abstract: DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system. / Dissertation/Thesis / Presentation for Lin Gan's thesis defense (orginally in pptx exported in PDF) / Doctoral Dissertation Chemistry 2015

abstract: There are many biological questions that require single-cell analysis of gene sequences, including analysis of clonally distributed dimeric immunoreceptors on lymphocytes (T cells and B cells) and/or the accumulation of driver/accessory mutations in polyclonal tumors. Lysis of bulk cell populations results in mixing of gene sequences, making it impossible to know which pairs of gene sequences originated from any particular cell and obfuscating analysis of rare sequences within large populations. Although current single-cell sorting technologies can be used to address some of these questions, such approaches are expensive, require specialized equipment, and lack the necessary high-throughput capacity for comprehensive analysis. Water-in-oil emulsion approaches for single cell sorting have been developed but droplet-based single-cell lysis and analysis have proven inefficient and yield high rates of false pairings. Ideally, molecular approaches for linking gene sequences from individual cells could be coupled with next-generation high-throughput sequencing to overcome these obstacles, but conventional approaches for linking gene sequences, such as by transfection with bridging oligonucleotides, result in activation of cellular nucleases that destroy the template, precluding this strategy. Recent advances in the synthesis and fabrication of modular deoxyribonucleic acid (DNA) origami nanostructures have resulted in new possibilities for addressing many current and long-standing scientific and technical challenges in biology and medicine. One exciting application of DNA nanotechnology is the intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell populations. DNA nanostructures can be transfected into individual cells to capture and protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing. This approach is highly modular and can be adapted to virtually any two (and possibly more) gene target sequences, and therefore has a wide range of potential applications for analysis of diverse cell populations such as understanding the relationship between different immune cell populations, development of novel immunotherapeutic antibodies, or improving the diagnosis or treatment for a wide variety of cancers. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2017

abstract: DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops. / Dissertation/Thesis / Ph.D. Biochemistry 2013

The microtubule cytoskeleton plays a vital role in the spatial-temporal organization of subcellular cargo required to maintain homeostasis and direct cell division. Cytoplasmic dynein and kinesin are opposite-polarity, microtubule-based motors that transport a wide variety of cargo throughout eukaryotic cells. While much is known about the stepping mechanism of kinesin from decades of study, cytoplasmic dynein's size and complexity has limited our understanding of its underlying motor mechanism. Here, a minimal, artificially-dimerized dynein motor was observed with two-color, near-simultaneous, high-precision, single-molecule imaging, which reveals the stepping pattern of each motor domain as dynein moves along the microtubule. Although the stepping behavior appeared highly irregular and erratic, with large variability in step sizes, side stepping behavior, and back stepping behavior, dynein did show evidence of tension-based, coordinated stepping. Furthermore, advances in DNA nanotechnology enabled us to engineer a synthetic motor-cargo system, referred to as a chassis, to investigate how multiple cytoskeletal motors work in teams to produce the myriad of motile behaviors observed in vivo. Specifically, the mechanisms that coordinate motor ensemble behavior was examined using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing control of motor type, number, spacing, and orientation in vitro. Ensembles of 1-7 identical-polarity motors displayed minimal interference with respect to directional velocity, while ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species. This experimental system allowed us to test directly the tug-of-war proposed to occur during dynein's delivery to the microtubule plus-end by the kinesin Kip2. This work led to the mechanistic understanding that Lis1/Pac1, CLIP170/Bik1, and EB1/Bim1 proteins function to enhance kinesin's processivity, allowing it to win a tug-of-war and transport dynein toward the microtubule plus-end. Overall, this work elucidated mechanisms of ensemble motor function and dynein's stepping mechanism in addition to building significant tools to further pave the way for future studies to elucidate how cytoskeletal motors function to organize cellular cargos.

