Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Genetic Code Expansion through Quadruplet Codon Decoding
Genetic code expansion enables the site-specific incorporation of unnatural amino acids (unAA) into proteins. A general approach to encode unAA in live cells is to suppress nonsense codons using an orthogonal aminoacyl-tRNA synthetase/tRNA pair. While amber stop codon (UAG) is commonly used to incorporate unAAs, quadruplet codon further expands the genetic code with up to 256 new “blank” codons for protein biosynthesis. In this thesis, we demonstrated the biomedical application and efforts to improve the decoding efficiency of quadruplet codons. In Chapter 1, a brief overview of genetic code expansion and quadruplet codon decoding is provided. Moreover, the recent advance of the application and engineering of the quadruplet codon decoding system is summarized. In Chapter 2, the quadruplet codon decoding efficiency was improved by using engineered tRNA mutants in mammalian cells. As a proof of concept, we reported a biomedical application of the unAA incorporation in response to quadruplet codons as a genetic switch to control the replication of HIV-1 viruses. In Chapter 3, we exploited a new approach to significantly improve the quadruplet codon decoding efficiency in bacteria by using recoding signals embedded in mRNA. This strategy will greatly enhance the utility of unAA mutagenesis based on quadruplet codon decoding. In Chapter 4, an efficient quadruplet codon decoding system was designed for mammalian cells. The identified recoding signals from Chapter 3 were examined in the mammalian system. In Chapter 5, we developed a quadruplet codon decoding system in yeast and conducted tRNA engineering in the yeast host. In Chapter 6, an engineered aminoacyl-tRNA synthetase was obtained via direct evolution to enable genetic incorporation of sulfotyrosine in mammalian cells, in collaboration with Dr. Niu and Xinyuan He. This engineered synthetase can be applied to the study of post-translational modification in biological processes. In collaboration with Dr. Erome Hankore, two unAAs were incorporated into a model protein in response to two quadruplet codons.
Chen, Yan, "Genetic Code Expansion through Quadruplet Codon Decoding" (2022). ETD collection for University of Nebraska - Lincoln. AAI29165578.