Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 66-71). Also available on the World Wide Web.

abstract: DNA nanotechnology is one of the most flourishing interdisciplinary research fields. Through the features of programmability and predictability, DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales, and more importantly, they can be used as scaffolds for organizing other nanoparticles, proteins and chemical groups. By leveraging these molecules, DNA nanostructures can be used to direct the organization of complex bio-inspired materials that may serve as smart drug delivery systems and in vitro or in vivo bio-molecular computing and diagnostic devices. In this dissertation I describe a systematic study of the thermodynamic properties of complex DNA nanostructures, including 2D and 3D DNA origami, in order to understand their assembly, stability and functionality and inform future design endeavors. It is conceivable that a more thorough understanding of DNA self-assembly can be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications. As a biocompatible nanoscale motif, the successful integration, stabilization and separation of DNA nanostructures from cells/cell lysate suggests its potential to serve as a diagnostic platform at the cellular level. Here, DNA origami was used to capture and identify multiple T cell receptor mRNA species from single cells within a mixed cell population. This demonstrates the potential of DNA nanostructure as an ideal nano scale tool for biological applications. / Dissertation/Thesis / Ph.D. Chemistry 2014

<p>Self-assembly is a pervasive natural phenomenon that gives rise to complex structures and functions. It describes processes in which a disordered system of components form organized structures as a consequence of specific, local interactions among the components themselves, without any external direction. Biological self-assembled systems, evolved over billions of years, are more intricate, more energy efficient and more functional than anything researchers have currently achieved at the nanoscale. A challenge for human designed physical self-assembled systems is to catch up with mother nature. I argue through examples that DNA is an apt material to meet this challenge. This work presents:</p><p>1. 3D self-assembled DNA nanostructures.</p><p>2. Illustrations of the simplicity and power of toehold-mediated strand displacement interactions.</p><p>3. Algorithmic constructs in the tile assembly model.</p> / Dissertation

