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Desoxyribonukleinsäure (DNS) ermöglicht die Selbstorganisation von nanoskopischen Elementen zu dreidimensionalen Einheiten mit vorgegebener Form, Zusammensetzung und Größe wie sie in der Nanoelektronik, Nanophotonik und Metamaterialien Verwendung finden. In dieser Arbeit werden DNS Origami Strukturen, in der Gestaltvon Nanoformen, Nanoblätchen und Nanoröhren, als Gerüste für den Aufbau von Nanodrähten und Metall/Halbleiter/Metall Heterostrukturen aus Goldnanoteilchen, Halbleiterquantenpunkten und Halbleiterstäbchen verwendet. Die so hergestellten Einheiten wurden mittels Elektronenstrahllithographie kontaktiert um ihre elektrische Leitwerte zwischen 4:2K und Raumtemperatur zu charakterisieren. Ein neues Konzept für die lösungsbasierte Herstellung von leitenden Goldnanodrähten mittels DNS-Templates wurde eingeführt: hierbei wurden DNS-Nanoformen eingesetzt in denen positionsspezifisch angedockte Goldkeime durch auÿenstromlose Goldabscheidung wachsen. Durch konfigurierbare Verbindungsstellen können sich die einzelnen Formen zu mikrometerlangen Strukturen verbinden. Während der folgendenden Abscheidung von Gold schränken die Wände der Gussformen über das Wachstum so ein, dass sehr homogene Nanodrähte gewonnen werden können. Goldnanodrähte wurden auch C-förmig hergestellt indem Goldnanoteilchen in der gewünschten Form auf DNS Origami-Nanoblättchen angeordnet und wiederum durch außenstromlose Goldabscheidung zu durchgängigen Drähten vergröbert wurden. Einige Abschnitte der DNS-Nanoform-geprägten Drähte zeigen metallische Leitfähigkeit, während andere durch Lücken zwischen den Goldkörnern deutlich höhere Widerstände aufweisen. Alle hergestellten C-förmigen Nanodrähte stellten sich als nicht-metallisch heraus, sie zeigten Eigenschaften von Hopping-, thermionischem und Tunneltransport in Abhängigkeit von der Temperatur. Die Anwesenheit dieser verschiedenen Transportmechanismen deutet darauf hin, dass die C-förmigen Nanodrähte aus metallischen Abschnitten bestehen welche aber nur schwach miteinander verbunden sind. Zwei verschiedene Metall/Halbleiter/Metall-Heterostrukturen wurden hergestellt: Metall/Halbleiternanstäbchen/Metall-Strukturen mittels DNS-Nanoformen und Metall/Quantenpunkt/Metall-Strukturen mittels DNS-Nanoröhren-Vorlagen Goldnanoteilchen konnten durch die DNA templates mit hoher Ausbeute neben den Halbleiterelementen platziert werden. Nach der erfolgter Anordnung wurden die Goldnanoteilchen gewachsen um durchgängige Heterostrukturen zu erhalten. Die Einflüsse des Inkubationsmediums und der -zeit, des Buffers, sowie der Quantenpunkt- und Goldnanopartikelkonzentrationen auf die Abscheidungseffzienz von Goldnanotailchen auf DNS Nanoröhren wurden systematisch untersucht. Zusätzlich zur Bestimmung der Morphologie der durch Selbstorganisation hergestellten Heterostrukturen, wurden auch ihre elektrischen Eigenschaften im Hinblick auf ihre Anwendung in nanelektronischen Bauelementen, wie Einzelelektronentransitoren untersucht.:1. Introduction 2. Overview on DNA Nanotechnology 2.1. Basic Concepts of DNA 2.1.1. Nanoscale Dimensions 2.2. Self-Assembled Architectures from DNA 2.3. DNA Origami: Nanomolds, Nanosheets and Nanotubes 2.3.1. DNA Origami Method 2.3.2. Nanomolds 2.3.3. Nanosheets 2.3.4. Nanotubes 2.4. DNA/DNA Origami-Templated Metallic Nanowire Fabrication 2.4.1. DNA/DNA Origami Templates 2.4.2. Metal Nanoparticle Attachment Yield 2.4.3. Metal Growth 2.5. Electron Transport Mechanisms of DNA-Templated Metallic Nanowires 2.5.1. Lithographically Defined Contacts and I-V Measurements of the DNA-Templated Metal Wires 2.5.2. Lithographically Defined Contacts and I-V Measurements of the DNA Origami-Templated Metal Nanowires 2.6. Applications 2.6.1. Introduction to Metamaterials: DNA-Templated Metamaterial Fabrication 2.6.2. Introduction to Single Electron Tunneling: A DNA-Templated Self-Assembly Concept 3. Experimental Details 3.1. Preparation of Substrates 3.2. DNA Origami Preparation and Deposition 3.2.1. DNA Nanomolds and Formation of linear mold superstructures 3.2.2. DNA Nanotubes 3.2.3. DNA Nanosheets 3.3. Metallization of DNA Origami Structures 3.3.1. DNA Nanomolds 3.3.2. DNA Nanotubes 3.3.3. DNA Nanosheets 3.3.4. Gold Growth on the DNA Origami Nanotube and Nanosheet 3.4. Semiconductor Nanoparticle Preparation and Assembly 3.4.1. CdS Semiconductor Quantum Rods for DNA Nanomold. 3.4.2. CdSe/ZnS Core-shell quantum Dots for DNA Nanotube 3.5. Deposition of DNA origami structures on SiO2 /Si surface 3.5.1. Deposition of DNA Nanomolds 3.5.2. Deposition of DNA Nanosheets and Nanotubes 3.6. Structural Characterization 3.6.1. Atomic Force Microscopy 3.6.2. Scanning Electron Microscopy 3.7. Electrical Characterization 4. Results and Discussion 4.1. DNA Nanomold-Templated Assembly of Conductive Gold Nanowires 4.1.1. Introduction 4.1.2. Results and Discussion 4.1.3. Conclusion 4.2. Conductance measurements on Gold/Semiconductor/Gold heterojunctions templated by DNA Nanomolds 4.2.1. Introduction 4.2.2. Results and Discussion 4.2.3. Conclusion 4.3. C-shaped Gold Nanowires Templated by DNA Nanosheet 4.3.1. Introduction 4.3.2. Results and Discussion 4.3.3. Conclusion 4.4. Self-Assembled Gold/Semiconductor/Gold heterojunctions templated by DNA Nanotube 4.4.1. Introduction 4.4.2. Results and Discussion 4.4.3. Conclusion 5. Conclusion and Future Work A. Supplement for DNA Nanomold-Templated Assembly of Conductive Gold Nanowires B. Conductance measurements on Gold/Semiconductor/Gold heterojunctions templated by DNA Nanomolds C. Supplement for C-shaped Gold Nanowires Templated by DNA Nanosheet D. Supplement for heterojunctions templated by DNA Nanotube / DNA allows self-assembly of nanoscale units into three dimensional nanostructures with definite shape and size in fields such as nanoelectronics, metamaterials and nanophotonics. Different DNA origami templates, such as: nanomold, nanosheet and nanotube templates have been used to assemble gold nanoparticles, quantum dots and semiconductor rods into nanowires and metal/semiconductor/metal