This thesis highlights novel synthetic routes towards the facile synthesis of lariat DNA and RNA oligonucleotides, and the utilities of branched (bNAs) and circular (i.e. dumbbell-shaped) nucleic acids for targeting biologically relevant processes (i.e. HIV proliferation, alternative RNA splicing) with potential therapeutic applications. / An innovative synthetic strategy for the synthesis and cyclization of a medium-sized (21-nucleotide) DNA lariat starting from a CPG-tethered, convergently synthesized branched DNA (bDNA) molecule was devised in Chapter 2. This synthetic route exploited the differential cleavage rates of two CPG-oligonucleotide tethers, namely the base-labile hydroquinone-O,O'-diacetate (Q-linker) and the more robust succinate (S-linker ) linkages, as well as phosphitylation of the 5'-oligonucleotide terminus and cyclization under standard phosphoramidite coupling conditions to effect new phosphodiester bond construction. The results clearly indicate a disadvantageous correlation between high branching efficiency on a densely loaded CPG and the production of dendrimeric (i.e. hyperbranched) oligonucleotide species rather than effective cyclization. / Given the entropic disadvantage of synthesizing medium-sized DNA lariats on solid-support using the method described in Chapter 2, unique intermolecular (i.e. DNA dumbbells) and intermolecular template-mediated approaches for lariat cyclization commencing with convergently and divergently synthesized bDNAs and bRNAs were developed in Chapter 3. Both methods lead to the exclusive and high-yielding formation of medium sized (46--57 nucleotides) DNA and RNA lariats. Parameters for successful phosphodiester bond construction were also elucidated in both systems. / A novel class of highly specific and potent oligonucleotide-based HIV-1 reverse transcriptase inhibitors, RNA dumbbells, comprising of a 10 base-pair stem and two flanking UUCG hairpin-loop motifs are described in Chapter 4. Explicitly, such constructs were capable of selectively hampering the RNase-H mediated activity of the retroviral enzyme without consequence to its DNA polymerase function with an IC50 in the 3 muM range. Its precise interaction with the RNase H domain of RT was authenticated via a UV-crosslinking assay. Furthermore, the RNA dumbbells did not inflict any effect on mammalian RNase H activity, suggesting that such compounds would not obstruct cellular RNase H function. / Chapter 5 describes the utility of synthetic bRNA for the inhibition and modulation of pre-mRNA splicing in yeast and mammalian in vitro systems. Most notably, synthetic bNAs can be suitably exploited as agents for the study of branchpoint recognition during in vitro splicing of a pre-mRNA transcript. The results clearly indicate the requirement for a fully formed branchpoint (i.e. 5 '-, 2'- and 3'-extensions; Y-shaped molecules) off the conserved branchpoint adenosine for efficient splicing inhibition. Specific methods for stabilizing bNAs against ubiquitous cellular exo- and endonucleases as well as the 2'-scissle (2'-debranching) activity present in the HeLa extract milieu are also described.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84488 |
| Date | January 2003 |
| Creators | Carriero, Sandra |
| Contributors | Damha, Masad J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002083309, proquestno: AAINQ98221, Theses scanned by UMI/ProQuest. |
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