L’ADN (acide désoxyribonucléique) est le support de notre génome, c'est aussi un biopolymère dont les propriétés d’hybridation de deux simples brins complémentaires en une double hélice permettent son utilisation comme brique élémentaire pour l’auto-assemblage de structures avec une résolution de quelques nanomètres. Parmi les différentes méthodes développées, l'origami d’ADN dans lequel un simple brin d’ADN issu du génome d’un phage est replié algorithmiquement par un ensemble de brins synthétiques plus petits s'est démontré très robuste pour l'assemblage de structures bi ou tridimensionnelles. La conception de ces origami est basée sur la thermodynamique à l'équilibre, c'est à dire sur l'optimisation de l'appariement complémentaire des bases. Cependant, bien que des outils interactifs qui facilitent la conception de structures aient été développés, très peu de recherches se sont focalisées sur le processus du repliement et sur son optimisation. Notre travail a consisté à étudier la thermodynamique de nanostructures d'ADN afin de mieux comprendre le processus d'assemblage et d'en identifier des étapes clés.Nous avons effectué des mesures en calorimétrie différentielle à balayage (DSC) sur des structures modèles et des origami d'ADN. Ainsi, nous avons pu identifier la présence d'étapes clés dans le repliement de nanostructures comportant un petit nombre de brins d'ADN. Nous montrons qu'en modifiant les séquences il est possible de changer la coopérativité et la stabilité de l'assemblage des nanostructures et donc de modifier le chemin de repliement.L'étude d'origami simplifiés comportant une ou deux agrafes nous a permis de mesurer l'influence de la position des agrafes, des tailles de boucles et de l'orientations des brins d'ADN sur la thermodynamique du repliement.Enfin, les mesures calorimétriques effectuées sur des origami d'ADN nous ont permis de résoudre l'hybridation collective d'ensemble d'agrafes. Cela nous permet de hiérarchiser l'assemblage de l'origami en domaines distincts.Notre travail de thèse a également consisté au développement de méthodes innovantes de nanocalorimétrie ultrasensible intégrant de la microfluidique. Ces méthodes calorimétriques permettront d'accéder aux paramètres cinétiques de l’assemblage en plus des paramètres thermodynamiques à l'équilibre.Nos résultats obtenus sur les nanostructures modèles montrent qu'il est possible d'optimiser la conception des nanostructures d'ADN en intégrant dans la conception le processus d'assemblage. Des nanostructures d'ADN à l'assemblage performant permettront peut-être à l'avenir le développement d'automates moléculaires synthétiques qui sont une des applications très prometteuses de ces systèmes. / DNA is the support of genetic information. The property of self-assembly of two complementary single strands to form a double helix enable the use of this biopolymer as a building block for nanofabrication. DNA origami are a method which enable the self-assembly of 2D or 3D nanostructures. In this method, a long single-stranded DNA taken from the genome of a phage is folded on itself in a programmable way thanks to a lot of short synthetic DNA strands. The design of origami is based on thermodynamic and on the optimization of the base pairing in the structures. However, although interactive tools that facilitate the design of DNA nano-structures have been developed, we know little about the folding process and its optimization. In this work, we study the thermodynamics of DNA nanostructures in order to have a better understanding of the folding process and to identify the key steps.We performed differential scanning calorimetry (DSC) on model structures and DNA origami. Thus, we have been able to identify the presence of key steps in the folding of small nanostructures. We show that by changing the sequences of the strands, it is possible to change the cooperativity and the stability of the assembly of the nanostructure and thus change the folding path.The study of small origami with one or two staples allowed us the see the influence of the position of the staples, of the sizes of the loops and of the orientation of the staples on the thermodynamic of the folding.Finally, the calorimetric measurements performed on origami allowed us to solve the collective hybridization of staple sets. This enable us to prioritize the origami assembly into separate domains.This work also consisted of the development of innovative methods of ultra-sensitive nano-calorimetry integrating microfluidics. These calorimetric methods will give us the access to the kinetic parameters of the folding and to the equilibrium thermodynamic parameters.Our results obtained on model nano-structures show that it is possible to optimize the design of DNA nanostructures by integrating the assembly process in the design of the structures. Such high-performance DNA nanostructures may allow in the future the development of molecular robot which is a very promising application of DNA nanostructures.

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.

<p>The manufacturing of molecular-scale computing systems requires a scalable, reliable, and economic approach to create highly interconnected, dense arrays of devices. As a candidate substrate for nanoscale logic circuits, DNA self-assembled nanostructures have the potential to fulfill these requirements. However, a number of open challenges remain, including the scalability of DNA self-assembly, long-range signal propagation, and precise patterning of functionalized components. These challenges motivate the development of theory and experimental techniques to illuminate the connections among the physical, optical, and thermodynamic properties of DNA self-assembled nanostructures. </p> <p>In this thesis, three tools are developed, validated, and applied to study the thermo-mechanical properties of DNA nanostructures: 1) a method to quantitatively measure the quality of DNA grid self-assembly, 2) a spectrofluorometer capable of capturing fluorescence and absorbance data under simultaneous multi-wavelength excitation, and 3) a Monte Carlo simulator that models the ensemble response of DNA nanostructures as simple harmonic oscillators. </p> <p>The broad contributions of this dissertation are as follows: 1) insight into the thermo-mechanical properties of DNA grid nanostructures, and 2) a categorization of self-assembly defects and their impact on proposed logic circuits. </p> <p>The results of the work presented in this dissertation show that: 1) the quality of self-assembly of DNA grid nanostructures can be quantitatively calculated to demonstrate the impact of changes in temperature or structure, 2) the optical absorbance of complex DNA nanostructures can be modeled to capture their thermo-mechanical properties (i.e., worst case within 10% of experimental melting temperatures and 70% of experimental thermodynamic parameters), 3) the structural resilience of DNA nanostructures can be quantifiably improved by chemical cross-linking with up to 60% retaining their original structure, and 4) DNA self-assembly introduces structural defects which create new fault models with respect to conventional technologies for logic circuits.</p> / Dissertation