heterostructures. Structures have been contacted using electron-beam lithography for electrical conductance characterization at temperatures between 4:2K and room temperature has been performed. A new concept has been introduced for the solution-based fabrication of gold nanowires. To this end, DNA nanomolds have been employed, inside which electroless gold deposition is initiated by site-specifically attached seeds. Using configurable interfaces, individual mold elements self-assemble into micrometer-long mold structures. During subsequent internal gold deposition, the mold walls constrain the metal growth, such that highly homogeneous nanowires are obtained. Gold nanowires have also been manufactured in a C-shape using gold nanoparticles arranged in the desired shape on a DNA origami nanosheet and enhanced to form a continuous wire through electroless gold deposition. Some sections of the DNA nanomold-templated wires show metallic conductance, while other sections of the wires have a much higher resistance which is caused by boundaries between gold grains. All C-shaped wires have been found to be resistive showing hopping, thermionic and tunneling transport characteristics at different temperatures. The different transport mechanisms indicate that the C-shaped nanowires consist of metallic segments which are weakly coupled along the wire. Two types of metal/semiconductor/metal heterostructures have been fabricated: Metal/semiconductor-rod/metal using DNA nanomolds and metal/quantum-dot/metal structures using DNA nanotube. AuNPs were assembled with high yield adjacent to the semiconductor material using origami templates. After the assembly, the gold nanoparticles were grown to produce continuous heterostructures. The influence of the incubation medium, time, buffer, quantum dot and gold nanoparticle concentration on nanoparticle attachment yield was systematically investigated for the nanotube templates. In addition to the determination of the self-assembled heterostructures' morphology, electrical properties were investigated to evaluate their applicability nanoelectronic devices such as single electron transistors.:1. Introduction 2. Overview on DNA Nanotechnology 2.1. Basic Concepts of DNA 2.1.1. Nanoscale Dimensions 2.2. Self-Assembled Architectures from DNA 2.3. DNA Origami: Nanomolds, Nanosheets and Nanotubes 2.3.1. DNA Origami Method 2.3.2. Nanomolds 2.3.3. Nanosheets 2.3.4. Nanotubes 2.4. DNA/DNA Origami-Templated Metallic Nanowire Fabrication 2.4.1. DNA/DNA Origami Templates 2.4.2. Metal Nanoparticle Attachment Yield 2.4.3. Metal Growth 2.5. Electron Transport Mechanisms of DNA-Templated Metallic Nanowires 2.5.1. Lithographically Defined Contacts and I-V Measurements of the DNA-Templated Metal Wires 2.5.2. Lithographically Defined Contacts and I-V Measurements of the DNA Origami-Templated Metal Nanowires 2.6. Applications 2.6.1. Introduction to Metamaterials: DNA-Templated Metamaterial Fabrication 2.6.2. Introduction to Single Electron Tunneling: A DNA-Templated Self-Assembly Concept 3. Experimental Details 3.1. Preparation of Substrates 3.2. DNA Origami Preparation and Deposition 3.2.1. DNA Nanomolds and Formation of linear mold superstructures 3.2.2. DNA Nanotubes 3.2.3. DNA Nanosheets 3.3. Metallization of DNA Origami Structures 3.3.1. DNA Nanomolds 3.3.2. DNA Nanotubes 3.3.3. DNA Nanosheets 3.3.4. Gold Growth on the DNA Origami Nanotube and Nanosheet 3.4. Semiconductor Nanoparticle Preparation and Assembly 3.4.1. CdS Semiconductor Quantum Rods for DNA Nanomold. 3.4.2. CdSe/ZnS Core-shell quantum Dots for DNA Nanotube 3.5. Deposition of DNA origami structures on SiO2 /Si surface 3.5.1. Deposition of DNA Nanomolds 3.5.2. Deposition of DNA Nanosheets and Nanotubes 3.6. Structural Characterization 3.6.1. Atomic Force Microscopy 3.6.2. Scanning Electron Microscopy 3.7. Electrical Characterization 4. Results and Discussion 4.1. DNA Nanomold-Templated Assembly of Conductive Gold Nanowires 4.1.1. Introduction 4.1.2. Results and Discussion 4.1.3. Conclusion 4.2. Conductance measurements on Gold/Semiconductor/Gold heterojunctions templated by DNA Nanomolds 4.2.1. Introduction 4.2.2. Results and Discussion 4.2.3. Conclusion 4.3. C-shaped Gold Nanowires Templated by DNA Nanosheet 4.3.1. Introduction 4.3.2. Results and Discussion 4.3.3. Conclusion 4.4. Self-Assembled Gold/Semiconductor/Gold heterojunctions templated by DNA Nanotube 4.4.1. Introduction 4.4.2. Results and Discussion 4.4.3. Conclusion 5. Conclusion and Future Work A. Supplement for DNA Nanomold-Templated Assembly of Conductive Gold Nanowires B. Conductance measurements on Gold/Semiconductor/Gold heterojunctions templated by DNA Nanomolds C. Supplement for C-shaped Gold Nanowires Templated by DNA Nanosheet D. Supplement for heterojunctions templated by DNA Nanotube

DNA Nanotechnology allows the synthesis of nanometer sized objects that can be site specifically functionalized with a large variety of materials. However, many DNA structures need a higher ionic strength than that in common cell culture buffers or in bodily fluids to maintain their integrity and can be degraded quickly by nucleases. The aim of this dissertation was to overcome this deficiency with the help of cationic PEG-poly-lysine block copolymers that can electrostatically cover the DNA nanostructures to form “DNA origami polyplex micelles” (DOPMs). This straightforward, cost-effective and robust route to protect DNA-based structures could therefore enable applications in biology and nanomedicine, where un-protected DNA origami would be degraded. Moreover, owing to high polarity, the DNA-based structures are restricted to the aque-ous solution based buffers only. Any attempt to change the favorable conditions, leads to the distortion of the structures. In this work it was demonstrated that, by using the polyplex micellization strategy, the organic solubility of DNA origami structures can be improved. The strategy was also extended to functional ligands that are otherwise not soluble in organic solvents. With this strategy, it