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The cytoskeletal molecular motors kinesin-1 and cytoplasmic dynein drive many diverse functions within eukaryotic cells. They are responsible for numerous spatially and temporally dependent intracellular processes crucial for cellular activity, including cytokinesis, maintenance of sub-cellular organization and the transport of myriad cargos along microtubule tracks. Cytoplasmic dynein and kinesin-1 are processive, but opposite polarity, homodimeric motors; they each can take hundreds of thousands of consecutive steps, but do so in opposite directions along their microtubule tracks. These steps are fueled by the binding and hydrolysis of ATP within the homodimer's two identical protomers. Individual motors achieve their processivity by maintaining asynchrony between the stepping cycles of each protomer, insuring that at least one protomer always maintains contact with the track. How dynein coordinates the asynchronous stepping activity of its protomers is unknown. We developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we found that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use "hand-over-hand" mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of both cytoplasmic dynein and kinesin-1. To investigate the mechanisms that coordinate the interactions between motors within an ensemble, we constructed programmable synthetic cargos using three-dimensional DNA origami. This system enables varying numbers of DNA oligonucleotide-linked motors to be attached to the synthetic cargo, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical- polarity motors, we found that motor number had minimal effect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

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A fast emerging technology in the microelectronics field is bottom-up self-assembly of computer circuitry. A promising method to develop nanoelectronic devices through bottom-up self-assembly is the implementation of DNA-based technologies. Using DNA to create nanoelectronic devices is advantageous because of its already well understood base-paring and annealing qualities. These base-pairing and annealing qualities can be used to design and construct DNA nanostructures called DNA origami. DNA origami are specially designed structures made from single stranded DNA. Short single stranded DNA oligonucleotides called staple strands attach to a large single stranded DNA called a DNA scaffold. DNA staple strands and DNA scaffold anneal to each other and fold into DNA origami. Constructing DNA origami is advantageous because structures can be made in a single folding step. In particular, bar-shaped DNA origami has proven to be a promising structure for nanoelectronics fabrication. Here, I present new research done to improve bar-shaped DNA origami design and fabrication for constructing bottom-up self-assembled templates for nanomaterial surface attachment. Furthermore, this work presents new methods for DNA origami agarose gel purification with the help of the DNA stabilizing molecule, 4'-aminomethyltrioxsalen (AMT). AMT is a photoreactive molecule that intercalates DNA and creates covalent crosslinks when irradiated by short wavelength ultraviolet light. Also, this work contains new research on a synthesized crosslinker and its role with AMT in nanoparticle surface seeding on DNA origami nanowire templates. Through its crosslinking properties, AMT serves as a DNA origami stabilizing molecule and also shows potential for seeding nanomaterials.

An important goal of nanoscience is the self-assembly of nanoscale building blocks into complex nanostructures. DNA is an important and versatile building block for nanostructures because of its small size, predictable base pairing, and numerous sequence possibilities. I use DNA origami to design and fold DNA into predesigned shapes, to assemble thin, branched DNA nanostructures as templates for nanoscale metal features. Using a PCR-based scaffold strand generation procedure, several wire-like nanostructures with varying scaffold lengths were assembled. In addition, more complex prototype circuit element structures were designed and assembled, demonstrating the utility of this technique in creating complex templates. My fabrication method for DNA-templated nanodevices involves a combination of techniques, including: solution assembly of the DNA templates, surface orientation and placement, and selective nanoparticle attachment to form nanowires with designed gaps for the integration of semiconducting elements to incorporate transistor functionality. To demonstrate selective surface placement of DNA templates, DNA origami structures have been attached between gold nanospheres assembled into surface arrays. The DNA structures attached with high selectivity and density on the surfaces. In a similar base-pairing technique, 5 nm gold nanoparticles were aligned and attached to specific locations along DNA templates and then plated to form continuous metallic wires. The nanoparticles packed closely, through the use of a high density of short nucleotide attachment sequences (8 nucleotides), enabling a median gap size of 4.1 nm between neighboring nanoparticles. Several conditions, including hybridization time, magnesium ion concentration, ratio of nanoparticles to DNA origami, and age of the nanoparticle solution were explored to optimize the nanoparticle attachment process to enable thinner wires. These small, branched nanowires, along with the future addition of semiconducting elements, such as carbon nanotubes, could enable the formation of high-density self-assembled nanoscale electronic circuits.

The technique of DNA origami utilizes the specific and limited bonding properties of DNA to fold single stranded DNA sequences of various lengths to form a predesigned structure. One longer sequence is used as a scaffold and numerous shorter sequences called staples, which are all complementary to the scaffold sequence, are used to fold the scaffold into intricate shapes. The most commonly used scaffold is derived by extracting the genome of the M13 phage and the staples are usually chemically synthesized oligonucleotides. Longer single stranded sequences are difficult to synthesize with high specificity, which limits the choices of scaffold sequences available. In this project two main methods of single stranded amplification, Rolling Circle Amplification (RCA) and the usage of helper phages, were explored with the goal to produce both a 378 nt scaffold and staple sequences needed for folding a DNA origami structure. To facilitate imaging by Transmission Electron Microscopy (TEM) of this small structure, the DNA origami structure was created to form a polymer structure. Production of the scaffold sequence in high yield was unsuccessful and no well-defined polymers were found in the folded samples, though a few results showed promise for further studies and optimizations. Due to time constraints of this project, only production of the scaffold sequence was tested. Unfortunately the scaffold produced by the helper phages was of the complementary strand to that used to design the DNA origami structure, and could therefore not be used for folding. The correct strand was produced by the RCA where the yield was too low when using Phi29 DNA polymerase for proper folding to take place, though small scale RCA by Bst DNA polymerase on the other hand showed promising results. These results indicate that the scaffold production may not be far off but still more experience in producing intermediate size oligonucleotides may be necessary before succeeding in high yield production of this 378 nt long sequence. The promise given by this production is to enable high yield, high purity, low cost and also an easily scalable process set-up. This would be an important step in future DNA nanotechnology research when moving from small scale laboratory research to large scale applications such as targeted drug delivery systems.

An outstanding challenge in modern medicine is the safe and efficient delivery of drugs. One approach to improve drug delivery yield and increase specificity towards diseased cells, is to employ a drug carrier to facilitate transport. Promising steps towards developing such a carrier have been taken by the nascent field of nanomedicine: nanometer-sized particles designed to evade premature excretion, non-specific absorption, and the body’s immune response, can reduce undesired drug loss, while also increasing specific drug uptake into diseased cells through targeting surface modifications. However, progress is limited by incomplete knowledge of the ‘ideal’ nanoparticle design as well as a lack of appropriate high resolution construction methods for its implementation. DNA origami, a modular, nanometer-precise assembly method that would enable the rapid testing of particle properties as well as massively parallel fabrication, could provide an avenue to address these needs. In this thesis, I employed the DNA origami method to investigate how nanoscale shape and ligand functionalization affect nanoparticle uptake into cultured endothelial cells. In the first part, I evaluated the uptake yield of a series of eight shapes that ranged from 7.5 nm to 400 nm in their individual dimensions. The best performing shape of that study, a 15 × 100 nm DNA origami nanocylinder, was internalized 18-fold better than a dsDNA control of the same molecular weight. In a follow up study, I decorated this nanocylinder with integrin-targeting cyclic RGD peptides. This surface functionalization increased cellular uptake another 13-fold. In addition, uptake yield and the ratio of internalized versus surface-bound particles depended on the number of ligands present on the nanoparticle surface. This work represents a significant first step towards attaining the ability to design and implement an 'ideal' nanoparticle drug carrier. In the future, the DNA origami method can be used as a platform technology to further expand our understanding of transport properties of drug carriers and achieve safer and more efficient drug delivery.

In this thesis, the folding process of the de novo designed polypeptide chignolin was elucidated through atomic-scale Molecular Dynamics (MD) computer simulations. In a series of long timescale and replica exchange MD simulations, chignolin’s folding and unfolding was observed numerous times and the native state was identified from the computed Gibbs free-energy landscape. The rate of the self-assembly process was predicted from the replica exchange data through a novel algorithm and the structural fluctuations of an enzyme, lysozyme, were analyzed. DNA’s structural flexibility was investigated through experimental structure determination methods in the liquid and gas phase. DNA nanostructures could be maintained in a flat geometry when attached to an electrostatically charged, atomically flat surface and imaged in solution with an Atomic Force Microscope. Free in solution under otherwise identical conditions, the origami exhibited substantial compaction, as revealed by small angle X-ray scattering. This condensation was even more extensive in the gas phase. Protein folding is highly reproducible. It can rapidly lead to a stable state, which undergoes moderate fluctuations, at least for small structures. DNA maintains extensive structural flexibility, even when folded into large DNA origami. One may reflect upon the functional roles of proteins and DNA as a consequence of their atomic-level structural flexibility. DNA, biology’s information carrier, is very flexible and malleable, adopting to ever new conformations. Proteins, nature’s machines, faithfully adopt highly reproducible shapes to perform life’s functions robotically.

<p>DNA origami is a novel self-assembly technique that can be used to form various </p><p>2D and 3D objects, and to position matter with nanometer accuracy. It has been </p><p>used to coordinate the placement of nanoscale objects, both organic and inorganic, to make molecular motor and walkers; and to create optically active nanostructures. In this dissertation, DNA origami templates are used to assemble plasmonic structures. Specifically, engineered Surface Enhanced Raman Scattering (SERS) substrates were fabricated. Gold nanoparticles were selectively placed on the corners of rectangular origami and subsequently enlarged via solution-based metal deposition. The resulting assemblies exhibited "hot spots" of enhanced electromagnetic field between the nanoparticles. These hot spots significantly enhanced the Raman signal from Raman molecules covalently attached to the assemblies. Control samples with only one nanoparticle per DNA template, which therefore lacked inter-particle hot spots, did not exhibit strong enhancement. Furthermore, Raman molecules were used to map out the hot spots' distribution, as the molecules are photo-damaged when experiencing a threshold electric field. This method opens up the prospect of using DNA origami to rationally engineer and assemble plasmonic structures for molecular spectroscopy.</p> / Dissertation