Traumatic brain injury (TBI) affects 70 million people each year. It places a burden on the healthcare system and the patient. TBI has primary and secondary injury phases. The secondary injury is triggered by mechanical damage of the primary injury and triggers a series of cellular cascades. Amongst them, the permeability of the blood-brain barrier (BBB) is affected. This can exacerbate the secondary injury but it also provides a therapeutic window where large payloads can be delivered. Research suggests that the permeability is dependent on sex and has time fluctuations. Nanoparticle (NP) systems can be utilized to tackle the complexities of TBI and take advantage of the BBB breakdown. Furthermore, targeting ligands can be utilized to increase the specificity and effectiveness of NP-based treatments. In this study, the temporospatial capabilities of a novel acute targeting peptide identified in house are validated. A NP system that displays the targeting peptide is developed to determine if it confers the NPs increased targeting and increased retention to the injured tissue on the acute phase of TBI. Furthermore, a low-cost nanoprecipitation system is developed to fabricate drug-loaded biodegradable NPs.Key findings from these studies include the following. (1) A dual conjugation NP protocol allows fabrication of NPs suitable for TBI research. (2) Acute peptide decorated NPs shows enhanced targeting the injured tissue compared to naïve tissue. (3) Acute peptide appears to confer enhanced retention to the ipsilateral hemisphere to the injury compared to the contralateral hemisphere. (4) The acute peptide showed increased targeting to the injury compared to the control peptide. (5) A low-cost nanoprecipitation system was characterized, and it can fabricate sub 100 nm NPs. (6) Lipid-based NPs can be loaded with high quantities of small drug molecules and show stability in vitro. The work presented here shows how NP systems can be tailored to study different aspects of TBI. The targeting NPs show novel targeting and retention patterns conferred by the acute peptide. The results show potential avenues for how the acute peptide can be utilized for subsequent studies. Additionally, a low-cost method for fabricating TBI relevant NPs was successful.

Nucleic acid nanotechnology has rapidly evolved as a field that exploits the addressability and programmability of DNA and RNA to construct intricate structures at the nanoscale. The principles of self-folding and self-assembly enable nucleic acids to form precise and functional architectures through programmed sequence design based on base pairing. However, nanostructures composed of single-stranded nucleic acids remain difficult to synthesize and apply. This dissertation focuses on the development of new methods for preparing single-stranded nucleic acid nanostructures and expanding their application potential.Firstly, a naturally inspired DNA structural motif, the DNA kissing loop, was utilized to facilitate intramolecular and intermolecular interactions in DNA nanostructures. The incorporation of DNA kissing loops enables the design of single-stranded DNA nanostructures with enhanced biostability. Next, a new design method for RNA nanostructures, termed double-stranded RNA bricks, was developed. This approach uses a polycistronic RNA precursor to generate multiple brick sequences via RNase H cleavage. The brick sequences fold into stable double-stranded tiles and self-assemble into large, highly complex RNA nanostructures. Finally, by incorporating base modifications during in vitro transcription, the immunogenicity of single-stranded RNA origami can be modulated, further increasing the safety in the application of RNA nanostructures. This dissertation significantly contributes to the design of novel functional nucleic acid nanostructures and expands their application possibilities. The future direction of this research is to develop artificial molecular machines in vivo, leveraging these nanostructures for applications in biotechnology and medicine.

Deoxyribonucleic acid (DNA) molecules serve as versatile building blocks for constructing programmable, addressable, and biocompatible nanostructures of predefined shapes and sizes beyond their role in genetic information storage. Various assembly techniques ranging from simple to complex, static to dynamic, such as tile-based self-assembly, DNA origami, and supramolecular DNA nanostructures, have been developed for diverse applications. Recent advancements in DNA nanotechnology focus on biomedical applications, including intelligent targeted drug delivery with controlled release mechanisms responsive to specific stimuli, bioimaging, biosensing, cell modulation, and theranostics. This thesis concentrates on developing an intelligent DNA nanostructure-based drug delivery platform called CytoDirect, designed for cancer treatment with target specificity, cytosolic uptake, deep tissue penetration, and controlled drug release. Initially, the design and characterization of curvature-controlled DNA origami nanoplatform are presented. The integration of disulfide modifications for cytosolic delivery and HER2 affibody for target specificity facilitates rapid cytosolic uptake in targeted cancer cells. Subsequent comprehensive cellular experiments validate its functionality within targeted cancer cells. Lastly, in vivo studies on a tumor-bearing mouse model demonstrate the potential of CytoDirect as an effective therapeutic drug delivery platform. Key findings include the successful synthesis of curvature-controlled DNA origami structures with efficient cytosolic uptake and various target specificity. Experimental results reveal that CytoDirect, with specific modifications, can effectively deliver therapeutic agents directly to the cytosol through thiol-mediated pathways, bypassing endosomal entrapment. Systematic biodistribution studies evaluate the target specificity, tissue penetration, and cytosolic uptake of CytoDirect in breast cancer tumor-bearing mice. The in vivo experiments confirmed selective tumor accumulation and cytosolic uptake of CytoDirect in the malignant breast cancer tumor-bearing mice, providing a foundation for future clinical applications. In summary, this thesis presents the development of CytoDirect, an intelligent DNA nanostructure-based drug delivery platform with promising therapeutic potential. Future directions include exploring the generality of CytoDirect for various disease treatments, optimizing the size, coating, and complexity (especially structure simplification) of the CytoDirect, enhancing its stability and biocompatibility, and reducing liver accumulation to maximize its potential for clinical translation. Additionally, evaluating the therapeutic efficacy and immunogenicity in vivo is necessary.

