Doctor of Philosophy, The Ohio State University, 2020, Biophysics

Control, and the degree thereof, is a critical factor to characterize for utilizing any technology. As the field of nanotechnology has progressed, the wonderful potential of technologies on the molecular scale have been held back by the difficulty in controlling the precise arrangement and interactions between molecules to create objects with desired properties and functions. The rise of DNA nanotechnology has provided a path to fully realize the potential of nanotechnology through the programmable self-assembly of DNA strands into complex geometries. Specifically, the more recently developed technology known as DNA origami offers a means to create nanoscale structures or mechanisms with nearly any imaginable geometry and with sub-nanometer precision. This thesis focuses on exploring methods for controlling DNA origami on multiple scales. Initially, this thesis will discuss methods for controlling the self-assembly of DNA origami, which can be used to form multiple nanostructures simultaneously from a complex mixture. The relative strengths of self-assembly reactions help illuminate how such processes can be so efficient in simpler contexts. We show that, not only is it possible to fold multiple nanostructures in a single pot but also that the relative yields of each structure can be tuned. We find that the kinetics of folding for small regions of a structure is a dominant factor in these yields. The intermediate portion of this thesis will discuss methods for control of dynamic DNA origami mechanisms. We develop a highly tunable control scheme combining gold nanoparticles and DNA origami hinge mechanisms which is rapid, robust, and repeatable without the need for material additives. We achieve an advancement over previously demonstrated control schemes by reducing the actuation times to the seconds timescale using temperature jump assays. We can more quickly control the hybridization of tunable latching strands compared to strand displacements methods and more re (open full item for complete abstract)

Committee: Carlos Castro Dr. (Advisor); Jessica Winter Dr. (Committee Member); Michael Poirier Dr. (Committee Member); Ezekiel Johnston-Halperin Dr. (Committee Member) Subjects: Biophysics

Master of Science, The Ohio State University, 2015, Mechanical Engineering

Scaffolded DNA origami has been used to construct objects with complex three-dimensional geometries via molecular self-assembly to create functional nanoscale devices in the 10-100 nm size range. The production of static DNA origami structures is well documented. Only a few designed DNA nanostructures utilize structural dynamics to achieve functionality. In contrast, there has been an emergence of work showing the functionalization of DNA nanostructures through the incorporation of functional molecules or nanoparticles. This work expands the scope of DNA origami applications by incorporating dynamic functional molecules to control dynamic components of DNA origami structures. An in depth investigation into controlling the dynamic motion of a DNA origami hinge through the integration of a transcription repressor protein is the main focus of this thesis. Investigation into the attachment of other nanoscale functional molecules to enable control over nanoscale DNA origami designs is also performed as a proof of concept. This work sets a foundation to create useful dynamic tools to explore complex nanoscale systems.

Committee: Carlos Castro Dr. (Advisor); Michael Poirier Dr. (Committee Member); Jonathan Song Dr. (Committee Member) Subjects: Biomedical Engineering; Engineering; Mechanical Engineering; Mechanics

Doctor of Philosophy, The Ohio State University, 2023, Biophysics

Most biomolecular systems are dependent on a complex interplay of forces and modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. I implemented a nanoscale force-sensing device using DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, I show that the opening force can be adjusted in real time by the addition of a single DNA oligonucleotide. These results establish the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties. In a secondary study in collaboration with Johnston-Halperin Lab and Winter Lab, we demonstrate an alternate approach to triggering of DNA base pair release by means of an optical trigger. Gold nanoparticles conjugated to short single stranded DNA oligonucleotides were used as a bridging component for two DNA tethers. We showed that the presence of the gold nanoparticle conferred a means of achieving localized surface plasmon resonance resulting in local heating of the environment. This heating resulted in an increased probability that the DNA linkage adjacent to the gold surface was disrupted. These results are a s (open full item for complete abstract)

Committee: Michael Poirier (Advisor); Carlos Castro (Committee Member); Ralf Bundschuh (Committee Member); Ezekiel Johnston-Halperin (Committee Member) Subjects: Biophysics

Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

DNA nanotechnology, specifically DNA origami, provides a platform that leverages complementary base-pairing to design nanoscale devices with a pre-defined geometry capable of precise motion and controlled mechanical properties. This work seeks to enable direct manipulation of complex higher order DNA nanodevice assemblies using DNA strand displacement and optical traps. Increased complexity of current actuation capabilities in the DNA nanotechnology space provides tools and methods spanning across multiple disciplines in engineering. We developed a 6-bar mechanism capable of shape transformations on the single device and multi-device scale using DNA strand displacement. We have also developed a 12-helix bundle, 4-bar linkage, and 7-bar linkage that are capable of higher order assembly with fluorescent readouts to measure force extension using dual optical trapping. Lastly, this work introduces DNA origami education modules that can be translated to middle school, high school, and undergraduate classrooms to enable a more accessible approach to teaching DNA nanotechnology. Creating bio-based tools for controlling more complex devices serves as a foundation for nano-or micro-scale robotic systems and provides platforms for multiplexing the control of nanomachines or molecular interactions. Translating DNA origami principles to classroom modules also allows for implementation of DNA nanotechnology to nontraditional research institutes.

Committee: Carlos Castro (Advisor); Hai-Jun Su (Committee Member); David Tomasko (Committee Member); Jessica Winter (Committee Member) Subjects: Biomedical Engineering; Chemical Engineering; Educational Technology; Engineering; Mechanical Engineering

Doctor of Philosophy, The Ohio State University, 2023, Mechanical Engineering

DNA origami technology allows for the design and fabrication of biocompatible and 3-D functional nanodevices via molecular self-assembly for various biological applications including cell function regulation, disease therapy and biomolecular detection. Biocompatibility and programmability of DNA molecules lead into the development of complex and functional DNA nanodevices that can work in biological media and living systems. Stability and functionality of DNA nanodevices compared to DNA molecules itself, make it an effective approach for cell modulation and payload delivery. The field of nanomedicine, has developed a path to detect and fight infectious disease using nanomaterials and DNA nanostructures are novel additions to the nanomaterial collection, providing great progress in diagnostics and therapy. Nucleic acids, proteins and whole pathogens are among the viral and bacterial targets which can be detected using DNA nanostructures. In this dissertation, we report four studies on biological applications of DNA origami in detection and modulation of biological cues. We establish the successful incorporation of DNA nanodevices on the cell membrane and report a robust method to monitor live cell interactions with biomolecules in their surrounding environment. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3-D environments under controlled biomolecular transport. Moreover, we propose a novel complex DNA platform for modulating the gap in intercellular junctions and finally establish three DNA nanodevices for the detection of viral and bacterial nucleic acids in human samples. Collectively, the findings of this dissertation establish a foundation on functionality of nanodevices for biological applications.

Committee: Carlos Castro (Advisor); Vishwanath Subramaniam (Committee Member); Comert Kural (Committee Member); Jonathan Song (Committee Member) Subjects: Biomedical Engineering; Biophysics; Mechanical Engineering; Nanotechnology

Doctor of Philosophy, The Ohio State University, 2021, Physics

The ability to apply and measure high forces (≥10pN) on the nanometer scale is critical to the ongoing development of nanomedicine, molecular robotics, and the understanding of biological processes such as chromatin condensation, membrane deformation, and molecular motors [1] [2] [3]. Current force spectroscopy techniques rely on micron-sized handles to apply forces, which can limit applications within nanofluidic devices or cellular environments [4]. To overcome these limitations, I used deoxyribonucleic (DNA) origami to self-assemble a nanocaliper, building on previous designs[5] [6]. I characterize the nanocaliper via a short double-stranded (ds)DNA with each strand attached to opposite arms of the device, via device equilibrium state, output force, and dynamics, to understand the effects of sequence, vertex design, and strut length on the device properties. I also produce nucleosomes, hexasomes, and an alternate dsDNA, which were then measured in the device, yielding mechanistic insight into the free energy landscape of each. I measure forces greater than 20 pN applied by the device with a nanometer dynamic range and 1 to 10 pN/nm stiffness. These high performing characteristics which expand the capabilities of existing force spectroscopy techniques as well as those of DNA origami devices.

