abstract: Deoxyribonucleic acid (DNA) has been treated as excellent building material for nanoscale construction because of its unique structural features. Its ability to self-assemble into predictable and addressable nanostructures distinguishes it from other materials. A large variety of DNA nanostructures have been constructed, providing scaffolds with nanometer precision to organize functional molecules. This dissertation focuses on developing biologically replicating DNA nanostructures to explore their biocompatibility for potential functions in cells, as well as studying the molecular behaviors of DNA origami tiles in higher-order self-assembly for constructing DNA nanostructures with large size and complexity. Presented here are a series of studies towards this goal. First, a single-stranded DNA tetrahedron was constructed and replicated in vivo with high efficiency and fidelity. This study indicated the compatibility between DNA nanostructures and biological systems, and suggested a feasible low-coast method to scale up the preparation of synthetic DNA. Next, the higher-order self-assembly of DNA origami tiles was systematically studied. It was demonstrated that the dimensional aspect ratio of origami tiles as well as the intertile connection design were essential in determining the assembled superstructures. Finally, the effects of DNA hairpin loops on the conformations of origami tiles as well as the higher-order assembled structures were demonstrated. The results would benefit the design and construction of large complex nanostructures. / Dissertation/Thesis / Ph.D. Biochemistry 2012

Nanostructuring has led to materials with novel and improved materials properties driving innovation across fields as varied as transportation, computing, energy and biotechnology. However, the benefits of nanostructured material have not widely been extended into large-scale, three-dimensional applications as deterministic pattern techniques have proven too expensive for devices outside of high value products. This thesis explores how colloidal self-assembly can be used to form macroscopic functional materials with short-range order for electronic, photonic and electrochemical applications at scale. DNA-functionalized nanoparticles are versatile models for exploring colloidal self-assembly due to the highly specific, tunable and thermally reversible binding between DNA strands. Gold nanoparticles coated with DNA were used to investigate the temperature-dependent interaction potentials and the gel formation in DNA-colloidal systems. The electronic conductivity and the plasmonic response of the DNA-gold gels were studied to explore their applicability as porous electrodes and SERS substrates, respectively. Subsequently, silica nanoparticles were assembled into nanostructures that preferentially scatter blue light using both DNA and polymer-colloid interactions. Finally, rod-sphere structures made from DNA-coated gold nanoparticles and viruses were explored, demonstrating how high-aspect ratio building blocks can create composite structures with increased porosity. The gold-virus gel structures inspired the design and assembly of a silicon-carbon nanotube composite material using covalent bonds that shows promise for high energy density anodes.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on August 9, 2009). Includes bibliographical references.

Thesis (M. S.)--Materials Science and Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Milam, Valeria; Committee Member: Boyan, Barbara; Committee Member: Li, Mo; Committee Member: McDevitt, Todd; Committee Member: Sandhage, Ken.

<p>A RET network consists of a network of photo-active molecules called chromophores that can participate in inter-molecular energy transfer called resonance energy transfer (RET). RET networks are used in a variety of applications including cryptographic devices, storage systems, light harvesting complexes, biological sensors, and molecular rulers. In this dissertation, we focus on creating a RET device called closed-diffusive exciton valve (C-DEV) in which the input to output transfer function is controlled by an external energy source, similar to a semiconductor transistor like the MOSFET. Due to their biocompatibility, molecular devices like the C-DEVs can be used to introduce computing power in biological, organic, and aqueous environments such as living cells. Furthermore, the underlying physics in RET devices are stochastic in nature, making them suitable for stochastic computing in which true random distribution generation is critical.</p><p>In order to determine a valid configuration of chromophores for the C-DEV, we developed a systematic process based on user-guided design space pruning techniques and built-in simulation tools. We show that our C-DEV is 15x better than C-DEVs designed using ad hoc methods that rely on limited data from prior experiments. We also show ways in which the C-DEV can be improved further and how different varieties of C-DEVs can be combined to form more complex logic circuits. Moreover, the systematic design process can be used to search for valid chromophore network configurations for a variety of RET applications.</p><p>We also describe a feasibility study for a technique used to control the orientation of chromophores attached to DNA. Being able to control the orientation can expand the design space for RET networks because it provides another parameter to tune their collective behavior. While results showed limited control over orientation, the analysis required the development of a mathematical model that can be used to determine the distribution of dipoles in a given sample of chromophore constructs. The model can be used to evaluate the feasibility of other potential orientation control techniques.</p> / Dissertation

