Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references. Also available in electronic version.

Thesis (Ph.D.)--Ohio University, November, 2004. / Includes bibliographical references (p. 202-211)

Thesis (Ph.D.)--Ohio University, November, 2004. / Title from PDF t.p. Includes bibliographical references (p. 202-211)

DNA cages are polyhedra self-assembled from synthetic oligonucleotides in a one-pot process. The main system described in this thesis is a reconfigurable, wire-framed DNA tetrahedron in nanometre-scale. On one of its vertices this tetrahedron has an overhang that can hybridise with a specific sequence of nucleic acids and open the cage. We describe the design of a reconfigurable cage that remained closed under physiological conditions and only opened in the presence of an appropriate signal in solution. Fluorescence techniques were employed to distinguish the open and closed states of the cage. We used flow cytometry and confocal microscopy to successfully established the open and closed states of the cage inside live cultured mammalian cells. Further experiments revealed that the DNA cage could be opened by a separately transfected signalling strand. Hybridisation between two separately transfected systems was possible. The DNA cage was then simplified to a DNA duplex so that the intracellular interactions between the two nucleic acids systems could be studied more efficiently. Microscopy images showed that the interaction occurred in membrane-bound compartments. We describe an investigation into the use of various cellular RNAs, including full-length mRNA and tRNA-RNA fusion, to actuate the DNA cages. Choosing an appropriate cellular opening signal remains a challenge. Analysis showed that bulky cellular RNA experienced steric hindrance with the rigid DNA cage. Finally, other potential biological applications of DNA cages, such as using DNA nanostructures as the carriers for genetic therapeutic agents, were also presented.

No description available.

The controlled organization of individual molecules and nanostructures with nanoscale accuracy is of great importance in the investigation of single-molecule events in biological and chemical assays, as well as for the fabrication of the next generation optoelectronic devices. In this regard, the precise patterning of individual molecules into hierarchical structures has attracted substantial research interest in recent years. DNA has been shown to be an ideal structural material for this purpose, due to the specificity of its programmability and outstanding chemical flexibility. DNA origami can display a high degree of positional and precise binding sites, allowing for complex arrangements and the assembly of different nanoscale architectures. In this project, we present a novel platform based on the use of DNA scaffolds for the organization of individual nanomoieties (with nanoscale spatial control), and their selective immobilisation on surfaces for single-molecule investigations. In particular, semiconductor quantum dots (QDs), fluorescence molecules, linear small peptides, and structural proteins were tethered with single-molecule accuracy on DNA origami; their subsequent organization in array configuration on nanopatterned surfaces allowed us to fabricate and test different platforms for single-molecule studies. In particular, we developed a Focused Ion Beam (FIB) nanofabrication strategy and demonstrated its general applicability for the assembly of functionalised DNA nanostructures in highly uniform nanoarrays, with single-molecule control. In addition, we further explored this nanofabricated platform for biological investigations at the single-molecule level, from protein-DNA interactions to cancer cell adhesion studies with single-molecule control. Investigations have been carried out via fluorescence microscopy, scanning electron microscopy (SEM), Focused Ion Beam (FIB) and atomic force microscopy (AFM). By and large, combining the programming ability of DNA as a scaffolding material with a one-step lithographic process, we have developed a platform of general applicability for the fabrication of nanoscale chips that can be employed in a variety of single-molecule investigations.

DNA nanotechnology has been developed in order to construct nanostructures and nanomachines by virtue of the programmable self-assembly properties of DNA molecules. Although DNA nanotechnology initially focused on spatial arrangement of DNA strands, new horizons have been explored owing to the development of the toehold-mediated strand-displacement reaction, conferring new dynamic properties to previously static and rigid structures. A large variety of DNA reconfigurable nanostructures, stepped and autonomous nanomachines and circuits have been operated using the strand-displacement reaction. Biological systems rely on information processing to guide their behaviour and functions. Molecular computation is a branch of DNA nanotechnology that aims to construct and operate programmable computing devices made out of DNA that could interact in a biological context. Similar to conventional computers, the computational processes involved are based on Boolean logic, a propositional language that describes statements as being true or false while connecting them with logic operators. Numerous logic gates and circuits have been built with DNA that demonstrate information processing at the molecular level. However, development of new systems is called for in order to perform new tasks of higher computational complexity and enhanced reliability. The contribution of secondary structure to the vulnerability of a toehold-sequestered device to undesired triggering of inputs was examined, giving new approaches for minimizing leakage of DNA devices. This device was then integrated as a logic component in a DNA-based computer with a retrievable memory, thus implementing two essential biological functions in one synthetic device. Additionally, G-quadruplex logic gates were developed that can be switched between two topological states in a logic fashion. Their individual responses were detected simultaneously, establishing a new approach for parallel biological computing. A new AND-NOT logic circuit based on the seesaw mechanism was constructed that, in combination with the already existing AND and OR gates, form a now complete basis set that could perform any Boolean computation. This work introduces a new mode of kinetic control over the operation of such DNA circuits. Finally, the first example of a transmembrane logic gate being operated at the single-molecule level is described. This could be used as a potential platform for biosensing.