Committee: Michael Poirier (Advisor); Ralf Bundschuh (Committee Member); Carlos Castro (Committee Member); Ezekiel Johnston-Halperin (Committee Member) Subjects: Biochemistry; Biophysics; Nanoscience; Physics

Doctor of Philosophy, The Ohio State University, 2021, Physics

Here I develop computationally efficient quantitative models to describe the behavior of DNA-based systems. DNA is of fundamental biological importance, and its physical properties have been harnessed for technological applications. My work involves each of these aspects of DNA function, and thus provides broad insight into this important biomolecule. First, I examine how DNA mismaches are repaired in the cell. Protein complexes involved in DNA mismatch repair appear to diffuse along dsDNA in order to locate a hemimethylated incision site via a dissociative mechanism. I study the probability that these complexes locate a given target site via a semi-analytic, Monte Carlo calculation that tracks the association and dissociation of the complexes. I compare such probabilities to those obtained using a non-dissociative diffusive scan, and determine that for experimentally observed diffusion constants, search distances, and search durations in vitro, both search mechanisms are highly efficient for a majority of hemimethylated site distances. I then examine the space of physically realistic diffusion constants, hemimethylated site distances, and association lifetimes and determine the regions in which dissociative searching is more or less efficient than non-dissociative searching. I conclude that the dissociative search mechanism is advantageous in the majority of the physically realistic parameter space, suggesting that the dissociative search mechanism confers an evolutionary advantage. I then turn to synthetic DNA structures, initially focusing on a composite DNA nano-device. In particular, manipulation of temperature can be used to actuate DNA origami nano-hinges containing gold nanoparticles. I develop a physical model of this system that uses partition function analysis of the interaction between the nano-hinge and nanoparticle to predict the probability that the nano-hinge is open at a given temperature. The model agrees well with experimental data and pre (open full item for complete abstract)

Committee: Ralf Bundschuh PhD (Advisor); Carlos Castro PhD (Committee Member); Michael Poirier PhD (Committee Member); Hirata Christopher PhD (Committee Member) Subjects: Biophysics; Nanotechnology; Physics; Polymers; Theoretical Physics

Master of Science, The Ohio State University, 2021, Mechanical Engineering

Scaffolded DNA origami has emerged as a prevalent technique for the design and construction of nanostructures of specific size, geometry, and function. Specifically, this technology enables programming nanostructure functionality by defining structural, mechanical and dynamic properties. Recent advancements have focused on integrating individual dynamic nanostructures to create reconfigurable supramolecular systems. Subsequently, these functional systems can be triggered by biological or environmental inputs to undergo conformational changes capable of reconfiguring other materials, providing measurable readouts, or influencing biological processes. This work aims to expand on dynamic functions by developing a reconfigurable assembly where local conformational changes can be physically communicated to other parts of the assembly through cascaded motion. We have designed a dynamic DNA nanostructure that can be assembled into arrays that can reach length scales ~10-100 times larger than the individual structure. We have demonstrated proof-of-concept for propagating conformational changes across nanodevices. DNA strands specific to one end of the array initiate motion for the “trigger” structure at that end, which in turn propagates motion to a neighboring structure, and so on in a sequential manner. This propagated motion is designed to transmit a signal across large distances. Creating programmable hierarchical assemblies capable of driving directional motion or signal has become a key goal in DNA nanotechnology. These systems could lead to customizable molecular transport systems, programmable circuits, and the catalysis of biochemical reactions.

Committee: Carlos Castro (Advisor); Jonathan Song (Committee Member) Subjects: Mechanical Engineering

