生物に着想を得た運動のモデリングと制御

東京電機大学201

Nanofluidics for Static and Dynamic DNA-Protein Interaction Studies - Repair of Double-Strand Breaks from a Single-Molecule Perspective

Double-strand breaks (DSBs) is one of the most lethal forms of DNA damage. A single DSB may result in stalling of vital cellular machineries, and thus, requires immediate measures by the cell. Although the main hallmarks of DSB repair mechanisms are known for most prokaryotes and eukaryotes, details on crucial intermediate steps are still to be explained, such as how the free DNA ends are kept in close proximity during the repair process.\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0 In the original work, upon which this Thesis is based, the two main DSB repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), have been studied from a molecular perspective. A fluorescence-based single-molecule nanofluidics assay has been developed and employed to characterize and visualize static biomolecular interactions between DNA and key DSB repairing proteins. Furthermore, a novel dynamic nanofluidic device has been developed to enable dynamic interaction studies in real time, allowing analytes to be introduced on-demand to stretched DNA molecules.\ua0\ua0\ua0\ua0\ua0\ua0\ua0\ua0 Single-molecule characterization of the NHEJ mechanism in Bacillus subtilis, comprising the Ku and Ligase D proteins, revealed that the end-joining activity is mediated by C-terminal protrusions on the homodimeric Ku complex. Using the novel dynamic nanofluidic device, the Ku homodimer was further demonstrated to stay bound to the DNA ends and junctions after completed repair, similar to the human Ku70/80 heterodimer. In addition, the traditional static nanofluidic device was used to identify a previously unknown potential DNA-bridging role of CtIP, a key protein in the human HR process. This could possibly explain how broken DNA ends are kept in close proximity during the initial steps of DSB repair through HR in humans. A similar method was employed to show that the Xrs2 component is indispensable for the end-joining activity of the Mre11-Rad50-Xrs2 complex of Saccharomyces cerevisiae

Integrating single-molecule visualization and DNA micromanipulation

MacKintosh, F.C. [Promotor]Peterman, E.J.G. [Copromotor]Wuite, G.J.L. [Copromotor

Single molecule studies of branched polymer dynamics

Polymer architecture plays a major role on the emergent physical and chemical properties of materials such as elasticity and wettability. Branched polymers exhibit strikingly different rheological behavior (e.g. enhanced stress dissipation and strain hardening) compared to linear polymers. In recent years, the dynamic properties of branched polymers have been studied using bulk rheological techniques (Chapter 1), but we still lack a full understanding of how molecular-scale interactions give rise to macroscopic properties for topologically complex polymers. Single molecule studies enable the direct observation of polymer chain dynamics at the molecular level; however, the vast majority of single polymer studies have only focused on linear DNA molecules (Chapter 2). In this dissertation, we extend single molecule techniques to study the dynamics of branched polymers, which effectively bridges the gap between bulk-scale rheological properties and molecular scale behavior. In particular, we explore the synthesis, characterization, single molecule dynamics, and Brownian dynamics simulations of DNA-based branched polymers. This approach enables us to interrogate the impact of distributions in molecular size and architecture, thereby holding the potential to fundamentally change our understanding of the rheological response of topologically complex polymers. We first developed a two-step synthesis method to generate branched polymers for single molecule visualization (Chapter 3). Here, we use a graft-onto synthesis method by linking side branches onto DNA backbones, thereby producing star, H-shaped, and comb-shaped polymers. In these experiments, DNA-based branched polymers are designed to contain short branches (1-10 kilobase pairs) and long backbones (10-40 kilobase pairs), where the branches and backbones are monodisperse and the branch distribution can be controlled in an average sense. Following synthesis and purification, we utilize single molecule fluorescence microscopy to observe the dynamics of these molecules, in particular by tracking the side branches and backbones independently (Chapter 4). In this way, this imaging method allows for characterization of these materials at the single molecule level, including quantification of polymer contour length and branch distributions for varying synthetic conditions. Moving beyond characterization, we study the dynamics of single branched polymers in flow using a molecular rheology approach. In one experiment, we study the dynamics of asymmetric star, H-shaped, and comb-shaped DNA polymers tethered to the surface in a microfluidic flow cell (Chapter 4). In this way, we study the impact of branch frequency and position on backbone chain relaxation from high stretch. In a second experiment, we utilize a microfluidic cross-slot device to hydrodynamically ‘trap’ branched DNA molecules in planar extensional flow, thereby studying the impact of branching on relaxation in solution, as well as transient and steady-state dynamics in flow (Chapter 5). We present results for branched polymer dynamics as functions of branch frequency and flow strength. We also conduct Brownian dynamics simulations based on a coarse-grained model for comb polymers (Chapter 6). Results from simulations and experiments agree qualitatively, and branched polymers exhibit a weaker dependence of relaxation on total polymer molecular weight in comparison to linear polymers. Overall, this work presents molecular-scale investigations of branched polymer dynamics. From a broad perspective, this research provides a molecular-based understanding of topologically complex polymers in flow, thereby holding the potential to advance the large-scale production of polymers. Importantly, this platform can be further extended to study branched polymers in alternate flow fields such as simple shear flow or linear mixed flows, semi-dilute solutions, and concentrated solutions. These experiments will provide a molecular basis for phenomena observed in branched polymers, from viscosity modification of blended branched polymer solutions to hierarchical relaxation mechanisms of entangled branched polymers to enhanced strain hardening of comb polymer melts

