Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603788
Title: Studies on the cell uptake and hybridisation properties of oligonucleotide derivatives
Author: Harrison, J. G.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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Abstract:
A series of 3'-linked conjugates between an oligodeoxyribonucleotide 13mer and cholic, folic, lipoic and pantothenic acids, cholesterol, vitamin E and a viral fusion peptide have been prepared in an attempt to enhance the cellular uptake properties of the nucleic acid. It was envisaged that the 4 biological acids may improve the cellular uptake of the oligonucleotide through natural receptor-mediated uptake mechanisms, whilst cholesterol, vitamin E and the fusion peptide may enhance interaction of the conjugate with cellular membranes. In the course of preparing conjugates involving cholic, folic, lipoic and panthothenic acids, a methodology was developed that allowed for simple and effective conjugation between moieties bearing a carboxylic acid group and an oligonucleotide sequence, achieving linkage through disulfide and thioether bonds. All of the 3'-linked conjugates were characterised by reverse phase HPLC and electrospray mass spectrometry, and were found to have increased stability towards nucleases present in serum. The solid phase synthesis of a small library of peptides composed of the cationic amino acids lysine, ornithine, histidine and arginine, the hydrophobic amino acid tryptophan, and alanine as a spacer, and their subsequent coupling through an N-terminal cysteine residue to a 5'-maleimide-modified 8mer was also accomplished. The preparation of 49 peptide-oligonucleotide conjugates was achieved in good yield and purity with a simple gel filtration employed as the only purification step. Melt analysis of the hybridisation properties of the constructs indicated that increasing the number of cationic residues within the peptide segment led to elevated Tm values on binding with a 16mer target. The binding affinities of the peptide segments were found to be stronger when bound to single-stranded rather than duplex DNA, and dependent on salt concentration and pH. Thermodynamic analysis revealed enhanced free energies of binding for cationic peptide-oligonucleotides compared to the unmodified 8mer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603788  DOI: Not available
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