is now also possible to perform organic solution reactions on the DNA-based structures, opening up the possibility to use hydro-phobic organic reagents to synthesize novel materials. The polyplex micellization strategy therefore presents a cheap, robust, modular, reversible and versatile method to not only solubilize DNA structures in organic solvents but also improve their stability in biological environments. A third project was based on the possibility to synthesize complementary sequences to single-stranded gap regions in the DNA origami scaffold cost-effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. The introduction of flexible gap regions resulted in fully collapsed or partially bent structures due to entropic spring effects. These structures were also used to demonstrate structural transformations with the help of DNA polymerases, expanding the collapsed bent structures to straightened tubes. This approach presents a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified or mechanically switched.:Abstract v Publications vii Acknowledgements ix Contents xiii Chapter 1 Introduction 1 1.1 Nanotechnology 1 1.1.1 History of nanotechnology 1 1.1.2 Phenomena that occur at nanoscale 4 1.1.3 Nature’s perspective of nanotechnology 4 1.1.4 Manufacturing nanomaterials 6 1.2 Deoxyribonucleic acid (DNA) 8 1.2.1 DNA, the genetic material, “The secret of life” 8 1.2.2 Structure of DNA 9 1.2.3 DNA synthesis 15 1.2.4 Stability of DNA 18 1.3 DNA nanotechnology 20 1.3.1 Historical development 20 1.3.2 DNA tile motifs 21 1.3.3 Directed nucleation assembly and algorithmic assembly 23 1.3.4 Scaffolded DNA origami and single-stranded DNA tiles 25 1.3.5 Expanding the design space offered by DNA 27 1.3.6 Assembling heterogeneous materials with DNA 30 1.3.7 Functional devices built using DNA nanostructures 35 Chapter 2 Motivation and objectives 40 Chapter 3 Block copolymer micellization as a protection strategy for DNA origami 42 3.1 Introduction 42 3.1.1 Cellular delivery of DNA nanostructures 42 3.1.2 The need for stability of DNA nanostructures 43 3.1.3 Non-viral gene therapy 44 3.2 Results and discussions 46 3.2.1 Strategy to form DNA origami polyplex micelles (DOPMs) 46 3.2.2 Optimizations 46 3.2.3 Decomplexation 53 3.2.4 Stability tests 55 3.2.5 Short PEG-PLys block copolymer 58 3.2.6 Compatibility with bulky ligands 59 3.2.7 Accessibility of handles on DOPMs 63 3.3 Conclusion 64 3.4 Outlook and state of the art 65 3.5 Methods 67 3.5.1 DNA origami folding 67 3.5.2 Preparation of ssDNA functionalized AuNPs 68 3.5.3 Agarose gel electrophoresis 69 3.5.4 Block copolymer preparation 70 3.5.5 DNA origami polyplex micelle preparation 70 3.5.6 Decomplexation of DOPM using dextran sulfate 73 3.5.7 Stability tests 74 3.5.8 tSEM characterization 75 3.5.9 AFM imaging 76 Chapter 4 Improving organic solubility and stability of DNA origami using polyplex micellization 77 4.1 Introduction 77 4.2 Results and discussions 79 4.2.1 Strategy for organic solubility of DNA origami 79 4.2.2 Proof of concept using AuNPs functionalized with ssDNA 80 4.2.3 Extending the strategy to DNA origami 82 4.2.4 Optimizations 86 4.2.5 Compatibility with functional ligands 88 4.2.6 Functionalization of DNA origami in organic solvent 94 4.3 Conclusion and outlook 95 4.4 Methods 97 4.4.1 Conjugation of functional ligands to DNA origami 97 4.4.2 Organic solubility 98 4.4.3 Reactions in organic solution on DOPMs 99 4.4.4 Fluorescence imaging using gel scanner 100 Chapter 5 Structural transformation of wireframe DNA origami via DNA polymerase assisted gap-filling 101 5.1 Introduction 101 5.2 Results and discussion 102 5.2.1 Design of the structures 102 5.2.2 Folding of gap-structures 105 5.2.3 Single-stranded DNA binding proteins 107 5.2.4 Gap filling with different polymerases 109 5.2.5 Gap filling with Phusion high-fidelity DNA polymerase 111 5.2.6 Optimization of the extension reaction using T4 DNA polymerase 115 5.2.7 Secondary structures 121 5.2.8 Folding kinetics of gap origami 124 5.2.9 Bending of tubes 125 5.3 Conclusion 126 5.4 Outlook 127 5.5 Methods 128 5.5.1 DNA origami folding 128 5.5.2 Gap filling of the wireframe DNA origami structures 128 5.5.3 Agarose gel electrophoresis 130 5.5.4 PAGE gel analysis 130 5.5.5 tSEM characterization 131 5.5.6 AFM imaging 131 5.5.7 AGE based folding-yield estimation 132 5.5.8 Gibbs free energy simulation using mfold 132 5.5.9 Staple list for folding the DNA origami triangulated structures 132 Appendix 134 A.1 Additional figures from chapter 3 134 A.2 Additional figures from chapter 4 137 A.3 Additional figures from chapter 5 149 Bibliography 155 Erklärung 171

Die rasant fortschreitende Digitalisierung in allen Bereichen des täglichen Lebens erzeugt einen immensen Bedarf an Mikrochips. Um diese Mikrochips zu erzeugen, durchlaufen Wafer eine Vielzahl an ressourcenaufwändigen Prozessschritten. In dieser Arbeit werden zwei Ansätze der reversiblen Immobilisierung von Biopolymeren verfolgt, welche als Grundlage für eine alternative Herstellung von Mikrochips fungieren könnten. Zum einen sollte die reversible Immobilisierung von Polyplexen auf Basis von 6HB DNA-Origami und kationischen Polymethacrylaten bzw. Poly(2-oxazolin)en auf strukturierten PDMAEMA-Bürsten nach Nawroth et al.[1],[2] realisiert werden. Zum anderen war die reversible Immobilisierung von Mikrotubuli nach Ionov et al.[3] unter Verwendung eines grafting-from Systems Ziel dieser Arbeit. Für die Immobilisierung von Polyplexen auf Polymerbürsten wurden die Polymere so gestaltet, dass sie mehrere Funktionen erfüllen können. Die Copolymere verfügten über einen kationischen Bereich mit 20 Wiederholeinheiten, welcher zur Anbindung an die 6HB diente, einen hydrophilen Spacer sowie mehrere Funktionalisierungsstellen. Das zuerst untersuchte methacrylische System auf Basis von HEMA stellte sich für den hier vorgesehenen Verwendungszweck als ungeeignet heraus. Für das Poly(2-oxazolin)-System wurden die nach Cesana et al.[4] und Hartlieb et al.[5] synthetisierten aminfunktionalisierten Monomere AmProOx, AmBuOx, AmPentOx und AmDecOx, sowie das nach He et al.