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Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

View ORCID ProfileRiley M. Bednar, Subhashis Jana, Sahiti Kuppa, Rachel Franklin, Joseph Beckman, Edwin Antony, View ORCID ProfileRichard B. Cooley, View ORCID ProfileRyan A. Mehl
doi: https://doi.org/10.1101/2021.04.12.439361
Riley M. Bednar
†Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural & Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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Subhashis Jana
†Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural & Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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Sahiti Kuppa
‡Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Edward A. Doisy Research Center, 1100 South Grand Blvd., St. Louis, MO 63104
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Rachel Franklin
†Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural & Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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Joseph Beckman
†Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural & Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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Edwin Antony
‡Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Edward A. Doisy Research Center, 1100 South Grand Blvd., St. Louis, MO 63104
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Richard B. Cooley
†Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural & Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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Ryan A. Mehl
†Department of Biochemistry and Biophysics, Oregon State University, 2011 Agricultural & Life Sciences Building, Corvallis, Oregon 97331-7305, United States
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  • For correspondence: ryan.mehl@oregonstate.edu
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Abstract

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 12, 2021.
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Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells
Riley M. Bednar, Subhashis Jana, Sahiti Kuppa, Rachel Franklin, Joseph Beckman, Edwin Antony, Richard B. Cooley, Ryan A. Mehl
bioRxiv 2021.04.12.439361; doi: https://doi.org/10.1101/2021.04.12.439361
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Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells
Riley M. Bednar, Subhashis Jana, Sahiti Kuppa, Rachel Franklin, Joseph Beckman, Edwin Antony, Richard B. Cooley, Ryan A. Mehl
bioRxiv 2021.04.12.439361; doi: https://doi.org/10.1101/2021.04.12.439361

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