Modified oligonucleotides and mimics have been developed with improved physicochemical and biological properties and are utilised in a wide range of applications including antisense and RNAi. Notably, new conjugation strategies have been developed to aid delivery and cellular localisation. Modified oligonucleotides are also now widely used for gene functional analysis, as probes for bioanalytical applications and as building blocks for assembly of higher-order nanostructures. The Micklefield group fIrst introduced pyrrolidine-amide oligonucleotide mimics (pOM) in 2000; the modifications involved the replacement of the ribose sugar of native RNA with a pyrrolidine ring and the phospho diester backbone by a rigid amide linker. The cationic nature of POM should also aid water solubility, cellular uptake and binding affinities due to electrostatic attraction between the cationic POMs and the anionic native DNAIRNA. , . Short POM homo-oligomers and longer mixed sequence POM oligonucleotides have shown' promising binding aff'mity towards both DNA and RNA. Homo-pyrimidine sequences have shown anti-parallel and parallel binding towards native'DNA and RNA. However, longer mixed sequence POM have also showed rates of association/dissociation that are noticeably slower than those typically observed with short oligonucleotides or PNA. Backbone extended pyrrolidine-amide oligonucleotide mimic (bePOM) monomers were prepared with the Boc protected backbone nitrogen and exocyclic amines of the nucleobases protected with the Cbz group. The bePOM oligomers were assembled using adapted PNA Boc/Cbz solid-phase peptide synthesis protocols. The bePOM oligomers were purified by reverse-phase HPLC and characterised by MALDI-ToF mass spectrometry. The DNAIRNA binding properties of the bePOMs have been studied by UV thermal denaturation, circular dichroism and isothermal titration calorimetry. Further to this a non-enzymatic DNA template directed synthesis approach has been developed that allows for the morpholino nucleoside extension of a PNA primer. Equilibration of the ribonucleosides with the template-primer complex, in a noncovalent fashion, was shown to be essential for high selectivity. The morpholino nucleotide extension products consisted of a seven membered amide backbone and the subsequent Fmoc protected thyminyl monomer was prepared for the assembly of homo-thymine oligomer via solid phase peptide synthesis. The morpholino oligomers were purifIed by reverse-phase HPLC and characterised by MALDI-ToF mass spectrometry. The DNAIRNA. binding properties of the amide linked morpholino oligomers were studied by UV thermal denaturation and circular dichroism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:506860 |
| Date | January 2009 |
| Creators | Bell, Neil Matthew |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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