Specificity of triple helix formation

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Triplex-forming oligonucleotides (TFOs) have been the subject of extensive \nresearch in recent years. They have potential applications in many areas; such as \ngene-based therapies, site-directed mutation and as biochemical tools. However, \ntriplex technology has been hampered by several problems, including low stability \ndue to electrostatic repulsion between strands. This thesis has investigated \ncombinations of four methods for stabilising triplex DNA; these include \nincorporation of the positively charged thymine analogues bis-amino-U and \npropargylamino-dU in TFOs. Also modified TFO’s containing anthraquinone \nderivatives have been tested. Further, the free-intercalating agent \nnaphthylquinoline has been used to modulate TFO binding. \nA TFO containing six consecutive BAU molecules has previously been \nshown to interact with non-target sites. The pH dependence of this TFO was \ninvestigated. These experiments showed that considerably higher TFO \nconcentrations were needed to generate a footprint as the pH was increased. The \nTFO had a high affinity for the exact template (tyrT) at pH 5.0 and 6.0 and showed \nsome evidence of binding even at 30 μM at pH 7.0. These gels also showed \nevidence of the secondary binding seen in previous studies; this was considerably \nmore evident at pH 5.0, however, suggesting that the secondary binding may be \nmore sensitive to pH than the primary binding. \nSecondary binding sites for TFOs were examined by ‘Restriction \nEndonuclease Protection, Selection and Amplification’ or REPSA. REPSA has \nbeen used to select for DNA templates that are bound by the 9mer TFO containing \nsix bis-amino-U residues. Fourteen of the sequences which emerged from \nREPSA were chosen for footprinting with TFOs containing BAU, propargylaminodU \nor T. The BAU-TFO produced clear footprints on all but one of the REPSA \ntemplates tested, indicating that the REPSA process was successful in selecting \nfor sequences which are bound by the TFO. Significantly higher concentrations of \nthe P-TFO were required, and magnesium chloride and / or the triplex binding \nligand naphthylquinoline were needed to promote binding. Despite the differences \nin template sequence there does not appear to be a strong pattern in the binding \nintensities of the TFOs on the different templates. However, all templates do \ncontain a run of four to eight A’s. Surprisingly it appears from these data that the \nBAU TFO discriminates better than the P-TFO against non-exact binding sites \nThe selectivity of TFOs containing anthraquinone modifications was also \ninvestigated. Anthraquinone intercalates between DNA bases in duplex DNA and \ncan be tethered to the end of a TFO to increase stability. The specificity of five \nTFOs with different anthraquinone modifications was examined by footprinting \nagainst fragments containing mismatches. A doubly modified TFO bound with the \nhighest affinity and was most tolerant of mismatches. Mismatches at the centre of \nthe template had a lesser effect on binding affinity than mismatches at the 3’ end. \nThe effect of a 3’ mismatch was also greater if the anthraquinone was at this end. \nThe presence of an S-base at the 3’ end allowing intercalation of the \nanthraquinone at a YpR step increased the binding affinity on the exact template in \ncomparison to TFO 3 which did not contain the S-base. The TFO containing the S \nbase did not bind quite as well as the doubly modified TFO however.