[6] synthetisierte MAMeOx verwendet. Die Synthese der verwendeten Monomere konnte optimiert bzw. AmProOx und AmDecOx im Zuge dieser Arbeit erstmalig synthetisiert werden. AmProOx, AmBuOx und AmPentOx konnten mittels LCROP erstmalig in dieser Arbeit mit Benzyltosylat direkt initiiert und homopolymerisiert werden. Diese AmOx-Derivate erlauben als Blockcopolymer mit MeOx eine effektive und gut kontrollierbare Polyplexbildung mit 6HB. Dies wurde mittels GEP und AFM verifiziert. Es zeigte sich, dass MAMeOx ungeeignet war, da es keinen Polyplex mit 6HB bildete. Als mögliche Ursachen sind der zu geringe Abstand zwischen den Aminen in der Seitenkette sowie der zu geringe Abstand zwischen dem Amin in der Seitenkette und dem Rückgrat zu nennen. Die AmOx konnten in einem Triblockcopolymer zusammen mit MeOx bzw. EtOx und PynOx copolymerisiert werden. Über den PynOx-Block ist eine Funktionalisierung mittels Click Chemie möglich.[7] Es konnte gezeigt werden, dass die Polyplexe in ihrer Peripherie vollumfänglich durch die Polymere zusätzlich funktionalisiert werden können. So wurden Fluoreszenzfarbstoffe ebenso wie CRP Initiatoren eingeführt. Die angeschlossene Pfropfungspolymerisation auf Basis der CuCRP zeigte, im Vergleich zur Literatur,8 einen bis zum Zeitpunkt der Erstellung dieser Arbeit unbekannten Zuwachs an Polymerhülle von bis zu 70 nm (240%) innerhalb von 15 min. Eine Immobilisierung der 6HB-POx Polyplexe nach Nawroth et al.[1],[2] auf PDMAEMA-Bürsten zeigte sich als nicht erfolgreich. Es konnte auf den nanostrukturierten PDMAEMA-Bürsten keine Abscheidung der Polyplexe beobachtet werden. In Zukunft könnten die entwickelten Polyplexe durch eine Funktionalisierung der PynOx Einheiten mit Metallisierungskeimen als Nanodrahttemplate verwendet werden. Außerdem ist die Nutzung der POx-DNA-Origami- Polyplexe als formstabile Wirkstofftransportsysteme denkbar, ebenso wie die Verwendung der kationischen POx als Ersatz für Polyethylenimin (PEI)[9]–[11] in der Gentherapie. Für die Immobilisierung von Mikrotubuli Gleit-Assays mit gepfropften PNIPAM-Bürsten auf Grundlage der Publikation von Ionov et al.[3] wurde erstmalig die SI-CuCRP mit PNIPAM auf Gold untersucht. Hierfür wurde zuerst eine geeignete Initiator-SAM ermittelt, wobei die Wahl auf BiB UD-SH fiel, welche eine Schichtdicke von ca. 1,2 nm bei einem Wasserkontaktwinkel von ca. 72° aufwies. Um die ideale PNIPAM Schichtdicke für die Immobilisierung zu ermitteln, wurden mehrere Gradienten mit unterschiedlichen Schichtdicken hergestellt. Als ideale Schichtdicke erwies sich eine Höhe von 10 bis 20 nm PNIPAM im kollabierten Zustand. Die generierten PNIPAM Schichten auf Gold zeigten in den Gleit-Assays ein effektives Verhalten bezüglich der Immobilisierung der Mikrotubuli. Dies zeigte sich dadurch, dass im gequollenen Zustand unterhalb der LCST der PNIPAM-Bürsten die Mikrotubuli von der Oberfläche gelöst wurden. Oberhalb der LCST konnten sich die Mikrotubuli frei auf der Oberfläche bewegen. Gleiches konnte auf im Anschluss präparierten UV-strukturierten Proben nachvollzogen werden. Die Mikrotubuli waren nicht in der Lage, sich unterhalb der LCST auf den PNIPAM-Strukturen zu bewegen, jedoch auf Bereichen ohne PNIPAM. Oberhalb der LCST war es den Mikrotubuli wieder möglich, sich frei zu bewegen. Das entwickelte System für die reversible Immobilisierung von Mikrotubuli könnte im nächsten Schritt auf die von Nicolau et al.[12] entwickelten Arrays übertragen werden. [1] Nawroth, J. F.; Neisser, C.; Erbe, A.; Jordan, R. Nanoscale 2016, 8 (14), 7513–7522. [2] Nawroth, J. F. Synthese nanostrukturierter Polymerbürsten zur reversiblen Immobilisierung von DNA Origami, Dissertation, TU Dresden, 2017. [3] Ionov, L.; Stamm, M.; Diez, S. Nano Lett. 2006, 6 (9), 1982–1987. [4] Cesana, S.; Auernheimer, J.; Jordan, R.; Kessler, H.; Nuyken, O. Macromol. Chem. Phys. 2006, 207 (2), 183–192. [5] Hartlieb, M.; Pretzel, D.; Kempe, K.; Fritzsche, C.; Paulus, R. M.; Gottschaldt, M.; Schubert, U. S. Soft Matter 2013, 9 (18), 4693–4704. [6] He, Z.; Miao, L.; Jordan, R.; S-Manickam, D.; Luxenhofer, R.; Kabanov, A. V. Macromol. Biosci. 2015, 15 (7), 1004–1020. [7] Luxenhofer, R.; Jordan, R. Macromolecules 2006, 39 (10), 3509–3516. [8] Tokura, Y.; Jiang, Y.; Welle, A.; Stenzel, M. H.; Krzemien, K. M.; Michaelis, J.; Berger, R.; Barner-Kowollik, C.; Wu, Y.; Weil, T. Angew. Chemie - Int. Ed. 2016, 55 (19), 5692–5697. 9Boussif, O.; LezoualC’H, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U. S. A. 1995, 92 (16), 7297–7301. [10] Horbinski, C.; Stachowiak, M. K.; Higgins, D.; Finnegan, S. G. BMC Neurosci. 2001, 2. [11] Dodds, E.; Piper, T. A.; Murphy, S. J.; Dickson, G. J. Neurochem. 1999, 72 (5), 2105–2112. [12] Nicolau, D. V; Lard, M.; Korten, T.; van Delft, F. C. M. J. M.; Persson, M.; Bengtsson, E.; Månsson, A.; Diez, S.; Linke, H.; Nicolau, D. V. Proc. Natl. Acad. Sci. 2016, 113 (10), 2591–2596.:Inhaltsverzeichnis Danksagung I Abkürzungsverzeichnis III 1. Einleitung 1 2. Grundlagen 3 2.1. DNA-Origami 3 2.2. Mikrotubuli als Nanoroboter 9 2.3. Poly(2-oxazolin)e 14 2.3.1. 2-Substituierte 2-Oxazoline 14 2.3.2. Lebende kationische ringöffnende Polymerisation 15 2.4. Oberflächenmodifikation mit Polymerbürsten 18 2.4.1. Selbstorganisierende Monoschichten 18 2.4.2. Polymerbürsten 19 2.4.3. Pfropfungspolymerisatonsarten 22 2.4.5. Thermoresponsive Polymerbürsten 27 2.5. Rasterkraftmikroskopie 29 3. Motivation 31 4. Ergebnisse und Diskussion 33 4.1. Kationische Bürsten auf Polymethacrylatbasis 33 4.1.1. Synthese der kationischen Copolymere 34 4.1.2. Funktionalisierung mit BiBB 38 4.1.3. Pfropfungspolymerisation 40 4.1.4. Polyplexbildung mit kationischen Polymethacrylaten 43 4.2. Synthese von 2-Oxazolinen mit Aminofunktionalität 45 4.3. Kationische Poly(2-oxazolin)e zur DNA-Origami-Komplexierung 50 4.3.1. Kinetik der Homopolymerisation der AmOx-Monomere 50 4.3.2. Erprobung der Click-Reaktion und der Pfropfung 53 4.3.3. Poly(2-oxazolin)e mit Aminfunktionalität zur Stabilisierung von DNA-Origami 59 4.3.4. Poly(2-oxazolin)e mit BiB-Typ Endfunktionalisierung 65 4.3.5. Kationische Poly(2-oxazolin)e für die Funktionalisierung von Polyplexen 69 4.3.6. Pfropfungspolymerisation auf immobilisierten Polyplexen 72 4.3.7. Pfropfungspolymerisation auf DNA-Origami in Lösung 74 4.3.8. Polyplex-gestütztes, gerichtetes Assemblieren von DNA-Origami 77 4.4. Reversible Immobilisierung von Mikrotubuli durch Polymerbürsten 81 4.4.1. Oberflächenmodifizierung 81 4.4.2. Mikrotubuli-Gleit-Assays auf PNIPAM-Gradienten 84 4.4.3. Mikrotubuli-Gleit-Assays auf strukturierten PNIPAM-Bürsten 89 5. Zusammenfassung und Ausblick 92 6. Experimentalteil 96 6.1. Geräte 96 6.2. Verbrauchsgüter 99 6.2.1. Chemikalien 99 6.2.2. 