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Title: An NMR study of a natural and a 3'-S-phosphorothiolate modified DNA triplex
Author: Evans, Kathryn
ISNI:       0000 0004 2744 2028
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Triple helical DNA is formed when purine or pyrimidine bases of a triplex forming oligonucleotide (TFO) occupy the major groove of a homopurine-homopyrimidine double helix, and interact with it via Hoogsteen hydrogen bonding. There is significant interest in triplex structures due to their prevalence in biology as they are the target complex for antigene therapeutic approaches. Triplex structures which comprise a DNA duplex and RNA TFO have previously been found to be more stable than the equivalent all DNA systems. There are synthesis and stability issues surrounding the use of RNA-based TFOs, hence there is interest in developing chemically-modified TFOs which show at least the same duplex-binding affinity. In this project, a 3�-S-phosphorothiolate linkage is the modification of interest, which has previously been shown to alter the conformation of deoxyribonucleic acid systems to that of ribonucleic acids. A 12 base pair homopurine-homopyrimidine hairpin duplex that is a target for a TFO was characterised by NMR spectroscopy and high resolution structures were generated. The hairpin was shown to adopt a B-type helix with predominantly south sugar puckers. Similar analysis was performed on a native triplex comprising the hairpin and a pyrimidine TFO. Binding of the third strand was found to cause little structural changes to the hairpin. Two non-adjacent 3�-S-phosphorothiolate modifications were then incorporated into the TFO and the effects determined by NMR analysis. The result was very localised conformational changes in the deoxyribose sugars attached to the modifications, as well as those 3� to it, from a DNA-like south to an RNA-like north pucker. Finally, UV thermal melting analysis of the target hairpin and triplexes was performed. The stability of the hairpin was found to be pH independent, whereas the triplex structures were only stable at acidic pH. The 3�-S-phosphorothiolate modification was found to stabilise the triplex by up to around 7 °C and to increase the pH range for triplex formation. It is thought that the increased stability is due to favourable base stacking interactions as a result of the modification.
Supervisor: Fisher, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available