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- Creators: Barrett, The Honors College
Description
DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.
ContributorsHostal, Anna Elizabeth (Author) / Anderson, Karen (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Yan, Hao (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
ContributorsTamirisa, Ritika Sai (Author) / Levitus, Marcia (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Windman, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Currently, treatment for multiple myeloma (MM), a hematological cancer, is limited to post-symptomatic chemotherapy combined with other pharmaceuticals and steroids. Even so, the immuno-depressing cancer can continue to proliferate, leading to a median survival period of two to five years. B cells in the bone marrow are responsible for generating antigen-specific antibodies, but in MM the B cells express mutated, non-specific monoclonal antibodies. Therefore, it is hypothesized that antibody-based assay and therapy may be feasible for detecting and treating the disease. In this project, 330k peptide microarrays were used to ascertain the binding affinity of sera antibodies for MM patients with random sequence peptides; these results were then contrasted with normal donor assays to determine the "immunosignatures" for MM. From this data, high-binding peptides with target-specificity (high fluorescent intensity for one patient, low in all other patients and normal donors) were selected for two MM patients. These peptides were narrowed down to two lists of five (10 total peptides) to analyze in a synthetic antibody study. The rationale behind this originates from the idea that antibodies present specific binding sites on either of their branches, thus relating high binding peptides from the arrays to potential binding targets of the monoclonal antibodies. Furthermore, these peptides may be synthesized on a synthetic antibody scaffold with the potential to induce targeted delivery of radioactive or chemotherapeutic molecular tags to only myelomic B cells. If successful, this would provide a novel alternative to current treatments that is less invasive, has fewer side effects, more specifically targets the cause of MM, and reliably diagnoses the cancer in the presymptomatic stage.
ContributorsBerry, Jameson (Co-author) / Buelt, Allison (Co-author) / Johnston, Stephen (Thesis director) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Division of Teacher Preparation (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity.
ContributorsHernandez, Victoria Joan (Author) / Wachter, Rebekka (Thesis director) / Chiu, Po-Lin (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The major goal of this large project is to develop a Recognition Tunneling Nanopore (RTP) device that will be used for determining the structure of glycosaminoglycans (GAGs). The RTP device is composed of a recognition tunneling junction that is embedded in a nanopore. In order to translocate the GAG molecule through the nanopore, researchers have designed a scheme in which the GAG molecule of interest will be attached to the 5’ end of a DNA primer (figure 1) and the DNA primer will be extended by a biotinylated Φ29 DNA polymerase that is anchored in the nanoslit using streptavidin. This research project specifically is part of a larger project with the main goal of comparing the activity of the wild-type Φ29 DNA polymerase which I have expressed and purified with the mutated Φ29 DNA polymerase devoid of 3’ - 5’ exonuclease activity which was made by Dr. Deng.
ContributorsDadkhah Tirani, Farbod (Author) / Wang, Xu (Thesis director) / Zhang, Peiming (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The purpose of my honors thesis project was to generate the tools needed for in vivo imaging by determining the optimal plasmid-fluorophore combination. To determine the optimal plasmid and fluorophore, asd plasmids were constructed with various promoters, origins of replications, and red fluorophores. The optimal asd plasmid for fluorescent in vivo imaging was determined by the plasmid stability, growth rate, and growth phase dependence on fluorescent intensity. The end goal is to be able to use the asd plasmid in vaccine strains for the purpose of in vivo imaging of the recombinant attenuated Salmonella vaccine (RASV).
ContributorsEudy, L. Adam (Author) / Curtiss, Roy (Thesis director) / Roland, Kenneth (Committee member) / Forbes, Stephen (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
Description
Curanderismo can be defined as a blend between ancient Aztec natural healing methods and modern day Catholicism. Today, it is practiced through various techniques, which can be observed in yerberias. Upon visiting these yerberias, the attendants were interviewed to understand which products are commonly sold, what they are used to treat, and the cultural significance behind the practice. After purchasing a number of products from each yerberia, a literature analysis of potential biochemical pathways was conducted to determine if these products have efficacy in what they treat. While potential pathways were found for a number of the products, it has been determined that further clinical research must be conducted to state whether these products are effective in treatment.
ContributorsDickey, Erin (Author) / Mullenmeister, William (Co-author) / Breitweiser, Mya (Co-author) / Holechek, Susan (Thesis director) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor)
Created2024-05
Description
Misincorporation of uracil bases into DNA can lead to mutations after transcription. Uracil-DNA glycosylase (UDG) is an enzyme that removes uracil bases from DNA, leaving an apurinic/apyrimidinic site. Different efficiencies of uracil base removal by UDG have been observed at different sites in DNA. A previous study found that UDG has a higher specificity constant for DNA sequences that are more flexible, specifically that those with uracil in a context of thymine adjacent on the 5’ side and adenine adjacent on the 3’ side (TUA sequence) bound UDG better than those with an adenine adjacent on the 5’ side and thymine adjacent on the 3’ side (AUT sequence) context. The purpose of this study is to expand the previous one by determining whether the ratios observed between TUA and AUT specificity constants within DNA sequences that are otherwise the same are also observed across a third sequence context that was not included in the first study. The hypothesis that same ratio would be observed is somewhat supported as the new sequence has a specificity constant of 1.24±0.043 ✕ 107 M-1s-1. However, conclusions to be drawn from this are limited by the wide margin of error seen among trials of the same concentrations.
ContributorsEngelken, Rylee (Author) / Levitus, Marcia (Thesis director) / Klein-Seetharaman, Judith (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / School of Human Evolution & Social Change (Contributor)
Created2024-05