Cellular encoding of Cy dyes for single-molecule imaging.

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.