<p>The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.</p><p>The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.</p><p>Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.</p><p>Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.</p><p>Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.</p><p>In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.</p> / Dissertation

In recent years, DNA nanotechnology has emerged as one of the most powerful strategies for bottom-up construction of nanomaterials. Due to the high programmability of DNA molecules, their self-assembly can be rationally designed. Engineered 3D DNA crystals, as critical products from the design of DNA self-assembly, have been proposed as the structural scaffolds for organizing nano-objects into three-dimensional, macroscopic devices. However, for such applications, many obstacles need to be overcome, including the crystal stability, the characterization methodology, the revision of crystal designs as well as the modulation of crystallization kinetics. My PhD research focuses on solving these problems for engineered 3D DNA crystals to pave the way for their downstream applications.<br>In this thesis, I started by enhancing the stability of engineered 3D DNA crystals. I developed a highly efficient post-assembly modification approach to stabilize DNA crystals. Enzymatic ligation was performed inside the crystal lattice, which was designed to covalently link the sticky ends at the crystal contacts. After ligation, the crystal became a covalently bonded 3D network of DNA motifs. I investigated the stability of ligated DNA crystals under a wide range of solution conditions. Experimental data revealed that ligated DNA crystals had significantly increased stability. With these highly stabilized DNA crystals, we then demonstrated their applications in biocatalysis and protein encapsulation as examples.<br>I also established electron microscope imaging characterization methods for engineered 3D DNA crystals. For crystals from large-size DNA motifs, they are difficult to study by X-ray crystallography because of their limited diffraction resolutions to no better than 10 Å. Therefore, a direct imaging method by TEM was set up. DNA crystals were either crushed or controlled to grow into microcrystals for TEM imaging. To validate the imaging results, we compared the TEM images with predicted models of the crystal lattice. With the advance in crystal characterization, DNA crystals of varying pore size between 5~20 nm were designed, assembled, and validated by TEM imaging.<br>The post-assembly ligation was further developed to prepare a series of new materials derived from engineered 3D DNA crystals, which were inaccessible otherwise. With the directional and spatial control of ligation in DNA crystal, I prepared new DNA-based materials including DNA microtubes, complex-architecture crystals, and an unprecedented reversibly expandable, self-healing DNA crystal. The integration of weak and strong interactions in crystals enabled a lot of new opportunities for DNA crystal engineering.<br>In the final chapter, I investigated the effect of 5’-phosphorylation on DNA crystallization kinetics. I found that phosphorylation significantly enhanced the crystallization kinetics, possibly by strengthening the sticky-ended cohesion. Therefore, DNA crystals can be obtained at much lower ionic strength after phosphorylation. I also applied the result to controling the morphology of DNA crystals by tuning the crystallization kinetics along different crystallographic axes. Together with previously methods to slow down DNA crystallization, the ability to tune DNA crystallization kinetics in both ways is essential for DNA crystal engineering.