Doctor of Philosophy, The Ohio State University, 2020, Mechanical Engineering

Major advances in DNA nanofabrication by the self-assembly process have occurred over the last decade to construct nano-devices for many applications of science and technology. However, advances in design methodology as well as advanced computational design tools have lagged behind, including computer-aided design (CAD) and coarse-grained models. Currently, for the majority of research in DNA nanotechnology, the design process is carried out using a bottom-up manual approach, which requires expertise and limits complexity. Recently developed top-down automated approaches that are limited to select types of static geometries, sacrificing the design flexibility for various applications. In addition, the integration between CAD and coarse-grained models require extra steps and limits the realization of virtual iterative design for engineering DNA assemblies in a robust manner. Here, we establish a versatile CAD tool that integrates top-down design automation with bottom-up control of component geometry and connectivity to build DNA nanomachines with various geometries (solid, shell, wireframe, or combinations), selected mobility (static, 1D, 2D, or 3D motion), large size via multi-structure assemblies. Based on this custom CAD tool, MagicDNA, we proposed a closed-loop integrated framework with MagicDNA and coarse-grained models, which enables the product design pipeline similar to macroscopic engineering (CAD and CAE) into nanoscale DNA assemblies for evaluating design parameters, rapid prototyping and eventually a robust design for experimental characterization and applications. Several structures were further fabricated to validate not only the target geometry but also the motion pathway, which in all cases generally agreed with simulation results. For these nanomachines, thermal fluctuation plays an important role to affect the component geometry and was quantified with hybrid coarse-grained models and kinematic variance analysis to predict the performance of the (open full item for complete abstract)

Committee: Hai-Jun Su (Advisor); Carlos Castro (Advisor); Shen Herman (Committee Member); Cho Hanna (Committee Member) Subjects: Engineering; Nanoscience; Nanotechnology

Doctor of Philosophy, The Ohio State University, 2018, Biophysics

Scaffolded DNA origami uses DNA as building blocks to create 3D nanostructures. Dynamic 3D DNA origami nanostructures have strong potential in biophysical experiments and applications. This includes measuring biomolecular forces, particularly on the nanoscale. Current biophysical methods lack the ability and/or specificity to measure on such a scale, in addition to being challenging and expensive. Dynamic DNA origami can address such a problem by providing a malleable material to accommodate shape and function on the 10-100nm length scale. The major goal of this work is to leverage DNA origami to develop tools to study biophysical properties of the biomolecular complexes. Nucleosomes, consisting of genomic DNA wrapped around a protein core, assemble into higher orders of chromatin structure to compact DNA. Tools to probe site-specific chromatin structure and dynamics at the 10-100nm length scale (relevant for gene regulation) and to apply tensile or compressive forces at targeted sites could greatly improve insight into how chromatin structural dynamics regulate DNA accessibility and processing. We designed, constructed, and implemented a nanocaliper via DNA origami. Our nanocalipers are hinge-like joints that consist of two 70nm rigid arms, each made up of bundled DNA helices, connected along one edge by single stranded DNA. For proof-of-concept, we first studied a single nucleosome by binding the two nucleosomal DNA ends to the ends of nanocaliper arms. Here, the caliper angle reports the nucleosome end-to-end distance. Moreover, the caliper angle is sensitive to nucleosome stability as a function of NaCl concentration. We demonstrated the nanocaliper can detect nucleosome conformational changes via transcription activator Gal4-VP16 binding. The caliper also significantly increases the probability of Gal4-VP16 occupancy by applying a tension to partially unwrap the nucleosome. This suggests that our DNA nanocalipers can report biologically relevant conformat (open full item for complete abstract)

Committee: Carlos Castro (Advisor); Michael Poirier (Committee Member); Marcos Sotomayor (Committee Member); Jonathan Song (Committee Member) Subjects: Biochemistry; Biomechanics; Biophysics; Mechanical Engineering

Doctor of Philosophy, The Ohio State University, 2017, Mechanical Engineering

Scaffolded DNA origami is a recently emerging technology that allows the construction of complex nanostructures via molecular self-assembly driven by Watson and Crick base-pairing, i.e. A-T, C-G. In the past decade, this approach has been successfully used to construct complex 2D or 3D static structures. Complex dynamic DNA origami mechanisms (DOM) have been fabricated and can achieve 1D, 2D and 3D motions. This research expanded scaffolded DNA origami nanotechnology to design dynamic nanomechanisms by following a design framework that parallels macroscopic compliant mechanism design. These compliant DNA origami mechanisms were referred as CDOM. The compliant components can be built by ssDNA connections or double-strand (dsDNA) bundles with small bending stiffness, which can be easily realized within DNA origami. In addition, a waterbomb base was designed and fabricated by scaffolded DNA origam, which demonstrated the origami of nanostructures.

