Abstract

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single molecule resolution in a cellular environment.

Article and author information

Author details

  1. Lilia Leisle

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Rahul Chadda

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. John D Lueck

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Daniel T Infield

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Jason D Galpin

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Venkatramanan Krishnamani

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Janice L Robertson

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5499-9943
  8. Christopher A Ahern

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    For correspondence
    christopher-ahern@uiowa.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7975-2744

Funding

National Institutes of Health (GM106569)

  • Daniel T Infield
  • Jason D Galpin
  • Christopher A Ahern

American Heart Association (A22180002)

  • Jason D Galpin
  • Christopher A Ahern

National Institutes of Health (GM087519)

  • Jason D Galpin
  • Christopher A Ahern

National Institutes of Health (GM101016)

  • Janice L Robertson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2016, Leisle et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Lilia Leisle
  2. Rahul Chadda
  3. John D Lueck
  4. Daniel T Infield
  5. Jason D Galpin
  6. Venkatramanan Krishnamani
  7. Janice L Robertson
  8. Christopher A Ahern
(2016)
Cellular encoding of Cy dyes for single-molecule imaging
eLife 5:e19088.
https://doi.org/10.7554/eLife.19088

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https://doi.org/10.7554/eLife.19088

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