The development of a general approach for the site-specific incorporation of unnatural amino acids into proteins in vivo, directly from the growth media, would greatly enhance the power of unnatural amino acid mutagenesis. For example, the ability to synthesize large quantities of proteins containing heavy atoms would facilitate protein structure determination, and the ability to site-selectively substitute fluorophores or photocleavable groups into proteins in living cells would provide powerful tools for studying protein function in vivo. Alternatively, one might be able to enhance the properties of proteins by providing building blocks with new functional groups, such as a keto-containing amino acid. To this end, the Schultz group has developed a general multistage approach for site specifically incorporating unnatural amino acids into proteins in vivo. The overall strategy consists of four elements: (1) The design and synthesis of the unnatural amino acid substrate; (2) The construction of an orthogonal amber suppressor tRNA (O-tRNACUA) that is not the substrate for any of the naturally occurring aminoacyl tRNA synthetases and which will eventually be used to deliver the unnatural amino acid in response to a UAG codon in the mRNA encoding the protein of interest; (3) the selection of aminoacyl tRNA synthase from a library of mutants that recognizes the O-tRNACUA but does not recognize any endogenous tRNAs; and (4) the screening of a library of mutants of this tRNA aminoacyl synthetase for the ability to acylate the O-tRNACUA with the unnatural amino acid but not a common amino acid.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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Jones, Warren
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Scripps Research Institute
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Young, Douglas D; Schultz, Peter G (2018) Playing with the Molecules of Life. ACS Chem Biol 13:854-870
Xuan, Weimin; Collins, Daniel; Koh, Minseob et al. (2018) Site-Specific Incorporation of a Thioester Containing Amino Acid into Proteins. ACS Chem Biol 13:578-581
Xuan, Weimin; Shao, Sida; Schultz, Peter G (2017) Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains. Angew Chem Int Ed Engl 56:5096-5100
Xuan, Weimin; Schultz, Peter G (2017) A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids. Angew Chem Int Ed Engl 56:9170-9173
Zambaldo, Claudio; Luo, Xiaozhou; Mehta, Angad P et al. (2017) Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids. J Am Chem Soc 139:11646-11649
Xuan, Weimin; Yao, Anzhi; Schultz, Peter G (2017) Genetically Encoded Fluorescent Probe for Detecting Sirtuins in Living Cells. J Am Chem Soc 139:12350-12353
Luo, Xiaozhou; Fu, Guangsen; Wang, Rongsheng E et al. (2017) Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria. Nat Chem Biol 13:845-849
Ma, Jennifer S Y; Kim, Ji Young; Kazane, Stephanie A et al. (2016) Versatile strategy for controlling the specificity and activity of engineered T cells. Proc Natl Acad Sci U S A 113:E450-8
Mehta, Angad P; Li, Han; Reed, Sean A et al. (2016) Replacement of 2'-Deoxycytidine by 2'-Deoxycytidine Analogues in the E. coli Genome. J Am Chem Soc 138:14230-14233
Rodgers, David T; Mazagova, Magdalena; Hampton, Eric N et al. (2016) Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proc Natl Acad Sci U S A 113:E459-68

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