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<p>  </p> <p>Architectured metamaterials are artificial systems with unique structural characteristics. They show distinct deformation behaviors and improved mechanical properties compared to regular materials. For example, mechanical metamaterials demonstrate negative Poisson's ratios, whereas regular materials have positive values. In theory, the auxetic behaviors arise from periodic cellular architectures regardless of the materials utilized. While this premise is mostly true for macroscopic metamaterials, it may not work well at a very small lengthscale since chemistry may play a critical role in nanostructures. However, this fundamental idea has not been addressed due to the lack of powerful manufacturing strategies at the nanoscale. The majority of architectured metamaterials are manufactured from top down with their unit size of microns or larger. On the other hand, there are also molecular auxetics which are natural crystals and thus are not designable. Therefore, there is a significant gap in lengthscale from 10 nm to 1 µm. DNA self-assembly is a bottom-up approach that can construct complex nanostructures based on sequence complementarity. Examples include DNA origami structures and DNA tile assemblies. This dissertation bridges the gap in the lengthscale by introducing nanoscale auxetic units from DNA and investigates relevant structural properties and mechanical behaviors. This study addresses the premise of metamaterials and elucidates the structure-property relation. The findings from this work formulate design principles for DNA based auxetic metastructures. </p> <p>In this work, we built several two-dimensional (2D) auxetic nanostructures from wireframe DNA origami. They serve as the model systems to demonstrate the feasibility of constructing nanoscale auxetics via DNA self-assembly. DNA origami structures are commonly constructed by a long ‘scaffold’ strand with many ‘staple’ oligonucleotides. Since the DNA metastructures are too small to directly apply external forces, we implemented chemical deformation by inserting ‘jack’ edges. Like a car jack, the length of the jack edges can be modulated via two-step DNA reactions: toehold-mediated strand displacement and annealing with a new set of jack staples. The DNA nanostructures reconfigure accordingly. To complement the experiment, we performed molecular dynamics (MD) simulations based on coarse-grained models using an open-source oxDNA platform. In the numerical computation, external loads were directly applied to deform the metastructures, providing details of structural deformation. We discovered that the auxetic behaviors of DNA metamaterials can be estimated by architectural designs, however the material properties are also crucial in the structures and deformations. Our mechanistic study provided general design guidelines for 2D auxetic DNA metamaterials. We also designed and constructed a Hoberman flight ring from DNA, a simplified planar version of Hoberman sphere. This structure consists of six equilateral triangles that are topologically organized into two layers, resembling a trefoil knot. The DNA flight ring deploys upon external forces, expanding (open state) or contracting (closed state) by sliding the two layers of triangles. This is the first synthetic deployable nanostructure and offers a versatile platform for topological research.</p> <p>This thesis also investigates 3D effects in DNA assemblies and related mechanics. We used a DNA origami tile designed with an intrinsic twist as a model system and explored its cyclization process using MD simulations. The numerical computation revealed the detailed process where the structure untwists and curves for cyclization simultaneously under external forces. The force and energy required to overcome the initial curvature and cause the 3D deformation were also calculated. The results agree well with the previous experiment and theory, further verifying the simulation method. Direct mechanical forces and DNA responses were realized experimentally with 3D DNA crystals built from triangular DNA tiles. Nanoindentation was performed on macroscopic ligated crystals using atomic force microscopy (AFM). MD simulations were performed in parallel, which revealed the full spectrum of several distinct deformation modes from linear elasticity to structural failure. The combined experiment, computation, and theoretical calculation showed that the complex behaviors can only be understood fully by considering the structure and its components. </p> <p>The scientific findings from this thesis should contribute to the construction of auxetic metastructures, the design methods for DNA based metamaterials as well as the prediction of their structural properties and mechanical behaviors. This thesis will pave the way for building architectured materials from DNA with tailored properties and functionalities, opening the door for new opportunities and unique applications.</p>

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24854号 / エネ博第463号 / 新制||エネ||87(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井, 孝, 教授 片平, 正人, 教授 佐川, 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

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