title: Cellular encoding of Cy dyes for single-molecule imaging.
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 theXenopus laevisoocyte 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.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Lilia
authors.initials: L
authors.lastname: Leisle
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Rahul
authors.initials: R
authors.lastname: Chadda
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: John D
authors.initials: JD
authors.lastname: Lueck
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Infield
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Galpin
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Venkatramanan
authors.initials: V
authors.lastname: Krishnamani
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Janice L
authors.identifier: 0000-0002-5499-9943
authors.initials: JL
authors.lastname: Robertson
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States.
authors.forename: Christopher A
authors.identifier: 0000-0002-7975-2744
authors.initials: CA
authors.lastname: Ahern
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000378:metabolism*; D015870:Gene Expression; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*; D014982:Xenopus laevis
chemical_list: D002232:Carbocyanines; D008164:Luminescent Proteins; D011994:Recombinant Proteins
keywords: TIRF; biochemistry; biophysics; cell-free protein synthesis; genetic code expansion; membrane proteins; orthogonal tRNA; single molecule imaging; structural biology; xenopus