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In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression

Rodriguez, Erik A. and Lester, Henry A. and Dougherty, Dennis A. (2006) In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression. Proceedings of the National Academy of Sciences of the United States of America, 103 (23). pp. 8650-8655. ISSN 0027-8424. PMCID PMC1482635.

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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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 2006 by the National Academy of Sciences Edited by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved April 21, 2006 (received for review December 15, 2005). Published online before print May 25, 2006, 10.1073/pnas.0510817103 E.A.R. is a National Science Foundation Predoctoral Fellow. This work was supported by National Institutes of Health Grants NS-34407 and NS-11756. Author contributions: E.A.R. designed research; E.A.R. performed research; E.A.R., H.A.L., and D.A.D. analyzed data; and E.A.R., H.A.L., and D.A.D. wrote the paper. Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office.
Funding AgencyGrant Number
NSF Predoctoral FellowshipUNSPECIFIED
Subject Keywords:nicotinic receptor; tRNA; quadruplet codon; stop codon; protein engineering
Issue or Number:23
PubMed Central ID:PMC1482635
Record Number:CaltechAUTHORS:RODpnas06
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5967
Deposited By: Archive Administrator
Deposited On:25 Nov 2006
Last Modified:02 Oct 2019 23:28

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