6-Helixbündel 6HB 99 6.2.3. Wafer 99 6.3. Synthese der Initiatoren 100 6.3.1. Benzyltosylat BnOTs 100 7.3.2. 2-Azidoethyl-2-bromoisobutyrat 100 6.4. Synthese der Monomere 103 6.4.1. Allgemeine Synthesevorschrift der AmOx Monomere 103 6.4.2. 2-(N-Methyl)aminomethyl-2-oxazolin MAMeOx 106 6.5. Polymersynthese 108 6.5.2. Homopolymerisation der AmOx Monomere 113 6.5.3. P(MeOx-grad-PynOx) P1 115 6.5.4. P(MAMeOx-co-MeOx-grad-PynOx) P2Boc 115 6.5.5. Synthese der P(AmOx-co-MeOx) 116 6.5.6. Allgemeine Synthesevorschrift der LCROP mit BiB-OH Terminierung 118 6.5.7. Allgemeine Synthesevorschrift der CuCRP mit einem POx-Makroinitiator 122 6.5.8. P(AmProOx-co-MeOx-grad-PynOx) P6Boc 126 6.5.10. P(AmPentOx-co-EtOx-grad-PynOx) P8Boc 128 6.6. Polymeranaloge Funktionalisierung 129 6.6.1. Allgemeine Bedingung der Funktionalisierung der HEMA-Gruppen mit BiBB 129 6.6.2. Pfropfungspolymerisation an PMETAC10-BiB mit HEMA 132 6.6.3. Allgemeine Vorschrift der Abspaltung der Boc-Schutzgruppe 133 6.6.4. Allgemeine Bedingungen der Huisgen 1,3-dipolaren kupferkatalysierten Azid-Alkin-Cycloaddition 137 6.6.5. Pfropfungspolymerisation an P1-BiB 141 6.6.6. Pfropfungspolymerisation an P2Boc-BiB 142 6.7. DNA-Origami-Polyplexe 143 6.7.1. Polyplexbildung 143 6.7.2. Präparation für AFM-Messung 143 6.7.3. Oberflächenbasierte Pfropfungspolymerisation 144 6.7.4. Lösungsbasierte Pfropfungspolymerisation 144 6.7.5. Polyplexabscheidung auf nanostrukturierten PDMAEMA Bürsten 144 6.8. Oberflächenmodifikation 145 6.8.1. Präparation der SiO2-Wafer mit PF-SAM 145 6.8.2. Selbst-initiierende Photopfropfung und Photopolymerisation SIPGP 145 6.8.3. Präparation Au-Wafer mit Thiol-SAM 145 6.8.4. UV-Lithografie 146 6.8.5. SI-CuCRP 146 6.9. Gleit-Assays 147 7. Quellen 149

DNA nanotechnology has developed a versatile set of methods to utilise DNA self-assembly for the bottom-up construction of arbitrary two- and three-dimensional DNA objects in the nanometre size range, and to functionalise the structures with unprecedented site-specificity with nanoscale objects such as metallic and semiconductor nanoparticles, proteins, fluorescent dyes, or synthetic polymers. The advances in structure assembly have resulted in the application of functional DNA-based nanostructures in a gamut of fields from nanoelectronic circuitry, nanophotonics, sensing, drug delivery, to the use as host structure or calibration standard for different types of microscopy. However, the analytical means for characterising DNA-based nanostructures drag behind these advances. Open questions remain, amongst others in quantitative single-structure evaluation. While techniques such as atomic force microscopy (AFM) or transmission electron microscopy (TEM) offer feature resolution in the range of few nanometres, the number of evaluated structures is often limited by the time-consuming manual data analysis. This thesis has introduced two new approaches to quantitative structure evaluation using AFM and super-resolution fluorescence microscopy (SRM). To obtain quantitative data, semi-automated computational image analysis routines were tailored in both approaches. AFM was used to quantify the attachment yield and placement accuracy of poly(3-tri(ethylene glycol)thiophene)-b-oligodeoxynucleotide diblock copolymers on a rectangular DNA origami. This work has also introduced the first hybrid of DNA origami and a conjugated polymer that uses a highly defined polythiophene derivative synthesised via state-of-the-art Kumada catalyst-transfer polycondensation. Among the AFM-based studies on polymer-origami-hybrids, this was the first to attempt near-single molecule resolution, and the first to introduce computational image analysis. Using the FindFoci tool of the software ImageJ revealed attachment yields per handle between 26 - 33%, and determined a single block copolymer position with a precision of 80 - 90%. The analysis has pointed out parameters that potentially influence the attachment yield such as the handle density and already attached objects. Furthermore, it has suggested interactions between the attached polymer molecules. The multicolour SRM approach used the principles of single-molecule high-resolution co-localisation (SHREC) to evaluate the structural integrity and the deposition side of the DNA origami frame “tPad” based on target distances and angles in a chiral fluorophore pattern the tPads were labelled with. The computatinal routine that was developed for image analysis utilised clustering to identify the patterns in a sample’s signals and to determine their characteristic distances and angles for hundreds of tPads simultaneously. The method excluded noise robustly, and depicted the moderate proportion of intact tPads in the samples correctly. With a registration error in the range of 10 -15 nm after mapping of the colour channels, the precision of a single distance measurements on the origami appeared in the range of 20 - 30 nm. By broadening the scope of computational AFM image analysis and taking on a new SRM approach for structure analysis, this work has presented working approaches towards new tools for quantitative analysis in DNA nanotechnology. Furthermore, the work has presented a new approach to constructing hybrid structures from DNA origami and conjugated polymers, which will open up new possibilities in the construction of nanoelectronic and nanophotonic structures.

