University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Student Research Projects, Dissertations, and Chemistry, Department of Theses - Chemistry Department 4-2017 Genetic Code Expansion in Biochemical Investigations and Biomedical Applications Nanxi Wang University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/chemistrydiss Part of the Biochemical and Biomolecular Engineering Commons, Biology Commons, and the Biomedical Engineering and Bioengineering Commons Wang, Nanxi, "Genetic Code Expansion in Biochemical Investigations and Biomedical Applications" (2017). Student Research Projects, Dissertations, and Theses - Chemistry Department. 79. http://digitalcommons.unl.edu/chemistrydiss/79 This Article is brought to you for free and open access by the Chemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Student Research Projects, Dissertations, and Theses - Chemistry Department by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. GENETIC CODE EXPANSION IN BIOCHEMICAL INVESTIGATIONS AND BIOMEDICAL APPLICATIONS by Nanxi Wang A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Chemistry Under the Supervision of Professor Jiantao Guo Lincoln, Nebraska April, 2017 GENETIC CODE EXPANSION IN BIOCHEMICAL INVESTIGATIONS AND BIOMEDICAL APPLICATIONS Nanxi Wang, Ph.D. University of Nebraska, 2017 Advisor: Jiantao Guo 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. Table of Contents List of Figures .............................................................................................................................................. iv List of Tables ............................................................................................................................................... vi Lists of Abbreviations ................................................................................................................................. vii CHAPTER 1 Genetic Code Expansion: An Overview and Recent Advances in Biochemical and Biomedical Research .................................................................................................................................... 1 1.1 Introduction: an overview of genetic code expansion ......................................................................... 1 1.2 Orthogonal aaRS/tRNA pairs.............................................................................................................. 2 1.2.1 MjTyrRS/tRNATyr ........................................................................................................................ 4 1.2.2 PylRS/tRNAPyl ............................................................................................................................. 4 1.2.3 EcTyrRS/BstRNATyr .................................................................................................................... 5 1.3 Nonsense Codons ................................................................................................................................ 6 1.3.1 Amber stop codon ........................................................................................................................ 6 1.3.2 Quadruplet codon ......................................................................................................................... 7 1.3.3 Rare codons .................................................................................................................................. 8 1.4 Unnatural Amino Acids ...................................................................................................................... 9 1.5 Approaches to improve the efficiency of unAA incorporation ......................................................... 12 1.5.1 Evolution of orthogonal aaRS/tRNA pairs ................................................................................ 12 1.5.2 Release factor 1 knockout and an amber-free host to enhance amber suppression ................... 14 1.5.3 Refining the interactions between the orthogonal tRNA and cellular components ................... 15 1.5.4 Orthogonal ribosomes for enhancing nonsense codon suppression efficiency .......................... 16 1.6 Application of genetic code expansion in vaccine development ...................................................... 17 1.6.1 Design therapeutic vaccines using genetic code expansion ....................................................... 17 1.6.2 Design preventive vaccines using genetic code expansion ........................................................ 18 1.7 Summary and remarks ...................................................................................................................... 19 1.8 Figures and tables ............................................................................................................................. 21 CHAPTER 2 Development of Live-attenuated HIV-1 Viruses via Genetic Code Engineering ............... 25 2.1 Introduction: A novel approach towards a HIV-1 live-attenuated vaccine ....................................... 25 2.2 Approaches ....................................................................................................................................... 26 2.2.1 Single-cycle amber suppression-dependent HIV-1 variants ...................................................... 26 2.2.2 Multi-cycle amber suppression-dependent HIV-1 variants ....................................................... 34 2.2.3 Attempts to improve the replication capacity of amber suppression-dependent HIV-1 variants ............................................................................................................................................................ 39 2.3 Summary and remarks ...................................................................................................................... 42 2.4 Figures and tables ............................................................................................................................. 45 CHAPTER 3 Systematic Evolution and Study of Quadruplet Codon Decoding tRNAs .......................... 70 3.1 Introduction: Quadruplet codon decoding tRNAs for genetic code expansion ................................ 70 3.2 Approaches ....................................................................................................................................... 71 3.2.1 tRNA (with an expanded anticodon loop) library construction and selection for UAGN (N = A, G, U, C) codon decoding. ................................................................................................................... 71 3.2.2 Characterization and validation of evolved tRNAs. .................................................................. 73 3.2.3 Incorporation of other unAAs using the evolved tRNAs. .......................................................... 74 3.2.4 Analysis of the evolved tRNA mutants. ..................................................................................... 75 3.2.5 Cross-decoding among UAGN codons. ..................................................................................... 75 3.2.6 tRNA (with regular 7-base anticodon loop) library construction and selection for UAGN (N = A, G, U, C) decoding. ......................................................................................................................... 76 3.2.7 Cross-decoding against UAG codons. ....................................................................................... 76 3.2.8 Quantification