This proposal is aimed at further developing and applying a general method that allows one to systematically add amino acids with novel physical, chemical and biological properties to the genetic codes of both prokaryotic and eukaryotic organisms. This methodology involves the generation of orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that uniquely recognize a noncoding codon and do not cross react with any of the endogenous tRNAs and aminoacyl-tRNA synthetases in the host organism. The amino acid specificity of the aminoacyl-tRNA synthetase is then modified such that it aminoacylates its cognate tRNA with only the desired unnatural amino acid and no endogenous amino acid. Using this method, a large number of structurally diverse unnatural amino acids have been incorporated efficiently and with high fidelity in response to nonsense and frameshift codons in bacteria, yeast and mammalian cells. We now propose to extend these studies in several directions. The first involves the evolution of tRNA-aaRS pairs in eukaryotes and/or prokaryotes specific for a series of biophysical probes/tools including fluorescent and photocaged amino acids, and amino acids that can be used to conformationally constrain the protein backbone. Another effort will focus on technical improvements to increase suppression efficiencies, protein yields and the number of orthogonal tRNA-aaRS pairs. The final direction will involve novel biomedical applications of this methodology including the generation of vaccines with enhanced immunogenicity, and the synthesis of novel antibody conjugates for imaging and therapeutic applications.

Public Health Relevance

The ability to genetically encode unnatural amino acids beyond the common twenty will significantly enhance our ability to manipulate protein structure. The result will be new tools to study protein function in vitro and in vivo. It should also be possible to create homogenous """"""""chemically mutated"""""""" therapeutic proteins and vaccines with enhanced activities and improved pharmacology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062159-09
Application #
7742614
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Jones, Warren
Project Start
2001-02-01
Project End
2012-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
9
Fiscal Year
2010
Total Cost
$450,252
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Xuan, Weimin; Schultz, Peter G (2017) A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids. Angew Chem Int Ed Engl 56:9170-9173
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
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
Luo, Xiaozhou; Zambaldo, Claudio; Liu, Tao et al. (2016) Recombinant thiopeptides containing noncanonical amino acids. Proc Natl Acad Sci U S A 113:3615-20
Liu, Yan; Wang, Ying; Zhang, Yong et al. (2016) Rational Design of Dual Agonist-Antibody Fusions as Long-acting Therapeutic Hormones. ACS Chem Biol 11:2991-2995
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
Mehta, Angad P; Li, Han; Reed, Sean A et al. (2016) Replacement of Thymidine by a Modified Base in the Escherichia coli Genome. J Am Chem Soc 138:7272-5
Cao, Yu; Rodgers, David T; Du, Juanjuan et al. (2016) Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer. Angew Chem Int Ed Engl 55:7520-4
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

Showing the most recent 10 out of 99 publications