Developing Nucleic Acid-Based Tools for Precision Medicine
Type of DegreePhD Dissertation
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Nucleic acid aptamers are new therapeutic tools that are growing in use in the bionanotechnology field. Understanding their behavior and engineering their structure is desirable for many applications due to unique advantages over antibodies and other current therapeutic methods. In this work, aptamer platforms compatible with hydrogels were designed and aptamers were explored as targeting and drug delivery molecules attached to the surface of gold nanoparticles and by utilizing a quartz crystal microbalance. We have shown that DNA structure and stability, which dictates function, can be engineered and maintained for multiple platforms and therapeutic purposes. The tear fluid in a human eye only contains one type of RNase, molecules that degrade RNA strands. Furthermore, this molecule, RNase 4, is known to only cut after the nucleotide Uracil. By designing a nucleic acid platform consisting of both a DNA anchor strand capable of binding to a hydrogel and RNA that can modified to regulate cut sites and additionally act as a drug, we were able to create a triggered release mechanism for aptamers within a hydrogel to release into the eye. This platform was made so that it is versatile and adaptable to a range of aptamers and other ligands. Results show that we can form a homogenous sample of our nucleic acid platform that maintains a structure capable of controlled release of aptamers from hydrogels into the eye for treatment of ocular diseases. A Quartz crystal microbalance with dissipation was used to characterize and optimize DNA behavior and binding to a gold surface in real time and with high sensitivity. In this work, it is desired to maximize drug loading onto a nanoparticle. Since the drug is binding to double stranded DNA in our system, this means it is essential to understand how to bind the most amount of double stranded DNA possible and maintain a DNA structure that has high affinity towards binding the drug. Dissipation monitoring tracked how flexible or rigid the DNA behaved when bound to the gold sensor, which in turn was used to determine if a highly dense surface is affecting DNA structure in a significant manner that will reduce drug binding affinity. The real time binding is based on traceable changes in frequency and was used to determine salt and DNA concentrations that maximize binding of double stranded DNA to the gold surface. If too much anchor DNA was bound, the surface area available decreased, which resulted in a decrease in the formation of double stranded DNA. Therefore, higher amounts of salt and increased DNA concentration are not always going to produce more double stranded complexes on the gold surface. Results indicate that different amounts of drug binding can be created by adjusting DNA and salt concentrations, while agreeing with similar experiments for DNA conjugation to gold nanoparticles, and dissipation data strongly indicates monolayer formation and behavior. DNA strands can also be used as drug delivery vehicles themselves. We designed a spherical DNA particle with potential for high drug loading and targeting capabilities. Modifications to DNA strands allow binding to the surfaces of many molecules, and in our case, gold nanoparticles. We used 15nm gold nanoparticles to bind approximately 101 nucleic acid strands vial Thiol chemistry. These strands can be a mixture of both drug delivery vehicles and targeting moieties. By combining research on optimizing nucleic acid intercalation of the cancer drug daunomycin with recently developed double stranded gold nanoparticles optimized with maximum DNA loading, we have engineered a therapeutic platform for treatment of cancer cells. Results are promising towards the goal of creating a new therapeutic tool for treating various types of cancers.
- Ricky Whitener PhD Dissertation.pdf