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Control of Dynamic DNA Origami Mechanisms Using Integrated Functional ComponentsMiller, Carl A. 20 May 2015 (has links)
No description available.
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DNA Origami Breadboard: A Platform for Cell Activation and Cell Membrane FunctionalizationMollica, Molly Y. 30 August 2016 (has links)
No description available.
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DNA Oligomers - From Protein Binding to Probabilistic ModellingAndrade, Helena 09 February 2017 (has links) (PDF)
This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein.
In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases.
To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.
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Structures and mechanisms for synthetic DNA motorsHaley, Natalie Emma Charnell January 2017 (has links)
DNA provides an ideal substrate for nanoscale construction and programmable dynamic mechanisms. DNA mechanisms can be used to produce DNA motors which do mechanical work, e.g. transportation of a substrate along a track. I explore a method for control of a DNA mechanism ubiquitous in DNA motor designs, toehold-mediated strand displacement, by which one strand in a duplex can be swapped for another. My method uses a mismatch between a pair of nucleotides in the duplex, which is repaired by displacement. I find that displacement rate can be fine-tuned by adjusting the position of the mismatch in the duplex, enabling the design of complex kinetic behaviours. A bipedal motor [1, 2] is designed to walk along a single-stranded DNA track. Previously the motor has only taken a single step, due to a lack of designs to extend the single-stranded track. I present a novel design for track held under tension using a 3D DNA origami tightrope, and verify its assembly. The bipedal motor design is adapted and a method to specifically place motors on tightropes is demonstrated. Motor operation is investigated on truncated tracks and tightrope tracks by electrophoresis and spectrofluorometry. The motor does not accumulate appreciably at the track end; this is tentatively attributed to rearrangement of the motor between track sites without interaction with fuel. Tightrope origami can hold single-stranded DNA under pN tension. I use tightropes to study hybridization kinetics under tension and find dramatic, non-monotonic changes in hybridization rate constants and dissociation constants with tension in the range ∼0-15 pN. Extended tracks for a 'burnt-bridges' motor which destroys its track as it moves [3] are created on the inside of DNA nanotubes, which can be polymerised to create tracks up to a few mm in length, and on tiles which I attempt to join in a specific order. Crossing of the motor between tubes is verified, and microscopy experiments provide some evidence that track is being cleaved by the motor, a requirement for movement along the track. Tile based tracks are imaged by super-resolution DNA PAINT [4], providing proof-of-principle for track observation to infer motor movement.
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DNA programmed assembly of active matter at the micro and nano scalesGonzalez, Ibon Santiago January 2017 (has links)
Small devices capable of self-propulsion have potential application in areas of nanoscience where autonomous locomotion and programmability are needed. The specific base-pairing interactions that arise from DNA hybridisation permit the programmed assembly of matter and also the creation of controllable dynamical systems. The aim of this thesis is to use the tools of DNA nanotechnology to design synthetic active matter at the micro and nano scales. In the first section, DNA was used as an active medium capable of transporting information faster than diffusion in the form of chemical waves. DNA waves were generated experimentally using a DNA autocatalytic reaction in a microfluidic channel. The propagation velocity of DNA chemical waves was slowed down by creating concentration gradients that changed the reaction kinetics in space. The second section details the synthesis of chemically-propelled particles and the use of DNA as a 'programmable glue' to mediate their interactions. Janus micromotors were fabricated by physical vapour deposition and a wet-chemical approach was demonstrated to synthesise asymmetrical catalytic Pt-Au nanoparticles that function as nanomotors. Dynamic light scattering measurements showed nanomotor activity that depends on H<sub>2</sub>O<sub>2</sub> concentration, consistent with chemical propulsion. Gold nanoparticles/Origami hybrids were assembled in 2D lattices of different symmetries arranged by DNA linkers. The third section details the design process and synthesis of nanomotors using DNA as a structural scaffold. 3D DNA Origami rectangular prisms were functionalised site-specifically with bioconjugated catalysts, i.e. Pt nanoparticles and catalase. Enzymatic nanomotors were also conjugated to various cargoes and their motor activity was demonstrated by Fluorescence Correlation Spectroscopy. In the final section, control mechanisms for autonomous nanomotors are studied, which includes the conformational change of DNA aptamers in response to chemical signals, as well as a design for an adaptive dynamical system based on DNA/enzyme reaction networks.
