Novel synthesis of branched nucleic acids : towards applications in chemical biology and nanotechnology

• Main Author: Mitra, Debbie.
• Format: Others
• Language: en
• Published: McGill University 2007
• Subjects: Nucleic acids > Synthesis.; Nucleic acid probes.; Nanostructured materials.
• Online Access: http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111880

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.