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNAbinding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and sitedirected modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria

The Characterization of Single DNA Molecules in Nanofluidic Devices

This dissertation investigates DNA extension and transport characteristics in nanochannel confinement. Experiments were performed with the goal of advancing DNA polymer physics theory in nanochannel confinement by determining the influence of nanochannel aspect ratio (width/depth) on extension. DNA extension lengths were measured in the extended de Gennes regime in devices with unique aspect ratio nanochannels. Contrary to past findings, the DNA extension length was found to scale in agreement with the theoretically predicted relationship (~^−2/3) and simulation results, with exponent values ranging from -0.67 to -0.70. In addition, the data suggests that modest aspect ratios do not appreciably affect scaling, while the smallest critical dimension of a nanochannel can strongly impact extension. Further research investigated DNA transport characteristics in nanofluidic optical mapping scenarios with the goal of quantifying and ultimately improving DNA transport efficiency or the number of fragments that transport without detrimental DNA-wall interactions. A method for quantitative analysis of DNA transport was developed to compare fluorescently-labeled DNA traces interrogated at three successive areas of a serpentine nanochannel. DNA fragments were identified with the use of cross-correlation, DNA sizing, size calibration, and lastly fragment assignment based on DNA transport time and size. The addition of Mg cofactors into experimental buffers inhibited DNA transport by increasing stick-slip motion and DNA-wall affinity, with Mg-DNA bridging hypothesized as the leading mechanism of decreased performance. Adding an oligonucleotide dynamic coating addition into the buffer decreased the number of DNA fragments with impacted transport and strongly improved transport efficiency. In addition, both transport efficiency and diffusivity of DNA fragments were observed to be size-dependent. Lastly, the design, fabrication, and testing of a single cell capture device is described. The purpose of the device was to enable cell capture, lysis, and DNA compartmentalization from single cells downstream for genetic analysis. The device demonstrated repeatable capture and lysis of individual cells, but was not able to successfully compartmentalize DNA for downstream analysis due to a number of failure modes during DNA extraction. The single cell capture work is included here to educate and inform future experiment in the area.Doctor of Philosoph

Engineered nanofluidic platforms for single molecule detection, analysis and manipulation

Since the pioneering studies on single ion-channel recordings in 1976, single molecule methods have evolved into powerful tools capable of probing biological systems with unprecedented detail. In this work, we build on the versatility of a type of nanofluidic devices, called nanopipettes, to explore novel modes of single molecule detection and manipulation with the aim of improving spatial and temporal control of biomolecules. In particular, a novel nanopore configuration is presented, where biomolecules were individually confined into a zeptoliter volume bridging two adjacent nanopores at the tip of a nanopipette. As a result of this confinement, the transport of biomolecules such as DNA and proteins was slow down by nearly three orders of magnitude, leading to an improved sensitivity and superior signal-to-noise performances compared to conventional nanopore sensing. Active ways of controlling the transport of biomolecule by combining the advantages of nanopore single-molecule sensing and Field-Effect Transistors are also presented. These hybrid platforms were fabricated in a simple two step process which integrates a gold electrode at the apex of a nanopipette. We show that these devices were effective in modulating the charge density of the nanopore and in actively switching "on" and "off" the transport of DNA through the nanopore. Finally, a nanoscale dielectrophoretic nanotweezer device has been developed for high resolution manipulation and interrogation of individual entities. Two closely spaced carbon nanoelectrodes were embedded at the apex of a nanopipette. Voltage and frequency applied to the electrodes generated a highly localized force capable of trapping and manipulating a broad range of biomolecules. These dielectrophoretic nanotweezers were suitable for probing complex biological environments and a new technique for minimally invasive single-cell nanobiopsy was established. Such study provides encouraging results on how nanopipettebased platforms can be integrated as a future tool for routinely interrogating molecules at the nanoscale.Open Acces

