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Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons

View ORCID ProfileJeffery M. Tharp, View ORCID ProfileOscar Vargas-Rodriguez, View ORCID ProfileAlanna Schepartz, View ORCID ProfileDieter Söll
doi: https://doi.org/10.1101/2020.12.07.415521
Jeffery M. Tharp
†Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520, United States
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Oscar Vargas-Rodriguez
†Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520, United States
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Alanna Schepartz
§Department of Chemistry, University of California, Berkeley, California 94705, United States
∥Department of Molecular & Cell Biology, University of California, Berkeley, California 94705, United States
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  • For correspondence: schepartz@berkeley.edu dieter.soll@yale.edu
Dieter Söll
†Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520, United States
‡Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
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  • For correspondence: schepartz@berkeley.edu dieter.soll@yale.edu
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ABSTRACT

We recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and tyrosine at elongating positions. Using itRNATy2AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes.

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Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 08, 2020.
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Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons
Jeffery M. Tharp, Oscar Vargas-Rodriguez, Alanna Schepartz, Dieter Söll
bioRxiv 2020.12.07.415521; doi: https://doi.org/10.1101/2020.12.07.415521
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Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons
Jeffery M. Tharp, Oscar Vargas-Rodriguez, Alanna Schepartz, Dieter Söll
bioRxiv 2020.12.07.415521; doi: https://doi.org/10.1101/2020.12.07.415521

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