Committee: Hai-Jun Su Prof./Dr. (Advisor); Carlos Castro Prof./Dr. (Committee Co-Chair); Jonathon Song Prof./Dr. (Committee Member); Hanna Cho Prof./Dr. (Committee Member) Subjects: Mechanical Engineering

Master of Science, The Ohio State University, 2016, Chemical Engineering

DNA origami nanostructure technology allows for the precise control of size and structure formation using the building blocks of life. Here, DNA was not used as the blueprint for protein formation but as a delivery vehicle for chemotherapeutic drugs, such as the anthracycline antibiotic, daunorubicin. By itself, daunorubicin has limited pharmacokinetics and biodistribution profiles when applied in vivo. In addition, daunorubicin, like most small molecule drugs, is ineffective against cancer cells that have acquired multi-drug resistance (MDR). By delivering the chemotherapeutic using DNA origami allows the drug to travel through the endolysosomal pathway, bypassing MDR mechanisms. Here, we were able to overcome MDR mechanisms in a liquid tumor cell line using the “Trojan Horse” DNA origami nanostructure as a drug delivery vehicle. Though promising, there are many barriers to pass before DNA origami nanostructures is a viable option for clinical use. This includes commercial level scale-up, target specificity and testing for immunogenicity and toxicity in vivo. Here, we discuss a method developed for the scale up of DNA origami production by 1500x standard volumetric reaction amounts. In addition, we were able to characterize a multitude of nanostructures for a more universal scaled process. Furthermore, we measured the effects that high concentrations of DNA origami nanostructures have in a mouse model. Lastly, since the binding and subsequent cellular internalization of DNA origami is non-specific, we were able to attached strategically located antibodies allowing for not only targeted drug specificity, but also blocking non-specific cell uptake. With these additions, the hope is that effective chemotherapeutics can be delivered to tumor sites while avoiding undesirable damage to healthy tissues in a clinical setting.

Committee: Carlos Castro (Advisor) Subjects: Biomedical Engineering; Biomedical Research; Chemical Engineering; Nanoscience; Nanotechnology; Oncology; Pharmaceuticals

Doctor of Philosophy, The Ohio State University, 2016, Mechanical Engineering

Mechanical forces in biological systems vary in both length and magnitude by orders of magnitude making them difficult to probe and characterize with existing experimental methodologies. From molecules to cells, forces can act across length scales of nanometers to microns at magnitudes ranging from picoNewtons to nanoNewtons. Although single-molecule techniques such as optical traps, magnetic tweezers, and atomic force microscopy have improved the resolution and sensitivity of such measurements, inherent drawbacks exist in their capabilities due to the nature of the tools themselves. Specifically, these techniques have limitations in their ability to measure forces in realistic cellular environments and are not amenable to in vivo applications or measurements in mimicked physiological environments. In this thesis, we present a method to develop DNA force-sensing nanodevices with sub-picoNewton resolution capable of measuring forces in realistic cellular environments, with future applications in vivo. We use a design technique known as DNA origami to assemble devices with nanoscale geometric precision through molecular self-assembly via Watson-Crick base pairing. The devices have multiple conformational states, monitored by observing a Forster Resonance Energy Transfer signal that can change under the application of force. We expanded this study by demonstrating the design of responsive structural dynamics in DNA-based nanodevices. While prior studies have relied on external inputs to drive relatively slow dynamics in DNA nanostructures, here we developed DNA nanodevices with thermally driven dynamic function. The device was designed with an ensemble of conformations, and we establish methods to tune the equilibrium distribution of conformations and the rate of switching between states. We also show this nanodynamic behavior is responsive to physical interactions with the environment by measuring molecular crowding forces in the sub-picoNewton range, whic (open full item for complete abstract)

Committee: Carlos Castro (Advisor); Michael Poirier (Committee Member); Soheil Soghrati (Committee Member); Jonathan Song (Committee Member) Subjects: Mechanical Engineering; Nanotechnology

