DNA origami is a method for constructing 2-dimensional nanostructures with arbitrary shapes, by folding a long piece of viral genomic DNA into an extended pattern (Rothemund, 2006). In this thesis DNA origami nanostructures that in- corporate active transport are developed, by combining rectangular DNA origami tiles with either synthetic DNA motors, or the protein motor F1-ATPase. The transport of an autonomous, unidirectional, and processive 'burnt-bridges' DNA motor across an extended linear track anchored to a DNA origami tile is demonstrated. Ensemble fluorescence measurements are used to characterise motor transport, and are compared to a simple deterministic model of stepping. The motor moves 100 nm along a track at 0.1 nms-1 Atomic force microscopy (AFM) is used to study the transport of individual motor molecules along the track with single-step resolution. A DNA origami track for a 'two-foot' DNA motor is also developed, and is characterised by AFM and ensemble fluorescence measurements. The burnt-bridges DNA motor is then directed through a track network with either 1 or 3 bifurcations. Ensemble fluorescence measurements demonstrate that the path taken can be controlled by the addition of external control strands, or pre-programmed into the motor. A method for attaching the rotary motor protein F1-ATPase to DNA origami tiles is developed. Different bulk and single-molecule methods for demonstrat- ing protein binding are explored. Single-molecule observations of rotation of the protein motor on a DNA origami substrate are made, and are of equivalent data quality to existing techniques.

Four-stranded DNA junctions (also known as Holliday junctions) are structural intermediates involved in a growing number of biological processes including DNA repair, genetic recombination, and viral integration. Although previous studies have focused on understanding the conformational variability and sequence-dependent formation of Holliday junctions in solution there have been relatively few insights into junction structure at the atomic level. Recent crystallographic studies have demonstrated that the more compact stacked-X junction form has an antiparallel alignment of DNA strands and standard Watson-Crick base pairs across the central crossover region. Junction formation within this crystallographic system was seen to be dependent on a common trinucleotide sequence motif ("ACC-triplet" at the 6th, 7th and 8th positions of the decanucleotide sequence d(CCnnnN₆N₇N₈GG)) containing a series of stabilizing direct and solvent-mediated hydrogen bonding interactions. This thesis addresses questions concerning the nucleotide sequence-dependent formation and conformational variability of DNA Holliday junctions as determined by single crystal x-ray diffraction. We have used the modified bases 2,6-diaminopurine and inosine to demonstrate that minor groove interactions adjacent to the trinucleotide junction core are not major contributors to overall conformation. In addition, incorporation of guanine into the sixth position of this core does not have a significant effect on junction geometry. Meanwhile, incorporation of 5-bromouracil into the eighth position perturbs the geometry in terms of the interduplex angle as well as the defined conformational variables, J[subscript roll] and J[subscript slide]. These novel junction structures demonstrate that the nucleotide sequence within the central core generates a position specific relationship between molecular interactions at the junction crossover and overall structural geometry. A systematic crystallographic screen of the trinucleotide core region is presented here as an unbiased, comprehensive, search for sequences that stabilize junctions. As the result of this screen, we can extend the core sequence motif to 'N₆Y₇C₈' where N₆ is an adenine, guanine, or cytosine nucleotide and Y₇ is either a cytosine or thymine (if N₆ = adenine) nucleotide. Using these novel junction structures, we demonstrate that base sequence within the central core has a significant effect on the overall geometry of the junction. Thus, this central region of the structure may serve as a linchpin for determining the local and global conformation and overall variability of the four-stranded DNA Holliday junction. These observations raise some interesting questions regarding the importance of this core region in biological processes such as genetic recombination. / Graduation date: 2005

DNA methylation is common in prokaryotes and eukaryotes and has been implicated in various biological roles including gene silencing, X-chromosome inactivation, and genomic imprinting. 5-methylcytosine the "fifth base" of the genetic code comprises 1-3% of the human genome and is primarily found on cytosines within the context of the CpG sequence. Although progress has been made in understanding the biological roles of 5-methylcytosine, we are only beginning to uncover how it changes the local structure and global conformation of DNA. This thesis deals with the local perturbations in structure and hydration and the global conformational changes induced by the presence of 5-methylcytosine in DNA as determined by single crystal x-ray diffraction. 5-methylcytosine induces a novel conformation in the structure of duplex DNA. This conformation has characteristics of both the A-DNA and B-DNA conformations as well as some unique defining characteristics. This distinct duplex provides a structural rationale for the increased rate of deamination in 5-methylcytosine relative to cytosine. In addition to this novel conformation, 5-methylcytosine stabilizes intermediates within the B-DNA to A-DNA transition pathway, thus providing a crystallographic map of the transition from B-DNA to A-DNA. 5-methylcytosine was also used as a tool to probe the stabilizing features of the DNA four-way junction (known as the Holliday junction). The first crystal structures of Holliday junctions were found serendipitously while studying duplex DNA. The DNA four-way junction formation in these crystals was thought to be stabilized by a network of sequence dependent hydrogen bonds at the junction crossover. In this thesis, 5-methylcytosine was used to perturb these hydrogen bonds; however, the junction persisted, suggesting that there is flexibility in the types of sequences that can accommodate junction formation in the crystal, as well as, flexibility in the global structure of the junction. Overall, this work describes the effects of 5-methylcytosine on the local and global structure and hydration of DNA structure, as well as raising some interesting questions regarding the biological impact of methylation induced DNA structure. / Graduation date: 2002

As genome sequencing projects begin to come to completion, the challenge becomes one of determining how to understand the information contained within the DNA. DNA is a polymorphic macromolecule; the A- B- and Z-DNA conformations have been observed by a variety of physical techniques. The magnitude of the energetic differences between these conformations suggests that these conformations may be important biologically and thus relevant in the analysis of genomes. A computer program, NASTE, was developed to evaluate the helical parameters of the set of Z-DNA crystal structures in order to determine the true conformation of Z-DNA and to understand the effects of various factors on the observed structure and stability. A thermodynamic method, elucidated in part with a genetic algorithm, was developed to predict the sequence-dependent propensity of DNA sequences for A- versus B-DNA in both the crystal and in natural DNA. Predictions from this method were tested by studying the conformation of short oligonucleotides using circular dichroism spectroscopy. Finally, the thermodynamic method was applied in an algorithm, AHUNT, to identify regions in genomic DNA with a high propensity to form A-DNA. Significant amounts of A-DNA were identified in eukaryotic and archeabacterial genes. E. coli genes have less A-DNA than would be predicted from their (G+C) content. These results are discussed with respect to the intracellular environment of the genomes. / Graduation date: 1998

Graduation date: 1994

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