Genome editing tools possess the potential to cure human genetic diseases. The most promising editing tools for addressing a significant portion of genetic diseases are adenine base editors (ABE). The complex consists of a deaminase domain fused to nCas9, guided by a sgRNA. Since no known adenine deaminase catalyzed DNA, E. coli tRNA deaminase (EcTadA) was evolved to ABE8e with 22 amino acid mutations to act on ssDNA substrates with extremely high efficiency. The cryoEM structure of ABE8e indicated that the evolved TadA is a dimer, and preliminary results show that wild-type TadA has weaker dimerization than TadA8e. This leaves us with the open question in the field: How does each amino acid introduced during directed evolution contribute to the dimerization strength and catalytic efficiency of ABE8e? In this experiment, we investigate the role of tyrosine 123 in dimerization strength as well as editing efficiency. With the reverse mutation of Y123 to H, the dimerization strength of TadA8e as well as the DNA editing efficiency of ABE8e were analyzed by ensemble FRET and in vitro single-turnover kinetics assays and were compared to ABE8e. The results from ensemble FRET reveal persistent dimer strength across varying protein concentrations indicating that Y123H does not impact the dimerization strength. The in vitro single-turnover kinetics assay revealed an editing efficiency and rate comparable to that of ABE8e which indicates that Y123H does not significantly impact the catalysis of ABE8e. Further experimentation, such as investigation of mutations that are close in proximity or introduced simultaneously during directed evolution, is required to understand the role of tyrosine 123 in ABE8e.

Cell immunotherapies have revolutionized clinical oncology. While CAR T cell therapy has been very effective in clinical studies, off-target immune toxicity limits eligible patients. Thus, NK cells have been approached with the same therapy design since NK cells have a more favorable safety profile. Therefore, the purpose of this research project is to explore DNA nanotech-based NK cell engagers (NKCEs) that force an immunological synapse between the NK cell and the cancer cell, leading to cancer death. DNA tetrabody (TB) and DNA tetrahedron (TDN) are fabricated and armed with HER2 affibody for tight adhesion to HER2+ cancer cell lines like SKBR3. Overall, relationship between TB-NK treatment and cancer cell apoptosis is still unclear. TB-NK treatment induces an apoptotic profile similar to PMA/IO stimulation. Pilot cell assay needs to be replicated with additional controls and a shortened treatment window. For DNA TDN fabrication, HER2 affibody polishing with Ni-NTA affinity chromatography achieves high purity with 20% to 100% high-imidazole elution gradient. ssDNA-HER2 affibody conjugation is optimal when ssDNA is treated with 40-fold excess sulfo-SMCC for 4 hours. In conclusion, the manufacturing of DNA-based NKCEs is rapid and streamlined, which gives these NKCEs the potential to become a ready to use immunotherapy.

