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Thesis advisor: Larry W. McLaughlin / Nucleic acids are essential biological molecules that encode and transfer genetic information from generation to generation. Intensive efforts have been made by scientists to study the properties of nucleic acids, looking for opportunities that could help diagnose, prevent, and cure disease, and/or gain a greater insight into the wonder of nature. Chapter 2 presents our synthetic attempts towards the rigidified nucleosides 2'-deoxy-6,3'-propanouridine and 2'-deoxy-6,3'-butanouridine. These nucleosides are constrained so that they mimic the native conformation in DNA duplex and are postulated to increase duplex stability, as well as increase the affinity of the nucleobase for its complementary partner. Chapter 3 presents work towards the synthesis of backbone extended nucleosides. These molecules have the potential to form a new type of helical structure when incorporated into a double helix. Through the investigation of these novel nucleic acids, we would like to gain a greater understanding of the properties that contribute to duplex stability. / Thesis (MS) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Larry W. McLaughlin / Thesis advisor: Mary F. Roberts / Nucleic Acids are unique biopolymers capable of encoding and transferring genetic information from one generation to the next for every form of life. This fascinating property has made them the topic of intense research from a variety of aspects. Some researchers try to understand how life might have started. Some try to elucidate how the whole process works. Some try to use the properties of nucleic acids as a tool for various purposes. The continuous effort over more than a century explored a lot about the structures and functions of nucleic acids. There is a lot to be discovered yet. This work began with the design and development of a new class of nucleoside analogue with the goal to study their ability to bind nucleic acids. The ongoing research will establish their application as therapeutics and as biomolecular tools. Along the way significant effort went into preparing these analogues. New methodology was developed to address some of the unanswered synthetic problems of nucleoside chemistry. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The purpose of this thesis was to study the nucleic acids of Capsicum annuum plastids during fruit ripening. The ultrastructural characteristics of Capsicum annuum plastids were studied during the transition between chloroplast and chromoplast, revealing the complex changes in membrane structure. Ploidy changes in ripening plastids was followed using a new experimental system, the Leitz MPV3 microphotometer. The relative changes in fluorescence of DAPI-stained plastids was measured. Chromoplasts proved to contain a UV-absorbing substance which reduced the level of fluorescence. The amount of plastid DNA decreases from leaf chloroplasts to green pepper plastids, and possibly decreases further in the chromoplast. It was concluded that the system was unsuitable for use with plastids of changing pigment content. A ripeness index of pepper fruit was formulated using a general pigment extraction. Ripeness was assessed by the ratio of absorbance measured at 470 and 430nm of this extract. The use of an in vitro ripening system was investigated to counter the problems of availibility and uneven ripening of green-house ripened fruit. Similarity between the chloroplast and chromoplast genome was examined by comparison of Bam Hl digestion patterns. The resolved gel bands were identical. Clones were created containing Capsicum annuum chloroplast DNA using a modified lambda bacteriophage, lambdaL47 vector. The lambdacap clones were investigated using Bam Hl restriction endonuclease digests. The enzyme digests revealed fragments in the 4-8.5kb region, and l8kb upwards. It was concluded that the 4-8.5kb fragments comprised the foreign DNA insertion. Hybridization of chloroplast DNA with lambdacap clones gave low levels of hybridization, which was concluded to be due to impurities in the plastid DNA. Hybridization of lambdacap clones to each other revealed that 17 of the 22 clones are closely related. l6s rRNA extract hybridized with certain lambdacap clones, although clones containing known chloroplast genes for LSU, beta and alpha subunits of ATPase and cytochrome f did not. Collation of this evidence, together with the Bam Hl fragment patterns led to the conclusion that the lambda cap clones map in the small single copy region and part of the inverted repeat region. Study of gene expression during the chloroplast-chromoplast transition was studied by hybridization of RNA, isolated from pepper fruit ripened in vitro, with lambda cap clones. Evidence showed changes in expression accompanied fruit ripening.

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A novel protocol for the study of protein-nucleic acid interactions is presented and demonstrated to be feasible. The protocol combines photochemical crosslinking techniques and mass spectrometric methods into a new strategy for identifying protein domains or amino acid residues that are in close contact with nucleic acid in protein-nucleic acid complexes. Identifying nucleic acid binding domains in proteins provides a starting point for understanding structure-function relationships in protein-nucleic acid complexes. The protocol can be divided into three parts: 1) Cross linking of the protein-nucleic acid complex by irradiation with ultraviolet light and subsequently verifying the crosslinking by mass spectrometry; 2) Mass spectrometric peptide mapping of crosslinked protein-nucleic acid complexes to identify crosslinked peptide-nucleic acid hybrids; 3) Tandem mass spectrometric sequencing of peptide-nucleic acid hybrids to localize the crosslinked amino acid residue(s). The experimental data described in this dissertation documents our efforts to establish and implement this analytical protocol. Using several different protein-nucleic acid systems and different crosslinking techniques, we have demonstrated the feasibility of a mass spectrometric based approach to structurally characterize UV-crosslinked protein-nucleic acid complexes. Matrix-assisted laser desorption/ionization mass spectrometry was for the first time demonstrated to be highly effective for detection and molecular weight determination of intact, UV-crosslinked protein-nucleic acid complexes and for molecular weight determination of synthetic and UV-crosslinked peptide-nucleic acid hybrids. Electrospray ionization mass spectrometry and tandem mass spectrometry was demonstrated to be effective for analysis of synthetic peptide-nucleic acid hybrids and, in conjunction with HPLC, for peptide mapping of a protein. The first application of MALDI mass spectrometry to the characterization of crosslinked peptide-nucleic acid hybrids isolated from a photochemically crosslinked protein-nucleic acid complex demonstrate that the new protocol can be used to identify nucleic acid binding domains in proteins. / Graduation date: 1995

The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle were investigated by studying the time-resolved fluorescence anisotropy decays of ethidium bromide intercalated into the DNA of the core particle. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. The anisotropy decays were collected using the method of time-resolved single-photon counting and were fit to a model developed by J. M. Schurr (Schurr, 1984) using a non-linear least squares fitting algorithm developed by the author for this purpose. It was shown that below a binding ratio of 0.1 there was no demonstrable change in the anisotropy as a function of binding ratio. Our results show, that the apparent torsional flexibility of DNA of to a nucleosome core particle is dependent on the number of base pairs of the DNA between points of attachment to the histone core. If this number is as high as 30 base pairs, then the torsional flexibility of DNA on a nucleosome core particle is as high or higher than DNA free in solution. Also, for reasonable values of N, the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the DNA on a core particle is highly constrained in its motions. The hydrogen ion concentration was shown to have a substantial effect on the fluorescent anisotropy decays, particularly in the early regions of the decay. These analyses indicated that the observed change could be attributed to either a loosening of the contacts between the DNA and the histone core, or a relaxing of the torsional flexibility of the DNA. / Graduation date: 1991

Thesis (Ph. D.)--School of Biology, Georgia Institute of Technology, 2004. Directed by Roger M. Wartell. / Vita. Includes bibliographical references (leaves 150-159).

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