Atomic Force Microscopy (AFM) has been envisioned as a means of nanodiagnostics due to its single molecule sensitivity. However, the AFM based recognition imaging lacks a multiplex capacity to detect multiple analytes in a single test. Also there is no user friendly wet chemistry to functionalize AFM tips. Hence, an uncatalyzed Click Chemistry protocol was developed to functionalize AFM tips. For multiplexed recognition imaging, recognition heads based on a C3 symmetrical three arm linker with azide functionalities at its ends were synthesized and the chemistry to attach them to AFM tips was developed, and these recognition heads were used in detecting multiple proteins simultaneously using AFM.
A bis-Angiopeptide-2 conjugate with this three-arm linker was synthesized and this was conjugated with anti-West Nile virus antibody E16 site specifically to target advanced West Nile virus infection in the Central Nervous System. The bis-Angiopeptide-2 conjugate of the antibody shows higher efficacy compared to a linear linker-Angiopeptide-2 conjugate of the antibody in in vitro studies and currently the efficacy of this antibody conjugate in studied in mice. Surface Plasmon Resonance imaging (SPRi) results indicate that the conjugation does not affect the antigen binding activity of the antibody very significantly.
A Y-shaped bisbiotin ligand was also prepared as a small sized antibody mimic. Compared to a monovalent biotin ligand, the y-Bisbiotin can cooperatively form a significantly more stable complex with streptavidin through intramolecular bivalent interactions, which were demonstrated by gel electrophoresis, SPR and AFM. Continuing on these lines, a four-arm linker was synthesized containing three single chain variable fragments (scFv) linked to the scaffold to form a tripod base, which would allow them to concomitantly interact with a trimeric Glycoprotein (GP) spike that has a “chalice” configuration. Meanwhile, a human IgG1 Fc is to be installed on the top of the tetrahedron, exerting effector functions of a monoclonal antibody.
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.
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.