The solution structure determination of DNA molecules has been an important part of structural biology. NMR solution structures are a complement to structures solved via X-ray crystallography; the two methods are the only ways of obtaining three dimensional coordinates of macromolecules. Because of the nature of the molecule, the solution structure determination of DNA has been a challenging task. Assignments are the first and most important part of NMR structures, and can be simplified for DNA with the use of the rotating frame Overhauser spectroscopy (ROESY) experiment. The ROESY technique can be used for unambiguous assignments of H2' and H2" protons and for distinguishing the three main forms of DNA duplexes: A-form, B-form and Z-form. Many types of DNA have been examined using NMR spectroscopy, including drug-bound DNA complexes. Most previous studies of complexes of the anti-cancer drug Actinomycin D (ActD) and DNA used self- complementary sequences to identify stabilizing features. The studies presented in this thesis use non-self-complementary DNA hexamers to identify the two orientations in the binding of the asymmetric ActD drug. The largest preference of asymmetric binding was found for the d(CCGCCG)•d(CGGCGG) sequence; however, NMR spectral complications prevented the structure elucidation of this complex. Instead the solution structure was determined for the complex with the next largest orientational preference, ActD:d(CTGCGG)•d(CCGCAG), which has 67% of ActD molecules intercalated with the benzenoid side of ActD in the first strand. The solved structure identifies unusual DNA features, which could be due to the bound drug inducing structural changes to the B-DNA duplex or the presence of conformational motion. For seven of the eight sequences, the orientation of ActD intercalation within the DNA duplex was identified. The largest preference occurs when the benzenoid intercalation site is followed by a guanine. When this guanine is replaced by an inosine, a reduction in the asymmetric binding of ActD is observed, indicating that the guanine NH₂ group plays a role in the intermolecular contacts. Thus, the two orientations of ActD binding are not present in equal concentrations although their structures are similar, and the preference of orientation is influenced by the asymmetric DNA sequence flanking the intercalation site. / Graduation date: 2002

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 156-165). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 144-154). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2005. Directed by Lawrence A. Bottomley. / William D. Hunt, Committee Member ; Nicholas V. Hud, Committee Member ; L. Andrew Lyon, Committee Member ; Lawrence A. Bottomley, Committee Chair ; Loren D. Williams, Committee Member. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 464-475). Available also in a digital version from Dissertation Abstracts.

Methods for delivery of genes and agents which bind to nucleic acids, and thereby modify the expression of genes, are areas of intensive research. The focus of the present study is the initial development and characterization of an enteric delivery system for DNA-binding drugs. Several fluorescent probes were screened as to their suitability for analysis of DNA in alginate beads. A UV transilluminator was used to identify SYBR I and SYBR II as two functional fluorescent probes for DNA within intact alginate beads. A methyl green-DNA complex was found to be useful for monitoring the dissolution of alginate beads and release of an intact drug-DNA carrier system. After dissolution of alginate beads containing DNA, addition of DNase I to the dissolution fluid resulted in the complete hydrolysis of DNA. This is a necessary condition for the release of a DNA binding drug from the DNA carrier system in that hydrolysis of the carrier by enteric nucleases must occur in the presence of alginate. Once released from a DNA carrier, protein binding plays an important role in the disposition of these agents. A phosphorothioate oligonucleotide complexed to a methidium-spermine affinity gel was used as a tool to study oligonucleotide binding proteins . Bovine serum albumin was used as a prototype binding protein and was found to elute as a single peak from the oligonucleotide affinity column. Elution was accomplished with a sodium chloride gradient. This approach may be useful for characterization of other oligonucleotide binding proteins.

Two diverse works regarding DNA-Drug Interaction are presented here. The first portion deals with covalent interactions between compounds that are derivatives of heterocyclic aza-enediynes and DNA (conventional Watson-Crick base paired double stranded DNA) and the second is related to non-covalent interactions of these compounds with G-quadruplex DNA. The aza-enediynes have been studied for their ability to undergo aza-variants of the Bergman and Myers cyclizations, and the potential role of the ensuing diradicals in DNA cleavage chemistry. The aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes has been recently reported. In the first part of the dissertation, the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-Npropargyl pyridine salts) are reported. Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. An optimized analog containing a p-methoxyphenyl substituent was prepared. Studies with radiolabeled DNA duplexes reveal that this analog generates nonselective frank DNA strand breaks, via deoxyribosyl 4'-hydrogen atom abstraction, and also leads to oxidation of DNA guanine bases. This is the first report of enediynelike radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization. The second part is based upon the growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for replication. A prototypical replicative helicase - SV40 large T-antigen (T-ag), a multifunctional protein with duplex DNA helicase activity is shown to also unwind G-quadruplex DNA structures. A series of G-quadruplex-interactive agents, particularly perylene diimide derivatives, is explored for inhibition of T-ag duplex and G-quadruplex DNA unwinding activities, and it is revealed that certain perylene diimides are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic Gquadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.S.)--Rochester Institute of Technology, 1990. / Includes bibliographical references, (leaves 108-110).

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