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In dieser Arbeit werden systematisch die bisher unerforschten grundlegenden physikalischen und chemischen Eigenschaften von DNA-Origami untersucht, die die Stabilität dieser aus doppelsträngiger DNA aufgebauten nanoskopischen Suprastrukturen bestimmen. In Analogie zu den zahlreichen Studien, die sich mit der Stabilität von Proteinen durch kontrollierte Denaturierung beschäftigen, spielen auch in dieser Arbeit die Denaturierungsbedingungen eine zentrale Rolle. Unter Verwendung von Guanidinium (Gdm+) als teilweise DNA-stabilisierendes, aber auch potentiell denaturierendes Kation steht dessen Wirkung auf DNA-Origami-Dreiecke im Mittelpunkt der Untersuchungen, wobei insbesondere die unerwartete Modulation der nanoskopischen Schädigung von DNA-Origami durch die begleitenden Gegenanionen zu Gdm+ im Vordergrund steht. Die Experimente zielen darauf ab, atomistische, molekulare, nanoskopische und thermodynamische Eigenschaften von DNA-Origami zu korrelieren und zu klären, wie diese vom Design des DNA-Origami selbst abhängen können. Die Ergebnisse zeigen einen unerwarteten Zusammenhang zwischen den spezifischen Gegenanionen des Denaturierungsmittels und der Stabilität der DNA-Origami-Dreiecke: Sulfat wirkt stabilisierend, während Chlorid die Superstruktur bereits unterhalb der globalen Schmelztemperatur destabilisiert. Statistische Analysen von Rasterkraftmikroskop (AFM)-Bildern und Zirkulardichroismus (CD)-Spektren zeigen Strukturübergänge auf nano-skopischer bzw. molekularer Ebene. Werden diese Techniken mit thermischer Denaturierung in Gegenwart von schwacher bis starker chemischer Denaturierung kombiniert, so zeigt sich, dass Änderungen der Wärmekapazität (ΔCp) während der strukturellen Veränderungen der DNA-Originale eine Schlüsselrolle bei der Bestimmung ihrer Empfindlichkeit gegenüber Temperatur und Denaturierungsmitteln spielen. Die Daten deuten darauf hin, dass Wasser auf apolaren DNA-Origami-Oberflächen der molekulare Ursprung der abgeleiteten Wärme-kapazitätsänderungen ist. Diese Hypothese wird durch Molekulardynamik-Simulationen (MD) unterstützt, die die Modulation von ΔCp durch die Hydratationshüllen der Anionen zeigen. Ihr unterschiedliches Potential, stabile Ionenpaare mit Gdm+ in konzentrierten Salzlösungen zu bilden, kann die experimentell beobachteten Variationen der strukturellen Stabilität erklären. Die Kopplung von strukturellen Übergängen an ΔCp wird somit als Schlüsselfaktor für die Destabilisierung von DNA-Origami sowohl bei höheren als auch bei niedrigeren Temperaturen identifiziert. Darüber hinaus weisen DNA-Origami nicht nur diese Eigenschaft auf, sondern ermöglichen auch die Beobachtung von kalten Denaturierungsprozessen auf nanoskopischer Ebene, bei denen kälteinduzierte Spannungen innerhalb der Superstruktur bei einem Bruch an vorherbestimmten lokalen Stellen freigesetzt werden, die in AFM-Bildern sichtbar sind. Dies ist die erste Beobachtung der kälteinduzierten Denaturierung von Nukleinsäuren bei Temperaturen über 0 °C sowie von DNA-basierten Superstrukturen. In dieser Arbeit wird die strukturelle Stabilität von sechs verschiedenen 2D- und 3D-DNA-Origami-Nanostrukturen in unterschiedlichen chemischen Umgebungen untersucht. Drei chaotrope Salze - Guanidiniumsulfat (Gdm2SO4), Guanidiniumchlorid (GdmCl) und Tetrapropylammoniumchlorid (TPACl) - werden als Denaturierungsmittel verwendet. Mittels Rasterkraftmikroskopie wird die Integrität der Nanostrukturen quantifiziert, wobei sich Gdm2SO4 als das schwächste und TPACl als das stärkste Denaturierungsmittel für DNA-Origami erweist, was sich auch in den Schmelztemperaturen widerspiegelt. Die Abhängigkeit der DNA-Origami-Stabilität von der Superstruktur wird besonders bei 3D-Nanostrukturen deutlich. Hier zeigen mechanisch flexible Designs sowohl in GdmCl als auch in TPACl eine höhere Stabilität als ihre starren Gegenstücke. Die Abhängigkeit der DNA-Origami-Stabilität von der Superstruktur wird besonders in 3D-Nanostrukturen deutlich, in denen mechanisch flexible Strukturen sowohl in GdmCl als auch in TPACl eine höhere Stabilität aufweisen als ihre steifen Gegenstücke. Dies begünstigt die Bildung von intramolekularen Verformungen, die sich entweder in 'weichen' Architekturen über die gesamte Superstruktur verteilen oder in ansonsten 'steifen' Strukturen in den weniger stabilen Regionen konzentrieren.:Table of contents Questions addressed in this thesis .................................................................................... I Abstract ................................................................................................................................ I Englisch .................................................................................................................................................. I Deutsch .................................................................................................................................................. II Acronyms ........................................................................................................................... III Substances .......................................................................................................................................... IV Physical and Chemical abbreviations ............................................................................................... IV Mathematical abbreviations ................................................................................................................ V 1 Introduction ................................................................................................................. 1 1.1 Deoxyribonucleic acid ................................................................................................................. 1 1.1.1 The structure of DNA ............................................................................................................ 1 1.1.2 Hydrogen bonds .................................................................................................................... 1 1.1.3 Base stacking ........................................................................................................................ 2 1.1.4 Water DNA interactions: A complex dance of stability and dynamics .................................. 5 1.1.5 The effect of ionic strength on DNA conformation ................................................................ 9 1.1.6 Conformational changes ....................................................................................................... 9 1.1.7 Forms of DNA ..................................................................................................................... 10 1.1.8 The role of apolar groups in DNA unfolding ........................................................................ 13 1.1.9 Energetics of DNA structural transitions ............................................................................. 14 1.1.10 Melting temperature ............................................................................................................ 15 1.2 Hofmeister series ....................................................................................................................... 17 1.2.1 Probing the Hofmeister series: Salt effects biomolecules ................................................... 17 1.2.2 Specific ion effects in electrolyte solutions .......................................................................... 19 1.3 DNA nanostructures................................................................................................................... 20 1.3.1 DNA origami ........................................................................................................................ 22 1.3.2 Challenges in DNA origami stability .................................................................................... 26 1.3.3 DNA origami in single molecule studies .............................................................................. 27 1.4 Circular dichroism ...................................................................................................................... 28 1.4.1 Circular dichroism spectroscopy for analyzing DNA conformations ................................... 30 1.4.2 Wavelength-dependent spectroscopic signatures of DNA conformation ........................... 32 1.5 Atomic force microscopy .......................................................................................................... 34 1.6 2D correlation spectroscopy ..................................................................................................... 36 1.6.1 2D correlation spectroscopy: Synchronous and asynchronous spectra analysis ............... 39 1.6.2 Perturbation-correlation moving-window 2D correlation spectroscopy ............................... 40 1.7 Multivariate analysis of spectral data using PCA and ITTFA ................................................ 41 1.8 Cold denaturation ....................................................................................................................... 42 2 Results and Discussion ............................................................................................ 44 2.1 Cold denaturation of the Rothemund DNA origami triangle .................................................. 45 2.2 Heat denaturation of the Rothemund DNA origami triangle .................................................. 50 2.2.1 Investigations by atomic force microscopy ......................................................................... 51 2.2.2 Circular dichroism spectroscopy and thermodynamic modelling ........................................ 56 2.2.3 Divergent effects of Cl- and SO42- on DNA origami stability ................................................ 62 2.3 Magnesium concentration modulation of DNA Origami heat denaturation ......................... 65 2.4 Assessing DNA origami stability in different chaotropic environments .............................. 66 2.4.1 DNA origami integrity influenced by Gdm2SO4 ................................................................... 67 2.4.2 DNA origami integrity influenced by GdmCl ........................................................................ 73 2.4.3 DNA origami integrity influenced by TPACl ........................................................................ 