Dynamic DNA complexes are able to undergo multiple hybridization and dissociation events through a process called strand displacement. This unique property has facilitated the creation of programmable molecular detection systems and chemical logic gates encoded by nucleotide sequence. This work examines whether the ability to selective exchange oligonucleotides among different thermodynamically-stable DNA complexes can be harnessed to create a new class of imaging probes that permit fluorescent reporters to be sequentially activated (“turned on”) and erased (“turned off”). Here, dynamic DNA complexes detect a specific DNA-conjugated antibody and undergo strand displacement to liberate a quencher strand and activate a fluorescent reporter. Subsequently, incubation with an erasing complex allows the fluorophore to be stripped from the target strand, quenched, and washed away. This simple capability therefore allows the same fluorescent dyes to be used multiple times to detect different markers within the same sample via sequential rounds of fluorescence imaging. We evaluated and optimized several DNA complex designs to function efficiently for in situ molecular analyses. We also applied our DNA probes to immunofluorescence imaging using DNA-conjugated antibodies and demonstrated the ability to at least double the number of detectable markers on a single sample. Finally, the probe complexes were reconfigured to act as AND-gates for the detection of co-localized proteins. Given the ability to visualize large numbers of cellular markers using dynamic DNA probe complexes, high-content proteomic analyses can be performed on a single sample, enhancing the power of fluorescence imaging techniques. Furthermore, dynamic DNA complexes offer new avenues to incorporate DNA-based computations and logic for in situ molecular imaging and analyses.

Kinetically controlled isothermal growth is fundamental to biological development, but it remains challenging to rationally design molecular systems that self-assemble isothermally into complex geometries via prescribed assembly and disassembly pathways. By exploiting the programmable chemistry of base pairing, sophisticated spatial and temporal control have both been demonstrated in DNA self-assembly, but largely as separate pursuits. This dissertation extends a new approach, called developmental self-assembly, that integrates temporal with spatial control by using a prescriptive molecular program to specify the kinetic pathways by which DNA molecules isothermally self-assemble into well-defined three-dimensional geometries. / Chemistry and Chemical Biology

<p>Biological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.</p> / Dissertation

DNA nanotechnology uses the Watson-Crick base-pairing of DNA to self-assemble structures at the nanoscale. DNA nanomachines are active structures that take energy from the system to drive a programmed motion. In this thesis, a new design for a reversible DNA motor and an automatically regenerating track is presented. Ensemble fluorescence measurements observe motors walking along the same 42nm track three times. A second new motor was designed to allow motors on intersecting tracks to block each other, which can be used to perform logical computation. Multiple design approaches are discussed. The chosen approach showed limited success during ensemble fluorescence measurements. The 'burnt bridges' motor originally introduced by Bath et al. 2005 was also sent down tracks placed along the inside of stacked origami tubes that are able to polymerise to micrometre lengths. Preliminary optical microscopy experiments show promise in using such a system for observing micrometre motor movement. Scaffold-based DNA origami is the technique of folding a long single-stranded DNA strand into a specific shape by adding small staple strands that hold it in place. Extended staple strands can be modified to functionalise the origami surface. In this thesis, the threading of staple extensions through a freely-floating origami tile was observed using single-molecule Förster resonance energy transfer (smFRET). Threading was reduced by bracing the bottom of the extension or by using a multilayered origami. smFRET was also used to investigate the process of staple repair, whereby a missing staple is added to a pre-formed origami missing the staple. This was found to be successful when the staple is single-stranded, and imperfect when partially double-stranded. Finally the idea for a new "DNA cage", a dynamic octahedron called the "Holliday Octahedron", is presented. The octahedron is made of eight strands, one running around each face. Mobile Holliday junctions at each face allow the stands to rotate causing a conformational change.