Proteins are among the important macromolecules in living systems, with diverse biological functions and properties that make them greatly interesting to study in both structure and function. The chemical synthesis of proteins allows researchers to incorporate a wide variety of post-translation modifications that can diversify protein functions. It also allows the incorporation of many noncanonical amino acids that enable the study of protein structure and function, as well as the control of their activity in living cells. The work presented in this dissertation focuses on two DNA-templated chemical synthesis approaches for the synthesis of proteins: i) DNA-templated native chemical ligation (NCL), and ii) DNA-templated click chemistry. NCL and its extended version has been used as a powerful tool to obtain proteins; however, it still struggles to make longer proteins due to aggregation and poor yield. To address these issues, a DNA-templated approach is being developed where two peptide fragments are brought into proximity by an oligonucleotide to facilitate the NCL reaction. The sequential ligation of the peptide fragments will result in full-length proteins with increased yield and improved solubility. This research involves synthesis of small molecule auxiliaries, thioester peptides, DNA-peptide conjugates, and ligation of peptides through NCL. This method has the potential to be applied to synthesize large hydrophobic proteins. A DNA-templated click chemistry method was also reported where duplex DNA was utilized as a template for enhancing the copper click reaction between peptide fragments into functional mini-proteins. As a proof of principle, peptide fragments were synthesized with click functional groups and conjugated with distinct DNA handles through a disulfide exchange bioconjugation reaction. The DNA-peptide conjugates were assembled with the template to bring the two peptides into proximity and enhance the effective molarities of the functional groups. The peptides were coupled efficiently using a copper click reaction. The designed DNA-templated method is being implemented to synthesize a designed mini-protein (called LCB1), which can bind tightly to the spike protein of SARS-CoV-2 and inhibit its interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor. This method allows researchers to introduce multiple non-natural amino acids in the protein and has the potential to extend to larger proteins, synthetic polymers, and DNA-peptide biomaterials.

In recent years, researchers have employed DNA and protein nanotechnology to develop nanomaterials for applications in the fields of regenerative medicine, gene therapeutic, and materials science. In the current state of research, developing a biomimetic approach to fabricate an extracellular matrix (ECM)-like material has faced key challenges. The difficulty arises due to achieving spatiotemporal complexity that rivals the native ECM. Attempts to replicate the ECM using hydrogels have been limited in their ability to recapitulate its structural and functional properties. Moreover, the biological activities of the ECM, such as cell adhesion, proliferation, and differentiation, are mediated by ECM proteins and their interactions with cells, making it difficult to reproduce these activities in vitro.Thus, the work presented in my dissertation represents efforts to develop DNA and protein-based materials that mimic the biological properties of the ECM. The research involves the design, synthesis, and characterization of nanomaterials that exhibit unique physical, chemical, and mechanical properties. Two specific aspects of the biomimetic system have been to include (1) a modular protein building block to change the bioactivity of the system and (2) to temporally control the self-assembly of the protein nanofiber using different coiled coil mechanisms. The protein nanofibers were characterized using atomic force microscopy, transmission electron microscopy, and super-resolution DNA Point Accumulation for Imaging in Nanoscale Topology. The domains chosen are the fibronectin domains, Fn-III10, Fn-III9-10, and Fn-III12-14, with bioactivity such as cell adhesion and growth factor binding. To extend this approach, these cys-nanofibers have been embedded in a hyaluronic acid scaffold to enable bioactivity and fibrous morphologies. Nanofiber integration within the HA gel has been shown to promote tunable mechanical properties and architectures, in addition to promoting a temporal display of the protein nanofibers. The hydrogels were characterized using scanning electron microscopy, mechanical compression testing, and fluorescence microscopy. The findings in this dissertation highlight the promise of biomimetic DNA and protein nanomaterials as a versatile approach for developing next-generation materials with unprecedented properties and functions. These findings continue to push the boundaries of what is possible in nanotechnology, leading to new discoveries that will have a significant impact on society.

Originally conceived as a way to scaffold molecules of interest into three-dimensional (3D) crystalline lattices for X ray crystallography, the field of deoxyribonucleic acid (DNA) nanotechnology has dramatically evolved since its inception. The unique properties of DNA nanostructures have promoted their use not only for X ray crystallography, but for a suite of biomedical applications as well. The work presented in this dissertation focuses on both of these exciting applications in the field: 1) Nucleic acid nanostructures as multifunctional drug and vaccine delivery platforms, and 2) 3D DNA crystals for structure elucidation of scaffolded guest molecules.Chapter 1 illustrates how a wide variety of DNA nanostructures have been developed for the delivery of drugs and vaccine components. However, their applications are limited under physiological conditions due to their lack of stability in low salt environments, susceptibility to enzymatic degradation, and tendency for endosomal entrapment. To address these issues, Chapter 2 describes a PEGylated peptide coating molecule was designed to electrostatically adhere to and protect DNA origami nanostructures and to facilitate their cytosolic delivery by peptide-mediated endosomal escape. The development of this molecule will aid in the use of nucleic acid nanostructures for biomedical purposes, such as the delivery of messenger ribonucleic acid (mRNA) vaccine constructs. To this end, Chapter 3 discusses the fabrication of a structured mRNA nanoparticle for more cost-efficient mRNA vaccine manufacture and proposes a multi-epitope mRNA nanostructure vaccine design for targeting human papillomavirus (HPV) type 16-induced head and neck cancers. DNA nanotechnology was originally envisioned to serve as three-dimensional scaffolds capable of positioning proteins in a rigid array for their structure elucidation by X ray crystallography. Accordingly, Chapter 4 explores design parameters, such as sequence and Holliday junction isomeric forms, for efficient crystallization of 3D DNA lattices. Furthermore, previously published DNA crystal motifs are used to site-specifically position and structurally evaluate minor groove binding molecules with defined occupancies. The results of this study provide significant advancement towards the ultimate goal of the field.