<p>Synthetic biology is an emerging technology field whose goal is to use biology as a substrate for engineering. Self-assembly is one of the many methods for fabricating such synthetic biosystems.</p><p>Self-assembly is a process where components spontaneously arrange themselves into organized aggregates by the selective affinity of substructures. DNA is an excellent candidate for making synthetic biological systems using self-assembly because of its modular structure and simple chemistry. This thesis describes several</p><p>techniques which use DNA as a nano-construction material and</p><p>explores the computational capabilities of DNA self-assembly.</p><p>For this dissertation, I set out to build a biomolecular computing device with several</p><p>useful properties, including compactness, robustness, high degrees of complexity, flexibility, scalability and easily characterized yields</p><p>and convergence rates. However, a unified device that satisfies all these properties is still many years away. Instead, this thesis presents designs, simulations,</p><p>and experimental results for several distinct DNA nano-systems, as</p><p>well as experimental protocols, each of which satisfies a subset of the above-mentioned properties. The hope is that the lessons learned from building all these biomolecular computational devices would bring us closer to our ultimate goal and would eventually pave the path for a computing device that satisfies all the properties. We experimentally demonstrate how we can reduce errors in tiling assembly using an optimized set of physical parameters. We propose a novel DNA tile design</p><p>which enforces directionality of growth, reducing assembly errors. We build simulation models to characterize damage in fragile nanostructures under the impact of various external forces. Furthermore, we investigate reversible computation as a means to provide self-repairability to such damaged structures.</p><p>We suggest two modifications of an existing DNA computing device,</p><p>called Whiplash PCR machine, which allow it to operate robustly outside of controlled laboratory conditions and allow it to implement a simple form of inter-device communication. We present analysis techniques which characterize the yields and time convergence of self-assembled DNA nanostructures. We also present an experimental demonstration of a novel DNA nanostructure which is capable of tiling the plane and could prove to be a way of building 3D DNA assemblies.</p> / Dissertation

<p>Nanoscale devices offer the technological advances to enable a new era in computing. Device sizes at the molecular-scale have the potential to expand the domain of conventional computer systems to reach into environments and application domains that are otherwise impractical, such as single-cell sensing or micro-environmental monitoring.</p><p>New potential application domains, like biological scale computing, require processing elements that can function inside nanoscale volumes (e.g. single biological cells) and are thus subject to extreme size and resource constraints. In this thesis we address these critical new domain challenges through a synergistic approach that matches manufacturing techniques, circuit technology, and architectural design with application requirements. We explore and vertically integrate these three fronts: a) assembly methods that can cost-effectively provide nanometer feature sizes, b) device technologies for molecular-scale computing and sensing, and c) architectural design techniques for nanoscale processors, with the goal of mapping a potential path toward achieving molecular-scale computing.</p><p>We make four primary contributions in this thesis. First, we develop and experimentally demonstrate a scalable, cost-effective DNA self-assembly-based fabrication technique for molecular circuits. Second, we propose and evaluate Resonance Energy Transfer (RET) logic, a novel nanoscale technology for computing based on single-molecule optical devices. Third, we design and experimentally demonstrate selective sensing of several biomolecules using RET-logic elements. Fourth, we explore the architectural implications of integrating computation and molecular sensors to form nanoscale sensor processors (nSP), nanoscale-sized systems that can sense, process, store and communicate molecular information. Through the use of self-assembly manufacturing, RET molecular logic, and novel architectural techniques, the smallest nSP design is about the size of the largest known virus.</p> / Dissertation

In this study we use AFM-based nanolithography technique to produce nanofeatures of the single strand DNA and LNA probe molecules which are prepared via thiolated nucleic acid self-assembled monolayers (SAMs) on gold substrates. The goal is to observe the topographic changes of the DNA film structures resulting from the formation of rigid double strand DNA when the target and probe DNAs bind together. The so-called hybridization depends strongly on the probe density on the substrate surface. To find the proper probe density for hybridization, we vary the concentration of the probe DNA and search for the optimal conditions for measuring the height changes of the nanofeatures. We also monitor the topographic changes of the DNA nanofeatures in the different target DNA concentrations as a function of time, and the binding isotherms are fitted with the Langmuir adsorption model to derive the equilibrium dissociation constant and maximum hybridization efficiency. In addition, we extend the nanoscale hybridization reaction detection to mismatched DNA:DNA and LNA:DNA hybridization, and observe that topographic change of mismatched hybridization is inconspicuous and rapidly reach equilibrium. The results reveal the apparent difference between the perfect match and mismatch conditions, and validate that this approach can be applied to differentiate the situations for both perfect match and mismatch cases, demonstrating its potentials in the gene chip technology.