Die Herstellung funktionaler und strukturell wohldefinierter Nanostrukturen ist eine Voraussetzung, um hochentwickelte Device im Bereich der Nanoelektronik oder Nanooptik zu entwickeln. Bottom-Up-Verfahren, welche auf biomolekularen Selbstassemblierungsprozessen basieren, haben sich hierfür, aufgrund ihrer hoch parallelen Synthese, als besonders effizient erwiesen. In dieser Arbeit wurde die DNA-Origami-Technik genutzt, um eine funktionale Nanostruktur für elektronische Schaltkreise oder optische Anwendungen zu assemblieren. Planare DNA-Origami-Strukturen können als molekulare Steckbretter dienen, um funktionale Objekte mit Nanometer-Präzision anzuordnen. Diese Arbeit verwendete als solch ein Objekt ein p-konjugiertes Polymer. Im Vergleich zu anorganischen Nanoobjekten, wie metallische Nanopartikel, zeichnen sich diese Polymere durch mechanische Flexibilität und ein leichtes Gewicht aus. Aufgrund ihres p-konjugierten Rückgrates sind diese Polymere optisch und elektronisch funktional. Diese Eigenschaften können über ihre molekulare Struktur eingestellt werden. Dotiert sind diese Polymere Halbleiter oder sogar Leiter. Ihre Funktionalität wurde in diversen optoelektronischen und elektronischen Bauteilen, wie z.B. organischen Feldeffekt-Transistoren, bewiesen. Für Anwendungen auf der Nanoskala sind die Polythiophenderivate des Typ P3RT besonders interessant. Deren Synthese, die Kumada Katalysatorenübertragungspolykondensation, folgt einem kontrollierten Kettenwachstumsmechanismus. Die Polymere zeichnen sich durch eine eng verteilte, einstellbare Molmasse und definierte Endgruppen aus. Im ersten Teil dieser Arbeit wurde das Polythiophenderivat designt und synthetisiert. Eine Oligoethylenglykol-Seitenkette gewährleistet die Löslichkeit in Wasser und Kompatibilität zur DNA. Über einen ex-situ Initiator wurde eine funktionelle Endgruppe eingeführt, um das Polymer zugänglich zur DNA-Origami-Assemblierung zu machen. Mittels verschiedener Charakterisierungen wurden die definierte Struktur und gute Löslichkeit in Wasser demonstriert. Im zweiten Abschnitt dieser Arbeit wurde die elektronische Aktivierung dieses Polythiophens durch molekulares Dotieren auf der Mikroskala untersucht. Der Einfluss des Dotierungmittels 2,3,5,6-Tetrafluoro-7,7,8,8-tetracyanoquinodimethan auf die optischen, morphologischen und elektronischen Eigenschaften des Polymers als dünner Film wurde untersucht, um optimale Dotierungskonditionen zu etablieren. Die dotierten Polymerfilme zeigten eine deutlich verbesserte Leitfähigkeit im Vergleich zu unbehandelten Filmen. Im dritten Abschnitt dieser Arbeit wurde die DNA-Origami-gelenkte Anordnung des Polythiophens untersucht. Hierfür wurde das Polymer zunächst an ein modifiziertes, synthetisches Oligonukleotid igebunden. Das resultierende Blockcopolymer wurde dann ortsspezifisch an DNA- Überhänge angebunden, welche sich eng aufgereiht auf einer planare DNA-Origami-DNA-Origami-Struktur befanden. Die Polymer-DNA-Hybridstrukturen wurden mittels hochauflösender Rasterkraftmikroskopie charakterisiert. Aufgrund von p-p Stapelwechselwirkungen der Polythiophenrückgrate kam es zur Ausbildung supramolekulare Polymerdrähte. Die Abmaße dieser Drähte wurde über der Anordnung der DNA-Überhänge gesteuert. Es wurde gezeigt, dass durch schrittweises Aufbrechen der p-p-Stapelwechselwirkung die Fluoreszenz dieser Polythiophendrähte verändert werden kann. Die Fähigkeit könnte nützlich sein, um die optischen Eigenschaften dieser Drähte für photonische Leitungen einfach auf Sender und/oder Empfänger abzustimmen. Des Weiteren sind diese Wechselwirkungen nötig, um Ladungsträger durch diese Drähte zu transportieren. Mittels Leitfähigkeitsrasterkraftmikroskopie wurden erste Untersuchungen getätigt um die Fähigkeit dieser Polythiophen-DNA-Hybridstrukturen als elektronischer Draht zu evaluieren. Im Rahmen dieser Arbeit konnte kein Ladungstransport festgestellt werden. Zusammenfassend wurde eine neuartige, funktionale Polythiophen-basierende Nanostruktur mittels der DNA-Origami-Technik synthetisiert. Solche Polymer-DNA-Hybridstrukturen versprechen eine vielfältige Anwendbarkeit als optische oder elektronische Bauteile in Schaltkreisen.:Kurzfassung i Acknowledgements iii List of Figures vii List of Tables ix 1 Introduction and Objectives 1 1.1 Introduction 2 1.2 Objectives of the Doctoral Thesis 4 2 Background 5 2.1 p-Conjugated Polymers 6 2.1.1 Fundamentals of Conjugated Polymers 6 2.1.2 Polarons and Molecular p-Doping of Polythiophenes 10 2.1.3 Synthesis of Polythiophene Derivatives 14 2.2 DNA-Based Templates for Confined, Functional Nanostructures 22 2.2.1 Structure and Properties of Deoxyribonucleic Acid 23 2.2.2 Linear, DNA-Templated Confined Nanostructures 25 2.2.3 DNA Origami as Molecular Breadboard 27 2.2.4 DNA Origami-Templated, Confined Nanostructures 30 2.3 Characterization Techniques for Conjugated Polymers and Functional Nanostructures 33 2.3.1 Structural Characterization 33 2.3.2 Spectroscopic Characterization 34 2.3.3 Imaging of Nanostructures 35 2.3.4 Electrical Characterization at the Nanoscale 36 3 Experimental Section 39 3.1 Materials 40 3.2 Synthesis 42 3.2.1 NH 2 -P3(EO) 3 T 42 3.2.2 N 3 -P3(EO) 3 T 44 3.2.3 P3(EO) 3 T-b-ON 44 3.2.4 P3(EO) 3 T@Origami 46 3.3 Methods and Instrumentation 47 4 Results and Discussion 57 4.1 Synthesis and Characterization of the Polythiophene Derivative 58 4.1.1 Introduction 58 4.1.2 Molecular Design of the Customized Polythiophene Derivative 59 4.1.3 Ex-Situ Initiated Kumada Catalyst-Transfer Polycondensation 60 4.1.4 Structural Characterization 62 4.1.5 Optical Characterization66 4.1.6 Summary 69 4.2 Electronic Functionality of P3(EO)3T as 2D Bulk 70 4.2.1 Introduction 70 4.2.2 Solution-Based Doping 70 4.2.3 Charge Transfer Reaction Upon Doping 71 4.2.4 Optical and Vibrational Spectroscopy 73 4.2.5 Microstructure and Morphology 78 4.2.6 Electrical Characterization 82 4.2.7 Summary 85 4.3 DNA Origami-Templated Formation of Polythiophene Filaments 86 4.3.1 Introduction 86 4.3.2 Block Copolymer Formation 87 4.3.3 Planar DNA Origami Template 93 4.3.4 Synthesis of P3(EO)3T@pad Hybrid Structure 96 4.3.5 Tunable Fluorescence of P3(EO)3T@pad Hybrid Structures 100 4.3.6 Potential as Conducting Wire 102 4.3.7 Summary 109 5 Conclusions and Future Perspectives 111 5.1 Conclusions 112 5.2 Future Perspectives 113 Appendix 115 A Supplementary Information 115 B DNA Origami Sequences 123 Abbreviations 131 List of Symbols 133 Bibliography 135 Publications and Conference Contributions 158

