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

Research output: Contribution to journalArticle

12 Scopus citations

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 wholecell 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 CyncAAs 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.

Original languageEnglish
Article numbere19088
JournaleLife
Volume5
Issue numberDECEMBER2016
DOIs
StatePublished - Dec 12 2016
Externally publishedYes

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    Leisle, L., Chadda, R., Lueck, J. D., Infield, D. T., Galpin, J. D., Krishnamani, V., Robertson, J. L., & Ahern, C. A. (2016). Cellular encoding of Cy dyes for single-molecule imaging. eLife, 5(DECEMBER2016), [e19088]. https://doi.org/10.7554/eLife.19088