The generation of synthetic oligonucleotides is dependent on an efficient solid-phase synthesis methodology. This thesis evaluates the 2'-O -levulinyl (Lv) and 2'-O-monomethoxytrityl (MMT) ribonucleosides, as possible synthons for RNA and branched RNA synthesis. A key feature of this RNA and bRNA synthesis procedure is their removal while still attached to the solid support and under conditions that prevent isomerization or cleavage of the nascent strands. For the first time, the stability of 3'-5'-internucleotide phosphate triesters (and diesters) adjacent to a ribose 2'-hydroxyl group was determined on a solid support. These studies are not only relevant to the proper assembly of branched and linear RNA species, but also to the stability of an unusual branched RNA species ("RNA X") proposed to form during the pre-mRNA splicing reactions in vitro. These studies are also important to the development of large quantities of native and chemically modified short interfering RNA (siRNA) for animal and human studies. / The 2'-O-Lv and 2'-O-MMT ribonucleoside monomers served as building blocks for the assembly of a series of branched nucleic acid species (bRNA, bDNA, msDNA and hyperbranched or "dendritic" DNA/RNA) with discrete length, base composition and structure. These structures were synthesized via an iterative divergent-growth strategy, which facilitates the regioselective branching, deblocking and chain lengthening steps from a branchpoint core. These structures served as useful materials (bio-probes) as demonstrated by the biological studies performed with E. coli RNaseH and the yeast lariat RNA debranching enzyme (yDBr1). These studies not only led to the identification of novel branched nucleic acid inhibitors of yDBR1 and RNase H, but also provided new insights about the substrate specificity of these important enzymes. / This thesis also describes the synthesis of a new nucleic acid form, the so-called "oxepane nucleic acids" (ONAs), in which the pentofuranose ring of DNA and RNA was replaced with a 7-membered heptose sugar ring. ONA were found to be much more resistant towards nuclease degradation than natural DNA, an important feature if these analogues are to be used in biological media. Furthermore, ONAs exhibited cross-pairing with complementary RNA and were found to elicit E. coli RNaseH mediated degradation of the RNA strand. These finding are significant because oligonucleotide-directed RNase H degradation of the target RNA is a key determinant for the gene-specific inhibitory potency of antisense oligonucleotides. When comparing the rates of RNase H-mediated degradation induced by 5 (furanose), 6 (2'-ene-pyranose) and 7 (oxepane) membered ring oligonucleotides, the following trend was observed: DNA > 2'-ene-pyranose NA > ONA. The implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme, particularly with regard to the required flexibility of the oligonucleotide strands that bind to the RNA target. Hence, ONAs are useful tools for biological studies and provide new insights into the structure/function of natural and alternative genetic systems.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.103291 |
| Date | January 2007 |
| Creators | Sabatino, David. |
| 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 | © David Sabatino, 2007 |
| Relation | alephsysno: 002665344, proquestno: AAINR38641, Theses scanned by UMI/ProQuest. |
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