<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

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.

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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