Cellular encoding of Cy dyes for single-molecule imaging
- University of Iowa Carver College of Medicine, United States
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
Janice L Robertson
Christopher A Ahern
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.
Reviewing Editor
- Olga Boudker, Weill Cornell Medical College, United States
Version history
- Received: June 24, 2016
- Accepted: December 9, 2016
- Accepted Manuscript published: December 12, 2016 (version 1)
- Accepted Manuscript updated: December 16, 2016 (version 2)
- Version of Record published: January 3, 2017 (version 3)
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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Cellular encoding of Cy dyes for single-molecule imaging
eLife 5:e19088.
https://doi.org/10.7554/eLife.19088
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