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On the interaction of DNA nanostructures with lipid bilayersJournot, Céline M. A. January 2017 (has links)
Much of our knowledge of cellular biology arises from direct observation of active cellular functions. Tools and techniques have steadily developed over the past several hundreds of years to aid in our understanding and control of the nanoworld and are referred to as nanotechnologies. In the context of nanotechnology, DNA is not used as a carrier for genetic information (as it is in cell), but as a construction material. DNA offers unprecedented control over the construction of simplified biomimetic models for the study of biological processes. This thesis first introduces and defines the field of DNA nanotechnology, with particular emphasis on the interaction of snthetic DNA nanostructures with biological membranes. Inspired by the protein clathrin, three-fold symmetric DNA tile made of eight, short DNA strands and capable of polymerising is described and studied, with the aim to interact with and controllably bend a membrane bilayer. This structure presented challenges during construction so an enhanced three-armed DNA structure built with DNA origami was designed. The succesful assembly of a rigid and functionalisable nanostructure is described. This origami structure was polymerised into large constructs in solution and on a supported lipid membrane. The shape of the structure was modulated to vary its curvature and apply a bending force to a lipid vesicle when anchored to it. Following the conclusion of this study, we present the construction of a small, unique DNA structure for enhanced electron microscopy imaging in cell lysate. This project is part of a developing technique to couple the interaction specificity of dyes in super-resolution microscopy and the high-resolution output of electron microscopy. Finally, the optimisation procedures and recommendations for TEM imaging of samples of DNA origami and lipid structures are discussed.
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Rolling circle transcription on smallest size double stranded DNA minicirclesKristoffersson, Anders January 2010 (has links)
The RNA polymerase T7 is utilized as a component of motor complexes in DNA nanotechnology due to its high promotor specificity, the lack of external transcription factors and its very high processivity, but there is no experience of its application on small double stranded DNA circles. Circular templates from 210 to 126 bp in circumference sharing a common promotor termination motif were synthesized and transcription was monitored at end point on gel and in real time with a 2’ O methyl RNA molecular beacon. The RNAP T7 was found to be able to utilize circular dsDNA templates down to 126 bp with moderate impact on transcription rate for saturated systems and rolling circle transcription products were evident with denaturizing PAGE gel electrophoresis for templates down to 167 bp.
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Design, Synthesis and Analysis of Self-Assembling Triangulated Wireframe DNA StructuresMatthies, Michael 18 November 2019 (has links)
The field of DNA nanotechnology offers a wide range of design strategies with which nanometer-sized structures with a desired shape, size and aspect ratio can be built. The most established techniques in the field rely on close-packed 'solid' DNA nanostructures produced with either the DNA origami or the single-stranded tile techniques. These structures depend on high-salt buffer solutions and require more material than comparable size hollow wireframe structures.
This dissertation explores the construction of hollow wireframe DNA nanostructures composed of equilateral triangles. To achieve maximal material efficiency the design is restricted to use a single DNA double helix per triangle edge. As a proof of principle, the DNA origami technique is extended to produce a series of truss structures including the flat, tetrahedral, octahedral, or irregular dodecahedral truss designs. In contrast to close packed DNA origami designs these structures fold at low-salt buffer conditions. These structures have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. The design process of these structures is simplified by a custom design software.
Next, the triangulated construction motif is extended to the single-stranded DNA tile technique. A collection of finite structures, as well as one-dimensional crystalline assemblies is explored. The ideal assembly conditions are determined experimentally and using molecular dynamics simulations. A custom design software is presented to simplify the design and handling of these structures.