This thesis forms part of an ongoing project in the DNA Group to build and operate a synthetic ribosome. We present two synthetic ribosome designs that can be combined with DNA-templated chemistry to generate libraries of functional synthetic small molecules. In Chapter 2 we use the DNA strand displacement technique to construct a mechanism that is capable of moving along a DNA track. We explore ways to control the speed and the driving force of the mechanism, and present a mathematical model of the system. We discuss the ability of the design to incorporate chemically-functionalised DNA strands. In Chapter 3 we use a 2D DNA origami tile as the basis of the synthetic ribosome mechanism. Functionalised DNA strands are arranged on the surface of the tile, and we demonstrate the ability to template reactions between the strands, and discuss the possibility of creating a library of distinct chemical products from a single origami tile.

In the past decade, DNA origami self-assembly has been widely applied for creating customised nanostructures with base-pair precision. In this technique, the unique chemical addressability of DNA can be harnessed to create programmable architectures, using components ranging from dye or protein molecules to metallic nanoparticles. In this thesis, we apply DNA nanotechnology for developing novel light-harvesting and optical voltage sensing nano-devices. We use the programmable positioning of dye molecules on a DNA origami plate as a mimic of a light-harvesting antenna complex required for photosynthesis. Such a structure allows us to systematically analyse optimal design concepts using different dye arrangements. Complementary to this, we use the resistive-pulse sensing technique in a range of electrolytes to characterise the mechanical responses of DNA origami structures to the electric field applied. Based on this knowledge, we assemble voltage responsive DNA origami structures labelled with a FRET pair. These undergo controlled structural changes upon application of an electric field that can be detected through a change in FRET efficiency. Such a DNA-based device could ultimately be used as a sensitive voltage sensor for live-cell imaging of transmembrane potentials.