An Exploratory Study to Bring Meaning of Haptic In Association with Human Emotion

The popularity of haptic technologies has permitted daily life, allowing intimate and emotional contact to be conveyed from sender to receiver. However there are weaknesses apart when haptic is being applied into an application, which can result misinterpreted, high complexity and confusion to the user. Research shows that emotion comprise close relationship with haptic feedback, this research project will investigate the effectiveness of emotion to bring haptic meaning. The project has predict the weaknesses of emotion in explore the absolute meaning of haptic, however with the present of multi-model technology the weaknesses could be reduce in order to identify the suitable definition of haptic with association to emotion

Synthesis of Metallic Nanowires Using Integrated DNA Molecules as Templates

The DNA double helix is inherently a nanoscale wire-like object, possessing a 2 nm diameter as well as a remarkable capability for molecular recognition and the interaction with other chemical compounds, thus making it an attractive material for biologically driven assembly of artificial nanostructures. In this work methods for the construction of functional electronic networks from single DNA molecules are presented. For this, (i) the generation of patterns of distinct interconnects between micro-fabricated contact pads are explored by stretching end-specifically thiol-functionalized, single-tethered DNA molecules using hydrodynamic flow as well as an electric field-induced thermal flow. (ii) These networks then serve as a template for a selective in-situ photoinduced nucleation and growth of platinum clusters of 4 nm diameter along the DNA molecules. In the synthesis exclusively platinum ions from an aqueous platinum nitrate solution bonded electrostatically to the backbone of the immobilized DNA can be reduced upon irradiation with UV light, while background metallization is inhibited. Furthermore, the metallization scheme is applied to DNA nanotubes and another photochemical deposition process is used to tune the interparticle gap space in a discontinuous platinum cluster chain to form conducting nanowires. The "process toolbox'' presented in this work offers a versatile alternative for the hierarchical patterning and incorporation of biotemplated nanomaterials into micro-/nanofabrication schemes.Ein doppelhelikaler DNA-Strang besitzt mit seinem hohen Aspektverhältnis von Natur aus Ähnlichkeit mit einem Kabel. Zusammen mit seinen einzigartigen Selbstassemblierungseigenschaften sowie der Fähigkeit, mit einer Vielzahl von chemischen Stoffen eine Verbindung einzugehen, macht dies ihn zu einem aussichtsreichen Baumaterial für den Aufbau von künstlichen Nanostrukturen. In dieser Arbeit werden deshalb verschiedene Methoden für den Bau von elektronischen Schaltkreisen aus einzelnen DNA-Strängen demonstriert. Dazu wird (i) die Herstellung von Verdrahtungsmustern zwischen lithographisch gefertigten Kontaktstrukturen untersucht. Endständig mit Thiolgruppen funktionalisierte DNA-Moleküle, die an nur einem Ende mit der Oberfläche verknüpft sind, werden mittels Strömung oder eines elektrothermisch induzierten Flusses zwischen Elektroden gespannt. (ii) Diese Netzwerke dienen im Weiteren als Vorlage für ein selektives, lichtinduziertes Wachstum von Platinpartikeln mit Durchmessern von 4 nm lokal entlang der DNA-Moleküle. Dabei werden unter UV-Bestrahlung nur solche Platinionen reduziert, die aus einer Platinnitrat-Lösung elektrostatisch an die immobilisierte DNA angebunden haben. Partikelwachstum in der umgebenden Lösung wird weitgehend verhindert. Darüber hinaus wird dieses Verfahren auch auf DNA-Nanoröhren angewendet und ein weiterer photochemischer Abscheideprozess eingesetzt, um unterbrochene Clusterkettern zusammenzuwachsen, mit dem Ziel, elektrisch leitfähige Nanodrähte zu erhalten. Die vorgestellten Verfahren stellen eine vielseitige Alternative zu herkömmlichen, hierarchischen Fabrikationsschemen der Mikro- und Nanotechnologie dar