At last, the cost-effective prototyping of triangulated wireframe DNA origami structures is explored. This is achieved through the introduction of single-stranded “gap” regions along the triangle edges. These gap regions are then filled using a DNA polymerase rather than by synthetic oligonucleotides. This technique also allows the mechanical transformation of these structures, which is exemplified by the transition of a bent into a straight structure upon completion of the gap filling.:Abstract v
Publications vii
Acknowledgements ix
Contents xi
Chapter 1 A short introduction into DNA nanotechnology 1
1.1 Nanotechnology 1
1.1.1 Top down 1
1.1.2 Bottom up 3
1.2 Deoxyribonucleic acid (DNA) 4
1.3 DNA Nanotechnology 6
1.3.1 Tile based assembly 9
1.3.2 DNA origami and single-stranded tiles 10
1.3.3 Some applications of DNA nanotechnology 12
1.3.4 Wireframe structures 15
1.3.5 Computational tools and DNA nanotechnology. 17
Chapter 2 Motivation and objectives 19
Chapter 3 Design and Synthesis of Triangulated DNA Origami Trusses 20
3.1 Introduction 20
3.2 Results and Discussion 21
3.2.1 Design 21
3.2.2 Nomenclature and parameters of the tube structures 23
3.2.3 Gel electrophoreses analysis 25
3.2.4 Imaging of the purified structures 26
3.2.5 Optimizing the folding conditions 28
3.2.6 Comparison to vHelix 29
3.3 Conclusions 29
3.4 Methods 30
3.4.1 Standard DNA origami assembly reaction. 30
3.4.2 Gel purification. 30
3.4.3 AFM sample preparation. 31
3.4.4 TEM sample preparation. 31
3.4.5 Instructions for mixing the staple sets. 31
Chapter 4 Triangulated wireframe structures assembled using single-stranded DNA tiles 33
4.1 Introduction 33
4.2 Results and Discussion 35
4.2.1 Designing the structures 35
4.2.2 Synthesis of test structures 37
4.2.3 Molecular dynamics simulations of 6-arm junctions 38
4.2.4 Assembly of the finite structures 40
4.2.5 Influence of salt concentration and folding times 42
4.2.6 Molecular dynamics simulations of the rhombus structure 43
4.2.7 1D SST crystals 44
4.2.8 Controlling the crystal growth 46
4.3 Conclusions 48
4.4 Methods 49
4.4.1 SST Folding 49
4.4.2 Agarose Gel Electrophoresis 49
4.4.3 tSEM Characterization 49
4.4.4 AFM Imaging 49
4.4.5 AGE-Based Folding-Yield Estimation 49
4.4.6 Molecular Dynamics Simulations 50
Chapter 5 Structural transformation of wireframe DNA origami via DNA polymerase assisted gap-filling 52
5.1 Introduction 52
5.2 Results and Discussion 54
5.2.1 Design of the Structures 54
5.2.2 Folding of Gap-Structures 56
5.2.3 Inactivation of Polymerase. 57
5.2.4 Secondary Structures. 58
5.2.5 Folding Kinetics of Gap Origami. 60
5.3 Conclusions 61
5.4 Methods 62
5.4.1 DNA origami folding 62
5.4.2 Gap filling of the wireframe DNA origami structures 63
5.4.3 Agarose gel electrophoresis 63
5.4.4 PAGE gel analysis 63
5.4.5 tSEM characterization 64
5.4.6 AFM imaging 64
5.4.7 AGE based folding-yield estimation 64
5.4.8 Gibbs free energy simulation using mfold 65
5.4.9 List of sequence for folding the DNA origami triangulated structures 65
Chapter 6 Summary and outlook 67
Appendix 69
A.1 Additional figures from chapter 369
A.2 Additional figures from chapter 4 77
A.3 Additional figures from chapter 5 111
Bibliography 127
Erklärung 138
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Generierung und Charakterisierung von Proteinderivaten zur gezielten Mineralisierung von DNA-KonstruktenGehlhar, Maria 14 June 2021 (has links)
Die Besonderheit der DNA-Nanotechnologie liegt in der Verwendung von DNA als Konstruktionsmaterial für die Herstellung von artifiziellen Strukturen im Nanometermaßstab (Seeman, 1982; Winfree et al., 1998). Diese DNA-Nanoobjekte, wie beispielsweise DNA-Nanoröhren, offerieren innovative und vielversprechende Anwendungsmöglichkeiten in verschiedenen Bereichen wie der Elektronik oder Medizin. Eine Herausforderung für die dauerhafte Verwendung von DNA-Nanoröhren stellt deren Stabilität dar. Einflussfaktoren wie die DNA-Nukleaseaktivitäten, die Ionenstärke und hohe Temperaturen können dabei eine langfristige Anwendung limitieren. Als ein Lösungsansatz für die Erhöhung der Beständigkeit wird eine gezielte Mineralisierung der DNA-Nanoröhren durch spezifische Fusionsproteine angestrebt.