Master of Science, The Ohio State University, 2016, Mechanical Engineering

Structural DNA Nanotechnology (“DNA origami”) techniques have enabled the design and synthesis of complex 3D nanostructures with dynamically controllable features that exploit molecular self-assembly principles. Any component that can be conjugated to an oligonucleotide (oligo) can be attached to a DNA nanostructure at a specific location and quantity with nanometer resolution. This includes some fluorescent dyes, quenchers, peptides, RNA, steroids, vitamins, and, by extension, all molecules capable of biotinylation. A DNA origami “breadboard” with 34 strategically located attachment points can therefore be functionalized with a wide variety of components and used for a multitude of purposes. In this thesis, the design, fabrication, purification, characterization, and application of a 68 x 25 x 6 nanometer honeycomb lattice DNA nanostructure will be presented for use in two distinct functions. In the first, a biotinylated antibody was added to the platform and used to better mimic a cell-to-cell receptor-ligand interaction with tunable antibody quantity, location, and flexibility (i.e. range of motion). This led to determination that ligand flexibility, which can be controlled using DNA origami, influences strength of cell activation. In the second, cholesterol-modified oligonucleotides were added to cells and used to anchor the nanostructures onto the cell surface. The ability to integrate DNA origami nanostructures into a cell membrane can enable a wide variety of applications such as a intracellular force sensing, programmed cell-cell adhesion, or triggered recruiting of biomolecules from solution.

Committee: Carlos Castro (Advisor); Jonathan Song (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Cellular Biology; Mechanical Engineering

Master of Science, The Ohio State University, 2013, Mechanical Engineering

This thesis establishes scaffolded DNA origami as a viable approach to designing and fabricating nanoscale mechanisms and machines. The work presented shows a fundamental proof-of-concept for the fabrication of DNA Origami Mechanisms and Machines (DOMM). The scope of the project is to design, fabricate, and analyze DNA origami joints, incorporate them into larger mechanisms, and actuate the mechanisms. This thesis starts with the initial step of creating DNA origami revolute joints (hinges). Revolute joints are single degree of freedom joints and demonstrate the design and construction of nanoscale kinematic joints with specific constrained motion. The revolute joint design was integrated into a DNA origami Bennett 4-bar mechanism. This mechanism has a 3D motion path that is specified by the dimensions of the links and the arrangement of the hinges. The motion of the DNA origami linkage agrees well with its rigid body counterpart. These linkages were further actuated using DNA inputs. This method of actuation proves to be an effective way to control DNA origami mechanisms. The ultimate goal of this project is to develop a library of DNA-based links and joints that can be widely used in the design and assembly of higher order controllable nanomachines. This work has the potential of initiating a new era of nanorobot design useful for applications including drug delivery, biosensing, and nanomanufacturing.

Committee: Carlos Castro (Advisor); Haijun Su (Committee Member) Subjects: Biomedical Engineering; Design; Engineering; Mechanical Engineering

Doctor of Philosophy, The Ohio State University, 2023, Chemical Engineering

DNA is commonly envisioned as the double stranded structure that encodes genetic information, but the specific binding pattern seen between the bases also makes DNA a highly specific building material. DNA nanostructures come in a variety of forms, such as tiles, wireframes, and scaffolded origami. Tile-based structures originated as cross-shaped, synthetic Holliday junctions designed by Seeman that were used for building 2D lattices from repeating units. Over time, DNA nanostructures have evolved into the more complex scaffolded origami pioneered by Rothemund3. These scaffolded structures were originally designed as 2D static objects that have evolved into dynamic materials with rotational, linear, and angular motion. Applications of DNA nanotechnology could include biosensing, multiplexed imaging, drug delivery, and nanorobotics. These applications seek to take advantage of the reconfiguration, biocompatibility, and functionalization potential of DNA nanostructures. Although DNA origami applications are currently limited by scaling issues, many fundamental studies in the fields of plasmonics and nanorobotics have been conducted. Many of these applications rely on the ability to attach nanoparticles (NPs) and biomolecules to DNA nanostructures. DNA origami-NP integration typically uses complementary single-stranded DNA (ssDNA) handles attached to the NP and DNA nanostructure, respectively, for binding. However, conjugation methods for modifying NPs with ssDNA are often lengthy and require precise salt and pH conditions that may destabilize NP colloids. ssDNA handles are easy to design into DNA origami and allow for precise organization of NPs for studying interactions of multiple particles. It is also possible to change the properties of bound NPs and even the DNA itself through metal growth. Metal growth can be performed from NP seeds or on the surface of the DNA origami, thus eliminating the need for NP binding to study emergent properties. In this work, I (open full item for complete abstract)

