Thesis advisor: Larry W. McLaughlin / The ability to control gene expression has traditionally been pursued at the protein level, using drugs designed to mimic a natural substrate or to disrupt a protein's active site. Traditional drug targeting by competitive and non-competitive inhibitors, however, requires a fairly detailed knowledge of the target protein's three-dimensional structure. More recently, focus has broadened to include alternative methods of genetic control, including the use of single-stranded DNA or RNA probe sequences which control gene expression by targeting the genes themselves. Within the last two decades, peptide nucleic acids (PNAs) – DNA mimics possessing natural bases linked to an N-(2-aminoethyl)-glycine (AEG) backbone – have proven as effective in gene-targeting as traditional synthetic DNA or RNA with the added advantages of tighter binding and greater specificity. Additionally, PNAs are not easily recognized by nucleases, proteases, and peptidases giving them greater resistance to enzyme degradation and making them even more favorable for gene targeting in vivo. Traditional PNA triplexes are composed of two polypyrmidine PNA strands bound to the Watson-Crick and Hoogsteen faces, respectively, of the polypurine strand of target DNA after displacing the polypyrimidine strand of the original DNA duplex. Janus Wedge (JW) residues, on the other hand, utilize unnatural bases linked to the AEG backbone, which are capable of hydrogen bonding to the Watson-Crick faces of both strands of a target DNA duplex. JW triplex formation, then, has a DNA2-PNA stoichiometry, and no Hoogsteen face interactions. The generalization of the DNA duplex targeting strategy by peptide oligomers requires substantial discoveries in the field of PNA research, including an understanding of the three-dimensional structure and folding pattern of these triple-stranded molecules. This report details the crystallization efforts on JW DNA-peptide-DNA triplexes using 11dC811-11T811 target sequences – with and without single base overhangs – and synthetic W8K peptide. Hanging drop vapor diffusion methods showed that while crystal formation was extremely elusive, in narrowing the optimal buffer conditions, 25% PEG concentration was consistently correlated with the most promising crystallization efforts for both the overhanged and non-overhanged sequences. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_102269 |
| Date | January 2005 |
| Creators | Hemak, Michael Joseph |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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