Mutational Analysis of HIV-1 Nucleocapsid Protein and Methods Development for Aptamer Discovery.

摘要

This dissertation focuses on structural and functional studies related to the interaction between proteins and nucleic acids and their applications. The work reported here includes two projects. The first part of the thesis is on the mutational and combinatorial analysis of HIV-1 nucleocapsid protein (NCp7). Sixteen mutants of NCp7 were constructed and over-expressed in E. coli. The dissociation constants (Kd) between the mutant proteins and the major packaging signal in HIV-1, SL3 RNA, were measured using tryptophan fluorescence titration assay and/or isothermal titration calorimetry (ITC) assay. Most mutants show tolerance of the mutation but some mutants, including F16A, I24A and K14E-E21K, suffered moderate to significant loss of affinity for SL3 RNA, indicating the roles of the affected residues in the interaction between NCp7 and SL3. The second part focuses on the development of methods for aptamer discovery. Two prototype platforms were set up to imitate the two main-stream next-generation sequencing (NGS) technologies, bridge amplification based and emulsion PCR based NGS technologies. Proof-of-principle experiments were carried out and optimized to evaluate the practicality of these platforms in high-throughput and multiplexed aptamer discovery.