 
    Article 

    Google Scholar
     

  21. 21.

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

    CAS 
    Article 

    Google Scholar
     

  22. 22.

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

    CAS 
    Article 

    Google Scholar
     

  23. 23.

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

    CAS 
    Article 

    Google Scholar
     

  24. 24.

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

    CAS 
    Article 

    Google Scholar
     

  25. 25.

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

    CAS 
    Article 

    Google Scholar
     

  26. 26.

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

    CAS 
    Article 

    Google Scholar
     

  27. 27.

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

    CAS 
    Article 

    Google Scholar
     

  28. 28.

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

    CAS 
    Article 

    Google Scholar
     

  29. 29.

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

    Article 

    Google Scholar
     

  30. 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.

Author information

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

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 or G. Ulrich Nienhaus or Andres Jäschke.

Ethics declarations

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

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Read More

Leave a Reply

Your email address will not be published. Required fields are marked *