Specificity of triple helix formation

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

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2010

Author dc:creator

• Cardew, Antonia

Advisor dc:contributor.advisor

• Fox, Keith R.