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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.

This thesis presents the development of novel methodologies in the template mediated chemical synthesis of lariat and branched nucleic acids. The synthetic branched DNA and RNA may be applicable as probes in the elucidation of the splicing mechanism or as potential therapeutic agents. Furthermore, this body of work describes the novel synthesis of Ru(II) branched DNA as building blocks in the supramolecular assembly of nano-motifs. In general, insight into the utilization of nucleic acids as biological molecules and as nanomaterials is presented at the interface of chemistry and biology. / Chapter 2 delineates the regioselective template directed synthesis of Y-RNA via chemical ligation at the branch point of a 5'-phosphate to a 2'-hydroxyl. The branched molecules resemble lariats as they possess the analogous branched architecture. The oligonucleotide components are synthesized from commercially available phosphoramidite building blocks through automated solid-phase synthesis. A unique template directed method in the synthesis of DNA and RNA lariats is proposed in Chapter 3. The regioselective chemical ligation affords wild-type DNA and RNA formed through assembly of a single oligonucleotide strand. A parallel DNA:RNA hybrid association was observed in the preorganized assembly and extensively characterized. Characterization of the Y-RNA and lariat nucleic acids were carried out through techniques such as thermal denaturation analysis, polyacrylamide gel electrophoresis, enzymatic degradation with the RNA lariat debranching enzyme, alkaline treatment as well as MALDI-TOF mass spectrometry. / The second part of the thesis exploits DNA as a nanomaterial in the convergent solid-phase synthesis of Ru(II)-DNA conjugates as branched building blocks in the assembly of nanostructures. Chapter 4 describes the synthesis of Ru branched DNA, utilizing cis-[(bpy)2Ru(imidazole) 2]2+ moiety as the vertex tethered to parallel DNA covalently through flexible hexamethylene linkers. Complete physical characterization and preliminary hybridization studies are conducted. The Ru-DNA conjugates presented were found to be unstable to the protocols required for synthesis of mixed sequence derivatives. The stability and scope of synthesis of these molecules are further discussed. / As an alternative, Ru-DNA branched complexes of mixed sequences, exhibiting greater stability, were synthesized. The transition metal building blocks of Chapter 5 employ a more rigid branch point, linking two parallel DNA strands through a one methylene spacer to the cis-[Ru(bpy)2 (4,4'-bis(hydroxymethyl)-2,2'-bipyridine)][PF6]2 vertex. Physical characterization and the intrinsic luminescent properties of the transition metal complex were confirmed in both the Ru-branched DNA and hybrized forms. A comparative study of the self-assembly behavior of the Ru-DNA conjugates to that of unmetallated branched DNA was also conducted. Interestingly, results indicate a metal-mediated assembly of almost exclusive formation of one discrete Ru-DNA dimeric cyclic nanostructure, where as unmetallated DNA building blocks produced an array of products. Complete confirmation of these products is presented through PAGE and enzymatic digestions. Finally the synthesis of novel Delta and Λ Ru-branched DNA diastereomers is presented as potential building blocks in the creation of chiral metallo-supramolecular constructs.

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