<p>  </p> <p>DNA nanotechnology has rendered programmable, bottom-up self-assembly of nanostructures in various morphology, versatile functionalization, and atomic level precision over the last forty years. DNA nanostructures are usually assembled in solution by the thermodynamic process in a specific solution. In recent years, DNA two-dimensional (2D) structures on the surface have been widely applied in semiconductors, electronic devices, and biomedical studies. My research mainly focused on novel DNA nanostructures assembly on the surface and their applications. I have developed an equilibrium-enabled flexibly curved DNA homopolymer. I have further developed a novel method to determine the interhelical angle of DNA secondary structure by DNA 2D-array.</p> <p>In this thesis, I have envisioned a strategy to prepare DNA linear polymers with flexible curvature and further assembled them into spiral or concentric rings on the surface. In DNA double crossover-like (DXL) homopolymers, an aromatic chemical group was introduced to the 3'-end in each strand. The planar group could stack into the DNA homopolymer, which increases the length on one side of the DXL polymer and further bend the structure by uneven-length stress. Moreover, the stacking in is under the equilibrium with flipping out, endowing the dynamic change and flexibility to the curvature of the DNA homopolymer, which could be a benefit in the surface-assisted construction of spirals or concentric rings.</p> <p>With the appropriate design, DNA could be self-assembled on the surface into 2D crystals in a certain periodicity. Such a structure could be applied in nucleic acid secondary structure determination as a crystallography-like method. In this work, I have successfully incorporated the 10-23 DNAzyme, a common-used RNA-cleavage DNA sequence into DNA 2D arrays. In the brick-wall-like DNA 2D arrays, the repeating distance determined the interhelical angle of the 10-23 DNAzyme flanks. By 2D fast Fourier transform (FFT), this repeating distance could be measured and calibrated, following the deduction of the target angle. This approach had been validated with well-known DNA secondary structures. </p>

The controlled positioning of DNA nanostructures on technologically relevant surfaces represents a major goal along the route toward the full-scale integration of DNA-based materials into nanoelectronic and sensor devices. Previous attempts to arrange DNA nanostructures into defined arrays mostly relied on top-down lithographic patterning techniques combined with chemical surface functionalization. Here we combine two bottom-up techniques for nanostructure fabrication, i.e., self-organized nanopattern formation and DNA origami self-assembly, in order to demonstrate the electrostatic self-alignment of DNA nanotubes on topographically patterned silicon surfaces. Self-organized nanoscale ripple patterns with periodicities ranging from 20 nm to 50 nm are fabricated by low-energy ion irradiation and serve as substrates for DNA origami adsorption. Electrostatic interactions with the charged surface oxide during adsorption direct the DNA origami nanotubes to the ripple valleys and align them parallel to the ripples. By optimizing the pattern dimensions and the Debye length of the adsorption buffer, we obtain an alignment yield of ~70%. Since this novel and versatile approach does not rely on any chemical functionalization of the surface or the DNA nanotubes, it can be applied to virtually any substrate material and any top-down or bottom-up nanopatterning technique. This technique thus may enable the wafer-scale fabrication of ordered arrays of functional DNA-based nanowires.