75 2.4.4 Quantitative comparison ..................................................................................................... 77 3 Critics ......................................................................................................................... 78 4 Conclusion ................................................................................................................. 79 5 Outlook ...................................................................................................................... 81 6 Material and Methods ................................................................................................ 82 6.1 DNA origami synthesis .............................................................................................................. 82 6.2 Sample preparation and AFM imaging ..................................................................................... 82 6.2.1 Anion-specific structure and stability of guanidinium-bound DNA origami & Cold denaturation of DNA origami nanostructures ...................................................................... 82 6.2.2 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 83 6.2.3 Cold denaturation of DNA origami nanostructures ............................................................. 83 6.3 CD spectroscopy and analysis ................................................................................................. 84 6.3.1 Anion-specific structure and stability of guanidinium-bound DNA origami ......................... 84 6.3.2 Pre-treatment of the CD data and calculation of melting temperatures .............................. 84 6.3.3 Cold denaturation of DNA origami nanostructures ............................................................. 84 6.3.4 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 84 6.4 Principal component analysis and iterative target test factor analysis ............................... 85 6.5 Thermodynamic modelling ........................................................................................................ 85 6.6 Molecular dynamics modelling ................................................................................................. 85 Appendix ........................................................................................................................... 88 Acknowledgment ............................................................................................................ 100 Bibliography .................................................................................................................... 101 List of Figures ................................................................................................................. 116 List of Tables ................................................................................................................... 118 Declaration of independence – Selbstständigkeitserklärung ...................................... 119 / This thesis undertakes the systematic study of hitherto unexplored fundamental physical and chemical properties of DNA origami that determine the stability of these designed nanoscopic superstructural assemblies of double-stranded DNA. In analogy to the vast number of studies addressing protein stability by controlled denaturation, denaturing conditions play a central role in this thesis as well. Using guanidinium (Gdm+) as a partly DNA-stabilizing but also potentially denaturing cation, its effect on DNA origami triangles is central to the study which particularly addressed the unexpected modulation of nanoscopic damage of DNA origami by the accompanying counter-anions to Gdm+. The experiments aim at correlating atomistic, molecular, nanoscopic and thermodynamic properties of DNA origami and at elucidating how these may depend on the DNA origami design itself. The results demonstrate an unexpected relationship between the specific counter-anions of the denaturant and the stability of DNA origami triangles: sulphate exhibits stabilizing effects and chloride induces destabilization of the superstructure already below the global melting temperature. Statistical analyses of both atomic force microscopy (AFM) images and circular dichroism (CD) spectra reveal structural transitions at the nanoscopic and molecular level, respectively. Combining these techniques with thermal denaturation in the presence of mild to strong chemical denaturation, changes in heat capacity (ΔCp) during DNA origami structural changes are shown to play the key role in determining their sensitivity to temperature and denaturants. The data suggest that water at apolar DNA origami surfaces is the molecular origin of the derived heat capacity changes. This hypothesis is substantiated by Molecular Dynamics (MD) simulations which shed light on the modulation of ΔCp by the hydration shells of anions. Their different potential to form stable ion pairs with Gdm+ in concentrated salt solutions can explain the experimentally observed variations of structural stability. The coupling of structural transitions to ΔCp is thus identified as a key factor in the destabilization of DNA origami at both elevated and lowered temperatures. Furthermore, DNA origami not only exhibit this property, but also enable the observation of cold denaturation processes at the nanoscopic level, where cold-induced strain within the superstructure is released upon breakage at predisposed local sites, visible in AFM images. This is the first observation of cold-induced denaturation of nucleic acids at temperatures above 0 °C, as well of DNA-based superstructures. Extending the scope, the work evaluates the structural stability of six different 2D and 3D DNA origami nanostructures in different chemical environments. Three chaotropic salts - guanidinium sulfate (Gdm2SO4), guanidinium chloride (GdmCl), and tetrapropylammonium chloride (TPACl) - are used as denaturants. Atomic force microscopy quantifies the nanostructural integrity, revealing Gdm2SO4 as the weakest and TPACl as the strongest DNA origami denaturant, which is also reflected in the melting temperatures. The dependence of DNA origami stability on its superstructure is particularly evident in 3D nanostructures, where mechanically flexible designs exhibit higher stability in both GdmCl and TPACl than rigid counterparts. This supports the buildup of intramolecular strain, which becomes either partitioned among the entire superstructure in “soft” architectures or accumulates at the least stable regions in otherwise “rigid” designs.:Table of contents Questions addressed in this thesis .................................................................................... I Abstract ................................................................................................................................ I Englisch .................................................................................................................................................. I Deutsch .................................................................................................................................................. II Acronyms ........................................................................................................................... III Substances .......................................................................................................................................... IV Physical and Chemical abbreviations ............................................................................................... IV Mathematical abbreviations ................................................................................................................ V 1 Introduction ................................................................................................................. 1 1.1 Deoxyribonucleic acid ................................................................................................................. 1 1.1.1 The structure of DNA ............................................................................................................ 1 1.1.2 Hydrogen bonds .................................................................................................................... 1 1.1.3 Base stacking ........................................................................................................................ 2 1.1.4 Water DNA interactions: A complex dance of stability and dynamics .................................. 5 1.1.5 The effect of ionic strength on DNA conformation ................................................................ 9 1.1.6 Conformational changes ....................................................................................................... 9 1.1.7 Forms of DNA ..................................................................................................................... 10 1.1.8 The role of apolar groups in DNA unfolding ........................................................................ 13 1.1.9 Energetics of DNA structural transitions ............................................................................. 14 1.1.10 Melting temperature ............................................................................................................ 15 1.2 Hofmeister series ....................................................................................................................... 17 1.2.1 Probing the Hofmeister series: Salt effects biomolecules ................................................... 17 1.2.2 Specific ion effects in electrolyte solutions .......................................................................... 19 1.3 DNA nanostructures................................................................................................................... 20 1.3.1 DNA origami ........................................................................................................................ 22 1.3.2 Challenges in DNA origami stability .................................................................................... 26 1.3.3 DNA origami in single molecule studies .............................................................................. 27 1.4 Circular dichroism ...................................................................................................................... 