Ziel dieser Arbeit ist die dafür notwendige Herstellung und Charakterisierung der DNA-bindenden Fusionsproteine mit Mineralisierungsdomänen vorzustellen. Das umfasst das Auswählen geeigneter DNA-Bindungsproteine als Bestandteil der Fusionsproteine. In dieser Arbeit wurden die DNA-Bindungsproteine MutH und SBB aus Escherichia coli (E. coli) sowie Yku70p aus Saccharomyces cerevisiae (S. cerevisiae) aufgrund ihrer spezifischen Bindungseigenschaften dafür identifiziert. Damit können die Fusionsproteine sequenzspezifische oder -unspezifische Bindungen mit doppelsträngiger (engl. double stranded DNA, dsDNA) oder einzelsträngiger DNA (engl. single stranded DNA, ssDNA) eingehen. Es ist mittels Klonierung gelungen, verschiedene Fusionskonstrukte mit den genannten DNA-Bindungsproteinen zu generieren. Diese beinhalten ebenfalls das enhanced green fluorescent protein sowie den His6-Tag für den Expressionsnachweis und die Proteinreinigung. Weitere Varianten der Fusionskonstrukte bestehen zusätzlich aus der tobacco etch virus-Protease-Erkennungssequenz zur Entfernung des His6-Tags und der Domänen R5-Peptid (R5P) oder Poly-L-Arginin (PLR) für die Mineralisierung.
Bestandteil dieser Arbeit sind Western-Blot-Analysen und mikroskopische Aufnahmen, welche die erfolgreiche heterologe Expression aller Fusionskonstrukte nachweisen. Aus den Ergebnissen der Expressions- und Löslichkeitsanalysen lässt sich schlussfolgern, dass insbesondere das Expressionslevel und die Synthese löslicher Proteine mit den Mineralisierungsdomänen eine Herausforderung darstellen. Ebenfalls in dieser Arbeit sind Versuche zur Optimierung mit verschiedenen Expressionsstämmen (E. coli und S. cerevisiae) und Expressionsparametern (Temperatur und Induktor-Konzentration) enthalten. Den Ergebnissen nach eignen sich besonders die Fusionsproteine MutH-EGFP-His6 und SSB-EGFP-His6 für die weiteren Experimente.
Die Untersuchungen der DNA-Bindungseigenschaften erfolgten mittels Electrophoretic mobility shift assay (EMSA) und Rasterkraftmikroskopie (engl. atomic force microscopy, AFM). Diese Methoden mussten für jedes Fusionsprotein zuvor etabliert und optimiert werden. Zu Beginn stand das MutH-Fusionsprotein im Focus, wobei die durchgeführten EMSA-Untersuchungen die Spezifität zur GATC-Erkennungssequenz sowie zur dsDNA betrachteten. Die Charakterisierung mittels AFM diente als weitere Möglichkeit zur Analyse der DNA-Bindungseigenschaften. Zusätzlich kam in dieser Arbeit eine Variante des CRISPR/Cas9-Systems als Fusionsprotein für eine sequenzspezifische Adressierung von dsDNA zum Einsatz. Die EMSA- und AFM-Analysen deuteten dabei auf eine Interaktion von dem dCas9-Fusionsproteins und dsDNA hin. Weiterhin war das SSB-Fusionsprotein Bestandteil der Untersuchungen. Die Bindungsanalysen mittels EMSA zeigten, dass es bevorzugt mit ssDNA interagiert und nur eine geringe Affinität zu dsDNA vorliegt. Die Bindung zu ssDNA konnte ebenfalls erfolgreich anhand von AFM-Untersuchungen gezeigt werden. Zusammenfassend bestätigen die Ergebnisse die Funktionalität des MutH- und SSB-Fusionsproteins. Es konnten zudem erste Hinweise erbracht werden, die eine spezifische Bindung der Fusionsproteine an dsDNA oder ssDNA belegen.
Mit dieser Arbeit ist es gelungen, Proteinderivate zu generieren und charakterisieren, wodurch eine entscheidende Grundlage für die gezielte Mineralisierung von DNA-Konstrukten geschaffen wurde.
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Block copolymer micellization, and DNA polymerase-assisted structural transformation of DNA origami nanostructuresAgarwal, Nayan Pawan 14 August 2019 (has links)
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
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