Elastic properties of carbon nanotubes (CNT), boron nitride nanotubes (BNNT), double stranded DNA (dsDNA), paranemic-juxtapose crossover (PX-JX) DNA and dendrimer bound DNA are discussed in this thesis. Structural phase transitions of nucleic acids induced by external force, carbon nanotubes and graphene substrate are also studied extensively. Electrostatic interactions have a strong effect on the elastic properties of BNNTs due to large partial atomic charges on boron and nitrogen atoms. We have computed Young’s modulus (Y ) and shear modulus (G) of BNNT and CNT as a function of the nanotube radius and partial atomic charges on boron and nitrogen atoms using molecular mechanics calculation. Our calculation shows that Young’s modulus of BNNTs increases with increase in magnitude of the partial atomic charges on B and N atoms and can be larger than the Young’s modulus of CNTs of same radius. Shear modulus, on the other hand depends weakly on the magnitude of partial atomic charges and is always less than the shear modulus of the CNT. The values obtained for Young’s modulus and shear modulus are in excellent agreement with the available experimental results. We also study the elasticity of dsDNA using equilibrium fluctuation methods as well as nonequilibrium stretching simulations. The results obtained from both methods quantitatively agree with each other. The end-to-end length distribution P(ρ) and angle distribution P(θ) of the dsDNA has a Gaussian form which gives stretch modulus (γ1) to be 708 pN and persistence length (Lp) to be 42 nm, respectively. When dsDNA is stretched along its helix axis, it undergoes a large conformational change and elongates about 1.7 times its initial contour length at a critical force. Applying a force perpendicular to the DNA helix axis, dsDNA gets unzipped and separated into two single-stranded DNA (ssDNA). DNA unzipping is a fundamental process in DNA replication. As the force at one end of the DNA is increased the DNA starts melting above a critical force depending on the pulling direction. The critical force fm , at which dsDNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the dsDNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base-pairs. Similar force-extension curve has also been observed when crossover DNA molecules are stretched along the helix axis. In the presence of mono-valent Na+ counterions, we find that the stretch modulus (γ1 ) of the paranemic crossover (PX) and its topoisomer juxtapose (JX) DNA structure is significantly higher (30 %) compared to normal B-DNA of the same sequence and length. When the DNA motif is surrounded by a solvent of divalent Mg2+ counterions, we find an enhanced rigidity compared to in Na+ environment due to the electrostatic screening effects arising from the divalent nature of Mg2+ counterions. This is the first direct determination of the mechanical strength of these crossover motifs which can be useful for the design of suitable DNA motifs for DNA based nanostructures and nanomechanical devices with improved structural rigidity. Negatively charged DNA can be compacted by positively charged dendrimer and the degree of compaction is a delicate balance between the strength of the electrostatic interaction and the elasticity of DNA. When the dsDNA is compacted by dendrimer, the stretch modulus, γ1 and persistence length, Lp decreases dramatically due to backbone charge neutralization of dsDNA by dendrimer. We also study the effect of CNT and graphene substrate on the elastic as well as adsorption properties of small interfering RNA (siRNA) and dsDNA. Our results show that siRNA strongly binds to CNT and graphene surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the CNTs and is maximum on graphene. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the CNT/graphene surface. However, dsDNA of the same sequence undergoes much less unzipping and wrapping on the CNT/graphene due to smaller interaction energy of thymidine of dsDNA with the CNT/graphene compared to that of uridine of siRNA. Unzipping probability distributions fitted to single exponential function give unzipping time (τ) of the order of few nanoseconds which decrease exponentially with temperature. From the temperature variation of unzipping time we estimate the free energy barrier to unzipping. We have also investigated the binding of siRNA to CNT by translocating siRNA inside CNT and find that siRNA spontaneously translocates inside CNT of various diameters and chiralities. Free en- ergy profiles show that siRNA gains free energy while translocating inside CNT and the barrier for siRNA exit from CNT ranges from 40 to 110 kcal/mol depending on CNT chirality and salt concentration. The translocation time τ decreases with the increase of CNT diameter having a critical diameter of 24 A for the translocation. After the optimal binding of siRNA to CNT/graphene, the complex is very stable which can serve as siRNA delivery agent for biomedical applications. Since siRNA has to undergo unwinding process in the presence of RNA-induced silencing complex, our proposed delivery mechanism by single wall CNT possesses potential advantages in achieving RNA interference (RNAi).

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