Triplex-forming oligonucleotides (TFOs) have been the subject of extensive research in recent years. They have potential applications in many areas; such as gene-based therapies, site-directed mutation and as biochemical tools. However, triplex technology has been hampered by several problems, including low stability due to electrostatic repulsion between strands. This thesis has investigated combinations of four methods for stabilising triplex DNA; these include incorporation of the positively charged thymine analogues bis-amino-U and propargylamino-dU in TFOs. Also modified TFO’s containing anthraquinone derivatives have been tested. Further, the free-intercalating agent naphthylquinoline has been used to modulate TFO binding. A TFO containing six consecutive BAU molecules has previously been shown to interact with non-target sites. The pH dependence of this TFO was investigated. These experiments showed that considerably higher TFO concentrations were needed to generate a footprint as the pH was increased. The TFO had a high affinity for the exact template (tyrT) at pH 5.0 and 6.0 and showed some evidence of binding even at 30 μM at pH 7.0. These gels also showed evidence of the secondary binding seen in previous studies; this was considerably more evident at pH 5.0, however, suggesting that the secondary binding may be more sensitive to pH than the primary binding. Secondary binding sites for TFOs were examined by ‘Restriction Endonuclease Protection, Selection and Amplification’ or REPSA. REPSA has been used to select for DNA templates that are bound by the 9mer TFO containing six bis-amino-U residues. Fourteen of the sequences which emerged from REPSA were chosen for footprinting with TFOs containing BAU, propargylaminodU or T. The BAU-TFO produced clear footprints on all but one of the REPSA templates tested, indicating that the REPSA process was successful in selecting for sequences which are bound by the TFO. Significantly higher concentrations of the P-TFO were required, and magnesium chloride and / or the triplex binding ligand naphthylquinoline were needed to promote binding. Despite the differences in template sequence there does not appear to be a strong pattern in the binding intensities of the TFOs on the different templates. However, all templates do contain a run of four to eight A’s. Surprisingly it appears from these data that the BAU TFO discriminates better than the P-TFO against non-exact binding sites The selectivity of TFOs containing anthraquinone modifications was also investigated. Anthraquinone intercalates between DNA bases in duplex DNA and can be tethered to the end of a TFO to increase stability. The specificity of five TFOs with different anthraquinone modifications was examined by footprinting against fragments containing mismatches. A doubly modified TFO bound with the highest affinity and was most tolerant of mismatches. Mismatches at the centre of the template had a lesser effect on binding affinity than mismatches at the 3’ end. The effect of a 3’ mismatch was also greater if the anthraquinone was at this end. The presence of an S-base at the 3’ end allowing intercalation of the anthraquinone at a YpR step increased the binding affinity on the exact template in comparison to TFO 3 which did not contain the S-base. The TFO containing the S base did not bind quite as well as the doubly modified TFO however.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:533338 |
| Date | January 2010 |
| Creators | Cardew, Antonia |
| Contributors | Fox, Keith |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/183845/ |
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