Abstract

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular ‘Riboglow’ platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescence turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small noncoding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye-binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

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Acknowledgements

The authors would like to acknowledge financial support from the Human Frontiers Science Project and NIH Director’s Pioneer Award GM114863 (to A.E.P.). We acknowledge support from the National Science Centre, SYMFONIA DEC-2014/12/W/ST5/00589 to D. Gryko and A.J.W., the National Institutes of Health (5R01 GM073850) to R.T.B., and NSF Physics Frontier Center at JILA (PHY1734006) to R.J.. We thank L. Lavis (Janelia Research Campus) for providing fluorophores JF585, SiR594 and JF646 for Halo staining, S. Jaffrey (Cornell University) and G. Matera (UNC Chapel Hill) for contributing plasmids and S. Shukla and J. Garcia for helpful discussions; T. Stasevich, D. Muhlrad, J. Lee and M. Lo for technical expertise; and to J. Eberhard and J. Gassensmith for helpful discussions. The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core, whose Nikon A1R microscope was acquired by the generous support of the NIST-CU Cooperative Agreement award number 70NANB15H226. R.J is a staff member in the Quantum Physics Division of the National Institute of Standards and Technology (NIST). Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Author information

Author notes

    • Jacob T. Polaski

    Present address: Computational Biology Program, Public Health Sciences Division, Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

  1. These authors contributed equally: Aleksandra J. Wierzba, Jacob T. Polaski.

Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO, USA

    • Esther Braselmann
    • , Jacob T. Polaski
    • , Zachariah E. Holmes
    • , Dilara Batan
    • , Joshua R Wheeler
    • , Roy Parker
    • , Ralph Jimenez
    • , Robert T. Batey
    •  & Amy E. Palmer
  2. BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, USA

    • Esther Braselmann
    • , Dilara Batan
    •  & Amy E. Palmer
  3. Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland

    • Aleksandra J. Wierzba
    • , Mikołaj Chromiński
    •  & Dorota Gryko
  4. JILA, University of Colorado and NIST, Boulder, CO, USA

    • Sheng-Ting Hung
    •  & Ralph Jimenez
  5. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    • Roy Parker

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Contributions

E.B., J.T.P., R.T.B. and A.E.P. conceptualized and designed the study. J.T.P. and R.T.B. rationally designed riboswitch variants. E.B., J.T.P., R.T.B., A.J.W., D.G. and A.E.P. designed organic probes. A.J.W. and M.C. synthesized organic probes. J.T.P. and Z.E.H. purified riboswitch variants for in vitro work. E.B. performed in vitro work, designed and performed cellular work, and analyzed data with input from all authors. D.B. constructed plasmids and assisted with cellular work. S.-T.H. performed in vitro fluorescence lifetime and bleaching experiments. J.R.W. made the Halo-G3BP1 U2-OS cell line. R.P. and R.J. provided critical advice. E.B. and A.E.P. wrote the manuscript with edits from all authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Amy E. Palmer.

Supplementary information

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  3. Supplementary Note 1

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DOI

https://doi.org/10.1038/s41589-018-0103-7