Committee: Jessica Winter (Advisor) Subjects: Chemical Engineering

PhD, University of Cincinnati, 2012, Engineering and Applied Science: Biomedical Engineering

Linear double-stranded DNA (dsDNA) viruses package its genome into a preformed procapsid fueled by the energy from ATP hydrolysis. The bacteriophage phi29 motor has a truncated cone shaped protein component, named connector, with a central channel of 3.6 nm at its narrowest part. The connector protein has been successfully inserted into an artificial lipid bilayer membrane, and the channel exhibited robust capability under various salt and pH conditions as revealed by single channel studies. This channel is suitable for extremely precise assessment of the transportation of small molecules, such as ions, DNA and RNA. There is an urgent need to development a highly sensitive detection system, for the applications in the area of pathogen detection, disease diagnosis, environmental monitoring, etc. The current challenges and limitations of these technologies are the sensitivity and accuracy issues arising from background noise and nonspecific reactions. The property of phi29 motor channel has been studied at various conditions, and was incorporated into lipid membrane. The motor channel exercised a one-way traffic property during the process of dsDNA translocation with a valve mechanism. In addition, the opening and closure of the channel also exhibit reversible and controllable. A modified version of the connector channel is founded to have a smaller channel size, which is able to detect the ssDNA and ssRNA. These findings have important implications since this artificial membrane-embedded channel would allow detailed investigations into the mechanisms of viral motor operation, as well as future applications for therapeutic molecule packaging, delivery, single molecule sensing and drug screening.

Committee: Jing-Huei Lee PhD (Committee Chair); Chong Ahn PhD (Committee Member); Peixuan Guo PhD (Committee Member); Jaroslaw Meller PhD (Committee Member); Marepalli Rao PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2012, Engineering and Applied Science: Biomedical Engineering

A powerful nanomotor is employed by the tailed dsDNA virus to package the genome into a preformed protein shell during the process of replication. The bacteriophage phi29 is an excellent model for investigating the viral DNA packaging mechanism. The phi29 DNA packaging motor is composed of three ring structures: the dodecameric connector ring, the hexameric pRNA ring and the hexameric ATPase gp16 ring. The connector is the central hub for the DNA to enter and to exit. There are four positively charged lysine rings scattered inside the highly negatively charged connector channel. It is speculated that these positive charged lysine rings may play active roles during DNA packaging in many models. To test this prevalent view, the basic lysine residues were mutated to neutral alanines and the pH environment was altered. Amazingly, the results were beyond expectation. Neither the DNA translocation nor the one-way traffic property of the channel were measurably influenced by the alteration of the charge of lysine residues when the basic lysine residues mutated to neutral alanines or the pH environment changed to acid or basic. The ATPase or the terminase is the central part of the viral DNA packaging motor. The phi29 ATPase is highly hydrophobic and tends to aggregate in solution. A green fluorescent protein tag (eGFP) fused to the N-terminus of gp16 enhanced its solubility and stability. The eGFP-gp16 showed similar activity to wild type gp16 and was easily detected by fluorescent instruments. The interaction between eGFP-gp16 and DNA in the various conditions were investigated by electrophoretic mobility shift assay, FRET and sucrose gradient. γ-S-ATP dramatically increased gp16 binding affinity to DNA and ATP, ADP, phosphate could release gp16 from gp16-DNA-γ-S-ATP complex. The sliding of gp16 out of the gp16-DNA-γ-S-ATP complex could be blocked by addition of Steptavidin to ends of dsDNA which is conjugated with biotins. Also, we found that six eGFP-gp16 molecules wer (open full item for complete abstract)

Committee: Carlo Montemagno PhD (Committee Chair); Peixuan Guo PhD (Committee Member); Jiukuan Hao PhD (Committee Member); Jaroslaw Meller PhD (Committee Member); Giovanni Pauletti PhD (Committee Member) Subjects: Biomedical Research