abstract: A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology. / Dissertation/Thesis / Ph.D. Chemistry 2011

abstract: Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial catalytic centers. PNA-polypeptide conjugates were synthesized directly using microwave assisted solid phase synthesis or alternatively PNA linkers were conjugated to biologically expressed proteins using chemical crosslinking. The PNA-polypeptides hybridized to the preassembled DNA nanocage at room tempera-ture or 11 &#8304;C and could be assembled in a stepwise fashion. Time resolved fluorescence anisotropy and gel electrophoresis were used to determine that a negatively charged az-urin protein was repelled outside of the negatively charged DNA nanocage, while a posi-tively charged cytochrome c protein was retained inside. Spectroelectrochemistry and an in-gel luminol oxidation assay demonstrated the cytochrome c protein remained active within the DNA nanocage and its redox potential decreased modestly by 10 mV due to the presence of the DNA nanocage. These results demonstrate the benign PNA assembly conditions are ideal for preserving polypeptide structure and function, and will facilitate the polypeptide-based assembly of artificial catalytic centers inside a stable DNA nanocage. A prospective application of assembling multiple cyclic &#947;-PNA-peptides to mimic the oxygen-evolving complex (OEC) catalytic active site from photosystem II (PSII) is described. In this way, the robust catalytic capacity of PSII could be utilized, without suffering the light-induced damage that occurs by the photoreactions within PSII via triplet state formation, which limits the efficiency of natural photosynthesis. There-fore, this strategy has the potential to revolutionize the process of designing and building robust catalysts by leveraging nature's recipes, and also providing a flexible and con-trolled artificial environment that might even improve them further towards commercial viability. / Dissertation/Thesis / Ph.D. Bioengineering 2014

