Date of Award

5-14-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Philip Borer

Second Advisor

Yan-Yeung Luk

Keywords

Aptamer, Biosensor, Branched Oligo Synthesis, CycT1-Tat, Drug Discovery, HIV-1 NCp7

Abstract

This dissertation focuses on the interaction of proteins and nucleic acids and their applications. It includes two projects: I: Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery and II. Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli.I: Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery Screening drug compounds targeting HIV-1 NCp7 provide attractive candidates for new anti-retroviral therapeutics because of the highly conserved nature of the zinc fingers in NCp71 in selecting and packaging RNA in the HIV-1 life cycle. The unique 3-segment, reversible switch for high throughput screening (HTS) drug targets will be for the HIV-1 nucleocapsid (NC) protein. The Probe is a natural binding element for the NCp7 protein target or, in our case, an aptamer hairpin with loop sequence, TGTGGT, having a nano-molar affinity (Kd=16nM).2 Toggle is a damaged probe where the target-binding sequences are replaced with other bases. Cover is a mostly complementary strand for the probe and the toggle. A hairpin loop forms around the 5Me-dC-brancher in both the ON and OFF forms. Using Visual OMPTM simulation, the following two potential switch molecules (NM-1 and NM-2) were designed and successfully synthesized using a one-step ligation method with at least 90% purity as judged by mass spectrometry. Then fluorescence measurements using NM-1 and NM-2 with NCp7 protein were analyzed to demonstrate proof-of-principle for 3-segment nucleic acid switches. Increasing [NC] causes a dramatic decrease in CY3 fluorescence. The ON/OFF contrast ratio of NM-1 and NM-2 are 2.6 and 3.2 showing the feasibility of 3-segment switches being used to further modify the design of HTS switches for the HIV-1 NC. II. Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli. Due to increasing drug resistance for current antiretroviral therapeutic (ART) treatments, it is important to add drugs targeting other aspects of HIV-1 infection to manage AIDS. Tat (trans-activator of transcription) protein up-regulates the transcription of viral-specific proteins by a factor of 1,0003, which makes Tat a very attractive drug target. High-level expression and purification of recombinant GST-CycT1(249-280)-linker(25aa)-Tat(1-101) fusion protein in Escherichia coli (E. coli) was challenging because CycT1-Tat forms inclusion bodies making it difficult to purify and obtain a high concentration of active CycT1-Tat. A commercially available pET-SUMO cloning vector was introduced for the high-level expression of four CycT1-Tat chimeras, F1-F4. The gene associated with the protein sequence was purchased from IDT and SUMO-CycT1-Tat (F1) fusion plasmid was prepared and transformed in E. coli BL21 (DE3). By inducing with IPTG, His6-SUMO-CycT1-Tat fusion protein was able to promote soluble expression making it more efficient to purify CycT1-Tat (F1) compared to GST fusion tag protein. The molecular weight of purified CycT1-Tat was confirmed with mass spectrometry (MALDI-TOF). For initial characterization of the TAR-CycT1-Tat complex, an Electrophoretic Mobility Shift Assay (EMSA) was carried out with partially purified CycT1-Tat protein using TAR-31 and truncated versions that altered the hairpin loop (TAR-H24) and deleted the bulge loop (TAR-B25). A TAR-CycT1-Tat complex was formed and a band shift was observed in the binding of CycT1-Tat to TAR-31 and a lower affinity complex with TAR-B25, but TAR-H24 did not bind.

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