Structure-Activity Relationships in the Biological Interactions of Spherical Nucleic Acids

Yamankurt, Gokay

doi:https://doi.org/10.21985/n2-3g9v-n035

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Structure-Activity Relationships in the Biological Interactions of Spherical Nucleic Acids

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Nucleic acid drugs promise to revolutionize the development of therapeutics. They offer a platform for digital medicine, where systematic changes to the nucleic acid sequence can be utilized to target the entire human genome. However, nucleic acids suffer from a number of drawbacks, such as negligible cellular uptake and rapid degradation. Recently, certain nanomedicines presented a way to overcome these challenges. One such nanomedicine, spherical nucleic acids (SNAs), consists of oligonucleotides radially conjugated to a nanoparticle core. This arrangement of nucleic acids gives rise to unique properties not observed with their linear counterparts, such as rapid cellular uptake and nuclease resistance. These emergent properties of SNAs arise from how the interactions of SNAs with the components of living systems differ from linear nucleic acids, which raises the question of how SNA mechanisms of action differ from linear nucleic acids. Additionally, SNAs have many design features that can be tuned independently unlike linear nucleic acids, and how these design features affect the biological activity of SNAs is not well understood. This thesis investigates these two questions. In Chapter 2, the manner in which SNAs interact with RNA interference machinery to effect gene silencing is assessed. A systematic analysis of siRNA biochemistry involving SNAs shows that Dicer cleaves the modified siRNA duplex from the surface of the nanoparticle, and the liberated siRNA subsequently functions in a way that is dependent on the canonical RNA interference mechanism. Importantly, this result demonstrates that siRNA-SNAs act as single-entity transfection and gene silencing agents, and should be designed with these differences in mind. In Chapter 3, this understanding was then leveraged to design a new class of SNAs, which increases the siRNA content by an order of magnitude through covalent attachment of each strand of the duplex. As a consequence of increased nucleic acid content, the new nanostructure architecture is more potent than conventional SNAs and exhibits less cell cytotoxicity. To understand how design features affect biological activity, structure-activity relationships of SNAs were determined for their use in ocular transport and as cancer vaccines. Chapter 4 shows that SNAs can travel from the anterior part of the eye to the retina in vivo, and can completely abrogate blood vessel formation \textit{in vitro}. However, these processes are drastically influenced by the SNAs structure. For example, SNAs synthesized from cholesterol-conjugated oligonucleotides dissociated during transport and required the design of a new SNA architecture using phospholipid-conjugated oligonucleotides to overcome. Finally, Chapter 5 investigates many design features at once through the development of a high-throughput synthesis and analysis pipeline. Using this pipeline, structure-activity relationships and design rules were determined for cancer-vaccine candidate SNAs by assessing the immunostimulatory potential of ~1,000 SNAs varying in 11 design features. Importantly, this methodology is general, can reduce the number of nanoparticles that need to be tested by an order of magnitude through the incorporation of machine learning tools, and serve as a screening tool for the development of nanoparticle therapeutics.

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