The biological lipid bilayer on cells or the cell membrane is a surface teeming with activity. Several membrane proteins decorate the lipid bilayer to carry out various functionalities that help a cell interact with the environment, gather resources and communicate with other cells. This provides a repertoire of biological structures and processes that can be mimicked and manipulated. Since its inception in the late 20th century deoxyribonucleic acid (DNA) nanotechnology has been used to create nanoscale objects that can be used for such purposes. Using DNA as the building material provides the user with a programmable and functionalizable tool box to design and demonstrate these ideas. In this dissertation, I describe various DNA nanostructures that can insert or interact with lipid bilayers for cargo transport, diagnostics and therapeutics. First, I describe a reversibly gated DNA nanopore of 20.4nm x 20.4nm cross sectional width. Controlled transport of cargoes of various sizes across a lipid bilayer through a channel formed by the DNA nanopore was demonstrated. This demonstration paves the way for a class of nanopores that can be activated by different stimuli. The membrane insertion capability of the DNA nanopore is further utilized to design a nanopore sensor that can detect oligonucleotides of a specific s equence inside a lipid vesicle. The ease with which the sensor can be modified to i dentify different diagnostic markers for disease detection was shown by designing a sensor that can identify the non small cell lung cancer marker micro ribonucleic acid -21 (miRNA21). Finally, I demonstrate the therapeutic capabilities of DNA devices with a DNA tetrabody that can recruit natural killer cells (NK cells) to target cancer cells. The DNA tetrabody functionalized with cholesterol molecules and Her2 affibody inserts into NK cell membrane leading it to Her2 positive cancer cells. This shows that inthe presence of DNA tetrabody, the NK cell activation gets accelerated.

Exoelectrogenic organisms transfer electrons from their quinone pool to extracellular acceptors over m-scale distances through appendages known as “biological nanowires”. These structures have been described as cytochrome-rich membrane extensions or pili. However, the components and mechanisms of this long-range electron transfer remain largely unknown. This dissertation describes supramolecular assembly of a tetraheme cytochrome into well-defined models of microbial nanowires and uses those structures to explore the mechanisms of ultra-long-range electron transfer. Chiral-induced-spin-selectivity through the cytochrome is also demonstrated. Nanowire extensions in Shewanella oneidensis have been hypothesized to transfer electrons via electron tunneling through proteinaceous structures that reinforce π-π stacking or through electron hopping via redox cofactors found along their lengths. To provide a model to evaluate the possibility of electron hopping along micron-scale distances, the first part of this dissertation describes the construction of a two-component, supramolecular nanostructure comprised of a small tetraheme cytochrome (STC) from Shewanella oneidensis fused to a peptide domain that self-assembles with a β-fibrillizing peptide. Structural and electrical characterization shows that the self-assembled protein fibers have dimensions relevant to understanding ultralong-range electron transfer and conduct electrons along their length via a cytochrome-mediated mechanism of electron transfer. The second part of this dissertations shows that a model three-component fiber construct based on charge complementary peptides and the redox protein can also be assembled. Structural and electrical characterization of the three-component structure also demonstrates desirable dimensions and electron conductivity along the length via a cytochrome-mediated mechanism. In vivo, it has been hypothesized that cytochromes in the outer surface conduit are spin-selective. However, cytochromes in the periplasm of Shewanella oneidensis have not been shown to be spin selective, and the physiological impact of the chiral-induced-spin-selectivity (CISS) effect on microbial electron transport remains unclear. In the third part of this dissertation, investigations via spin polarization and a spin-dependent conduction study show that STC is spin selective, suggesting that spin selectivity may be an important factor in the electron transport efficiency of exoelectrogens. In conclusion, this dissertation enables a better understanding of long-range electron transfer in bacterial nanowires and bioelectronic circuitry and offers suggestions for how to construct enhanced biosensors.