28 1.4.1 Circular dichroism spectroscopy for analyzing DNA conformations ................................... 30 1.4.2 Wavelength-dependent spectroscopic signatures of DNA conformation ........................... 32 1.5 Atomic force microscopy .......................................................................................................... 34 1.6 2D correlation spectroscopy ..................................................................................................... 36 1.6.1 2D correlation spectroscopy: Synchronous and asynchronous spectra analysis ............... 39 1.6.2 Perturbation-correlation moving-window 2D correlation spectroscopy ............................... 40 1.7 Multivariate analysis of spectral data using PCA and ITTFA ................................................ 41 1.8 Cold denaturation ....................................................................................................................... 42 2 Results and Discussion ............................................................................................ 44 2.1 Cold denaturation of the Rothemund DNA origami triangle .................................................. 45 2.2 Heat denaturation of the Rothemund DNA origami triangle .................................................. 50 2.2.1 Investigations by atomic force microscopy ......................................................................... 51 2.2.2 Circular dichroism spectroscopy and thermodynamic modelling ........................................ 56 2.2.3 Divergent effects of Cl- and SO42- on DNA origami stability ................................................ 62 2.3 Magnesium concentration modulation of DNA Origami heat denaturation ......................... 65 2.4 Assessing DNA origami stability in different chaotropic environments .............................. 66 2.4.1 DNA origami integrity influenced by Gdm2SO4 ................................................................... 67 2.4.2 DNA origami integrity influenced by GdmCl ........................................................................ 73 2.4.3 DNA origami integrity influenced by TPACl ........................................................................ 75 2.4.4 Quantitative comparison ..................................................................................................... 77 3 Critics ......................................................................................................................... 78 4 Conclusion ................................................................................................................. 79 5 Outlook ...................................................................................................................... 81 6 Material and Methods ................................................................................................ 82 6.1 DNA origami synthesis .............................................................................................................. 82 6.2 Sample preparation and AFM imaging ..................................................................................... 82 6.2.1 Anion-specific structure and stability of guanidinium-bound DNA origami & Cold denaturation of DNA origami nanostructures ...................................................................... 82 6.2.2 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 83 6.2.3 Cold denaturation of DNA origami nanostructures ............................................................. 83 6.3 CD spectroscopy and analysis ................................................................................................. 84 6.3.1 Anion-specific structure and stability of guanidinium-bound DNA origami ......................... 84 6.3.2 Pre-treatment of the CD data and calculation of melting temperatures .............................. 84 6.3.3 Cold denaturation of DNA origami nanostructures ............................................................. 84 6.3.4 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 84 6.4 Principal component analysis and iterative target test factor analysis ............................... 85 6.5 Thermodynamic modelling ........................................................................................................ 85 6.6 Molecular dynamics modelling ................................................................................................. 85 Appendix ........................................................................................................................... 88 Acknowledgment ............................................................................................................ 100 Bibliography .................................................................................................................... 101 List of Figures ................................................................................................................. 116 List of Tables ................................................................................................................... 118 Declaration of independence – Selbstständigkeitserklärung ...................................... 119

L' objet d'étude de cette thèse est l' origami d' ADN. Le nombre important d'applications utilisant cette technique, et leurs diversités, sont des preuves que ces structures présentées en 2007 sont une avancée importante pour la technologie ADN. Dans cette thèse nous présentons dans un premier temps les résultats de simulations sur les propriétés mécaniques de ces nouvelles structures. Les résultats d'expériences et de simulations sur le processus de formations pour de petites structures d'ADN et sur des origamis d'ADN sont présentés dans une deuxième partie. Finalement une application de ces structures à la détection SERS est proposée, et les résultats expérimentaux concernant la réalisation de cette structure sont discutés. / DNA origami are new nanostructures (2006) whose physical properties are still to be understood. In this work we were first interested in their mechanical properties. The first approach of this study was through the use of polymer physics, as it is the classical way to study DNA. We then used computer simulations to model the system in a more detailed manner and to extract general rules on the mechanical behaviour of DNA constructs. The other aspect that we studied is the process of formation of DNA origamis. We first realised an experimental study of the process of formation of the simplest origami that we could envision. This study was intended to investigate basic principles on the process of formation of DNA structures. A coarse grain model is then developed to have a first insight onto the formation process. Then an experimental study on large origamis follows with a modeling of the annealing and melting curves based on the principles determined from the study of the simplest origami. We also worked on the development of a SERS platform.

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.

This work examines the deposition of metallic and semiconductor elements onto self-assembled DNA templates for the fabrication of nanodevices. Biological molecules like DNA can self-assemble into a variety of complex 2-D and 3-D architectures without the need for expensive patterning tools. In addition, self-assembled DNA templates can be designed to controllably place functional nanomaterials with molecular precision. These characteristics make DNA an attractive template for fabricating electronic circuits from biological molecules. However, electrically conductive structures are required for electronic applications. While metallized DNA nanostructures have been demonstrated, the ability to make thin, continuous wires that are electrically conductive still represents a formidable challenge. DNA-templated wires have generally been granular in appearance with a resistivity approximately two to three orders of magnitude higher than that of the bulk material. An improved method for the metallization of DNA origami is examined in this work that addresses these challenges of size, morphology and conductivity of the metallized structure. Specifically, we demonstrated a metallization process that uses gold nanorod seeds followed by anisotropic electroless (autocatalytic) plating to provide improved morphology and greater control of the final metallized width of conducting metal lines. Importantly, growth during electroless deposition occurs preferentially in the length direction at a rate that is approximately four times the growth rate in the width direction, which enables fabrication of narrow, continuous wires. The electrical properties of 49 nanowires with widths ranging from 13 nm to 29 nm were characterized, and resistivity values as low as 8.9 x 10-7 Ω-m were measured, which represent some of the smallest nanowires and the lowest resistivity values reported in the literature. The metallization procedure developed on smaller templates was also successfully applied to metallize bigger DNA templates of tens of micrometers in length. In addition, a polymer-assisted annealing process was discovered to possibly improve the resistivity of DNA metal nanowires. Following metallization of bigger DNA origami structures, controlled placement of gold nanorods on a DNA breadboard (~100 x 100 nm2) to make rectangular, square and T-shaped metallic structures was also demonstrated. For site-specific placement of nanorods to a DNA template, we modified the surface of the gold nanorods with single-stranded DNA. The rods were then attached to DNA templates via complementary base-pairing between the DNA on the nanorods and the attachment strands engineered into the DNA "breadboard" template. Gaps between the nanorods were then filled controllably via anisotropic plating to make 10 nm diameter continuous metallic structures. Finally, controlled placement of metal (gold) - semiconductor (tellurium) materials on a single DNA origami template was demonstrated as another important step toward the fabrication of DNA-based electronic components. The combination of molecularly directed deposition and anisotropic metallization presented in this work represents important progress towards the creation of nanoelectronic devices from self-assembled biological templates.