Deoxyribonucleic acid (DNA) is arguably the most studied and most important biological molecule. Recently, it has also been established as a potential candidate for nanoconstruction. Self-assembly of DNA molecules has emerged as a simple yet elegant technique to organize matter with sub-nanometer precision. The unique base-pairing properties which helps DNA to carry genetic information, also makes it a suitable building block for creating stable and robust nanostructures. Recent decades have witnessed a major revolution in the synthesis of different topological structures made of DNA molecules at nanoscale like, two dimensional arrays, nanotubes, polyhedra, smiley faces, three dimensional crystals etc. Due to their easier design, high fidelity and automated chemistry, DNA nanostructures have proposed applications in diverse fields of bio-nanotechnology and synthetic biology. The field of structural DNA nanotechnology is just entering in adulthood and offer paramount challenge towards the journey of DNA-based nanostructures from the laboratory to their practical implementation in the real world. The aim of my dissertation is to develop a de novo computational framework to investigate the nanoscale structure and properties of DNA-based nanostructures. This will help to understand the molecular origin of interaction governing the structure and stability of DNA nanostructures. In this thesis, we have studied the in-solution behavior of self-assembled DNA nanostructures. The state of art all atom molecular dynamics (MD) simulation has been extensively implemented to understand the various thermodynamic properties of these self-assembled soft matter systems. We expect that the results presented here will lead to better design of self-assembled DNA nanostructures to address the real world challenges. In particular, we have developed algorithms to build very accurate atomistic models of various DNA nanostructures like crossover DNA molecules, DNA nanotubes (DNTs) and DNA icosahedron (IDNA). Further, we discuss a computational framework to understand the in situ structure and dynamics of these DNA nanostructures using state-of-art MD simulation. We carried out several hundred nanosecond long MD simulations on these systems which sometimes contains close to one million atoms. Following the trajectories of nanostructures in physiological conditions, we predicted numerous properties like equilibrium solution structure behavior and elastic properties which are difficult to measure in experiments. DNTs are self-assembled tubular templates where the circular double helical domains, kept at the vertices of a polygon, are connected at crossovers junctions. Ned Seeman and co-workers at New York University have synthesized different kind of DNTs using tile-based self-assembly of oligonucleotides. To investigate their microscopic structure, stability and mechanical properties, we have come up with 3d atomistic models of various DNTs which will facilitate further studies of these nanotubes towards their proposed nanotechnological and biological applications. In chapter 3 of this thesis, we discuss the analysis of several nanoseconds long all-atom MD simulation trajectories of various DNTS in the presence of explicit salt solution. We conclude that 6-helix DNT (6HB) structures are most stable and well behaved due to the better crossover designs and geometry. There has been considerable interest to investigate and enhance the mechanical strengths of DNTs to create rigid motifs. One simple way to increase the rigidity is to add further helices to the 6HB, which is known to be the most stable design of DNT, with the same tile-based crossover method. In chapter 4, we report atomistic models of 6HB flanked symmetrically with two double helical DNA pillars (6HB+2) and 6HB flanked symmetrically by three double helical DNA pillars (6HB+3). From the fluctuation analysis of the equilibrium MD simulation trajectories, we calculated the stretch modulus and persistence length of these DNTs. The measured persistence lengths of these nanotubes are ∼10 μm, which is 2 orders of magnitude larger than that of dsDNA. We also find a gradual increase of persistence length with an increasing number of pillars, in quantitative agreement with previous experimental findings. We also carried out non-equilibrium Steered-Molecular-Dynamics (SMD) to measure the stretch modulus from the force-extension behavior of these pillared DNTs. The values of the stretch modulus calculated using contour length distribution of equilibrium MD simulations are similar to those obtained from non-equilibrium SMD simulations. The addition of pillars makes these DNTs very rigid. Engineering the synthetic nanopores through lipid bilayer membrane to access the interior of a cell is a long standing challenge in biotechnology. Recently, a new class of DNA nanopores through the lipid bilayer membranes has been characterized using advanced imaging techniques and transmembrane ionic current recordings. In chapter 5 of the thesis, we present a MD simulation study of 6HB embedded in POPC lipid bilayer membranes. The analyses of 0.2 µs long equilibrium MD simulation trajectories demonstrate that structure is stable and well behaved. We observe that the head groups of the lipid molecules close to DNT cooperatively tilt towards the hydrophilic sugar-phosphate backbone of DNA to form a toroidal structure around the patch of DNT protruding in the membrane. Based on this observation, we propose a new mechanism, which has been largely overlooked so far, to explain the stability to this DNA-lipid molecular self-assembly. We further explore the effect of monovalent ionic concentrations to the in-solution structure and stability of the nanocomposite. Transmembrane ionic current measurements during the constant electric field simulation provide the I-V characteristics of the water filled DNT lumen in lipid membrane. The conductivity of the DNT lumen turns out to be several nS and increases with ionic concentration. Recently, Krishnan’s research group at NCBS Bangalore and Chicago University have characterized DNA icosahedra (IDNA) using advance imaging techniques and validated it for biological targeting and bioimaging in vivo. A high resolution structural model of such polyhedra would be critical to widening their applications in both materials and biology. In chapter 6 of this thesis, we discuss an atomistic model of this well-characterized IDNA to study the in-solution behavior using MD simulation. We provide quantitative estimate of the surface and volume of the equilibrium structure which is essential to estimate its maximal cargo carrying capacity. Importantly, our simulation of gold nanoparticles (AuNP) encapsulated within DNA icosahedra (IAuNP) revealed enhanced stability of the AuNP loaded structure as compared to the empty icosahedra. This is consistent with experimental results that show high yields of cargo-encapsulated DNA icosahedra that have led to its diverse applications for precision targeting. These studies reveal that the stabilizing interactions between the cargo and the DNA scaffold powerfully positions DNA polyhedra as targetable nanocapsules for payload delivery. The insights from our study can be further exploited for precise molecular display for diverse biological applications. Finally, in chapter 7, we give a summary of the main results presented in this dissertation. We also briefly discuss the ongoing research work and the bright future of this emerging field of DNA nanotechnology. We believe that this thesis deepens the microscopic understanding of the recent experimental observation and provides impetus in the real world application of DNA nanostructures in vitro and well as in vivo.

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

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19525号 / 理博第4185号 / 新制||理||1601(附属図書館) / 32561 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19668号 / 薬科博第56号 / 新制||薬科||7(附属図書館) / 32704 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 橋田 充, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM