The creation of molecular machines has been one of the goals of modern nanotechnology for a few decades. Such machines can be assembled from small molecules, as well as DNA. Of particular interest are mechanically interlocked nanoconstructs – catenanes and rotaxanes. These structures offer developments such as nanoswitches and rotational motors. DNA nanotechnology has produced numerous systems that consist of catenanes that could perform programmable switching and stimuli-responsive behaviour, as well as switching between stations in a semi-autonomous, rotary, motor-like behaviour. Energy transduction and the speed of such Brownian ratchet motors are negligible when compared to natural enzymatic activity. Bridging the gap between enzymology and structural DNA nanotechnology, we propose a method to assemble and operate a prototype system of fully complementary interlocked ssDNA rings that can roll against each other as a pair of gears with a ratio 1:2. The directionality and force is proposed to be generated by a strand-displacing polymerase enzyme performing a rolling circle amplification reaction on one of the members of this catenane, generating torque. Computer modelling of this system using oxDNA script package has been carried out, enabling both the topological visualisation and structural inquiry into the system prior to experimental development. Variations to the system such as changing the overall size, gearing ratio and developments towards integration into larger assemblies have also been described and are discussed in detail. Several experimental assembly strategies are described, together with experimental evidence of their outcomes. A method for operation of the single-stranded DNA catenane as a pair of continuously rolling gears has been investigated using strand displacing polymerases. Applications and suggestions for future developments is provided, addressing integration into complex systems. Additional methods of assembly and operation are discussed and compared.

Differential scanning calorimetry (DSC) melting analysis was performed on 27 short double stranded DNA duplexes containing 15 to 25 base pairs and short single stranded overhangs from one to 10 bases, on both ends. Molecules have two 5' dangling ends or one 5' and one 3' dangling end. For these molecules the duplex region was incrementally reduced from 25 to 15 base pairs with increased length of the dangling ends from one to 10 bases. A third set of molecules contained 21 base pair duplexes with a four base dangling end on either the 5' or 3' end. Blunt ended duplexes from 15 to 25 base pairs were also examined and served as control duplexes. DSC melting curves were measured in solution containing 85 mM, 300 mM or 1.0 M Na+. From these measurements, thermodynamic parameters for 5' and 3' dangling ends as a function of end length were evaluated. Results showed the 5' ends were slightly stabilizing, and this stability was essentially constant with end length, while the 3' ends were generally destabilizing with increasing length of the end. This finding of lower stability for the 3' ends is consistent with results of published studies that have found 5' dangling ends to be more than or equally as stabilizing as 3' dangling ends. Our finding that 3' dangling ends are actually destabilizing for duplex DNA contrasts with published results. The 3' ends also display a stronger dependence on the [Na+]. In the lower Na+ environment the 3' ends are more destabilizing than at the higher salt environments. Analysis of the thermodynamic parameters of the dangling-ended duplexes as a function [Na+] indicated the 3' dangling end molecules behave differently compared to 5' dangling ended and blunt ended duplexes. The net counterion release per phosphate upon melting the molecules having one 5' and one 3' end was approximately 15% smaller as a function of end length compared to the duplex having two 5' ends. Further analysis of the DSC evaluated thermodynamic transition parameter, ΔHcal, and its relationship to the measured transition temperatures of the DNA molecules, provided an estimate on the excess heat capacity differences, ΔCp, between duplex and melted single strands for the dangling-ended molecules. The analysis revealed the molecules with one 5' and one 3' dangling end had very different ΔCp values compared to the blunt-ended molecule; while the molecules with two 5' ends have ΔCp that are essentially the same as the blunt-ended duplex. These observations are interpreted as differences in the interactions with Na+, solvent and the terminal base pairs of the duplex for the 5' versus 3' dangling ends.

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

Thesis Erasmus University Rotterdam. / ook verschenen in gedrukte versie. With bibliogr., with a summary in Dutch.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, 2002. / Includes bibliographical references (p. 157-158). Also available on the Internet.

Not available / text

No description available.

Thesis (Ph.D.)--University of Melbourne, Dept. of Paediatrics, Faculty of Medicine, 2006. / Typescript. Includes bibliographical references (leaves 287-313).

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, Aug. 2008. / Title from title screen. Includes bibliographical references.