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Genetic code expansion provides a powerful tool for site-specific incorporation of unnatural amino acids (unAAs) with novel biochemical and physiological properties into proteins in live cells and organisms. To achieve this, a nonsense codon suppression system, which consists of an orthogonal aminoacyl-tRNA synthetase (aaRS) and tRNA pair that specifically decodes a nonsense codon (e.g., amber codon and quadruplet codon) with an unAA but do not “cross talk” with their endogenous counterparts, was established. This Ph.D. thesis presents our efforts on evolution and application of nonsense codon suppression systems for biochemical and biomedical investigations.
In Chapter 1, a brief overview of genetic code expansion technique and recent advances in this area of research was given. To improve unAA incorporation efficiency, we focused on systematic evolution of two most commonly used orthogonal aaRS/tRNA pairs: PylRS/tRNAPyl and MjTyrRS/tRNATyr. We enhanced quadruplet codon decoding efficiency of PylRS/tRNAPyl pairs by completely randomizing the anticodon-stem loop of tRNAPyl (Chapter 3). In addition, we improved amber suppression efficiency of MjTyrRS/tRNATyr derivatives by engineering the anticodon binding pocket of MjTyrRS (Chapter 4). All these efforts lead to a further improvement in current nonsense codon suppression systems and may expand their applications in unAA mutagenesis. Next, we reported the application of an amber suppression system as an unnatural genetic switch to manipulate the expression of essential HIV-1 proteins, which resulted in either single-cycle or multicycle live-attenuated HIV-1 viruses (Chapter 2). These genetically modified viruses can be potentially used as preventive vaccines to protect against HIV-1 infection. Our methodology can also be applied to the generation of vaccines against other pathogens.
Advisor: Jiantao Guo