Cellular encoding of Cy dyes for single-molecule imaging

Lilia Leisle; Rahul Chadda; John D Lueck; Daniel T Infield; Jason D Galpin; Venkatramanan Krishnamani; Janice L Robertson; Christopher A Ahern

doi:10.7554/eLife.19088

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Cellular encoding of Cy dyes for single-molecule imaging

Journal article

Open access

Peer reviewed

Cellular encoding of Cy dyes for single-molecule imaging

Lilia Leisle, Rahul Chadda, John D Lueck, Daniel T Infield, Jason D Galpin, Venkatramanan Krishnamani, Janice L Robertson and Christopher A Ahern

eLife, Vol.5, e19088

12/12/2016

DOI: 10.7554/eLife.19088

PMCID: PMC5207767

PMID: 27938668

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

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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 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.

Gene Expression

Recombinant Proteins - metabolism

Animals

Microscopy, Fluorescence - methods

Xenopus laevis

Carbocyanines - metabolism

Luminescent Proteins - genetics

Recombinant Proteins - genetics

Single Molecule Imaging - methods

Luminescent Proteins - metabolism

Details

Title: Subtitle: Cellular encoding of Cy dyes for single-molecule imaging
Creators: Lilia Leisle - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Rahul Chadda - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
John D Lueck - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Daniel T Infield - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Jason D Galpin - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Venkatramanan Krishnamani - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Janice L Robertson - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Christopher A Ahern - Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
Resource Type: Journal article
Publication Details: eLife, Vol.5, e19088
DOI: 10.7554/eLife.19088
PMID: 27938668
PMCID: PMC5207767
NLM abbreviation: Elife
ISSN: 2050-084X
eISSN: 2050-084X
Publisher: England
Grant note: U54 GM087519 / NIGMS NIH HHS R01 GM120260 / NIGMS NIH HHS R00 GM101016 / NIGMS NIH HHS R01 GM106569 / NIGMS NIH HHS
Language: English
Date published: 12/12/2016
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984070152702771

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