title: In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.
abstract: Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more "orthogonal" to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Rodriguez
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D018389:Codon, Nonsense / Q000235:genetics*; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D016153:Genes, Suppressor; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry; D013489:Suppression, Genetic / Q000235:genetics*; D046249:Transfer RNA Aminoacylation / Q000235:genetics; D014981:Xenopus / Q000235:genetics
chemical_list: D000596:Amino Acids; D018389:Codon, Nonsense; D012343:RNA, Transfer