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Cellular encoding of Cy dyes for single-molecule imaging.

Lilia Leisle1, Rahul Chadda1, John D Lueck1

  • 1Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.

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Summary

Researchers developed a method to genetically encode cyanine dyes (Cy-ncAAs) into proteins. This technique allows for the fluorescent labeling of proteins in living cells, enabling single-molecule analysis.

Keywords:
TIRFbiochemistrybiophysicscell-free protein synthesisgenetic code expansionmembrane proteinsorthogonal tRNAsingle molecule imagingstructural biologyxenopus

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Site-specific protein labeling is crucial for studying protein function.
  • Incorporating non-canonical amino acids (ncAAs) offers a powerful tool for protein engineering.
  • Cyanine dyes are valuable fluorescent probes but their in vivo incorporation is challenging.

Purpose of the Study:

  • To establish a general method for the site-specific genetic encoding of cyanine dyes as ncAAs (Cy-ncAAs) into proteins.
  • To demonstrate the utility of Cy-ncAAs in a eukaryotic expression system for cellular analysis.
  • To enable single-molecule resolution studies of protein function in living cells.

Main Methods:

  • Utilized an improved nonsense suppression technique with in vitro misacylated orthogonal tRNA.
  • Engineered proteins with cyanine dyes (Cy3 and Cy5) as ncAAs.
  • Expressed Cy-ncAA-containing proteins in *Xenopus laevis* oocytes and analyzed them using TIRF microscopy.

Main Results:

  • Cyanine dye ncAAs were tolerated by the eukaryotic ribosome in cell-free and whole-cell systems.
  • Soluble and membrane proteins successfully incorporated Cy-ncAAs.
  • Encoded Cy-ncAA ion channels were trafficked to the plasma membrane, exhibited robust function, and displayed distinct fluorescent signals.

Conclusions:

  • This study presents the first demonstration of encoded cyanine dyes as ncAAs in a eukaryotic expression system.
  • The developed method allows for the site-specific incorporation of fluorescent labels into proteins.
  • This opens new avenues for analyzing protein behavior with single-molecule resolution within cellular environments.