Stabilisation of DNA triple helices

Oligonucleotide-directed triple helix formation offers the possibility of acheiving sequence recognition of DNA and may be useful for designing compounds targeted at individual genes as antiviral or anticancer agents. In this strategy, a synthetic oligonucleotide binds within the DNA major groove, forming specific hydrogen bonds with substitutes on the duplex purine residues. Structures in which the third strand runs parallel to the duplex purine strand are characterised by T·AT and C+·GC triplets and those in which the third strand runs in an antiparallel orientation are characterised by G·GC, A·AT and T·AT.In this thesis, DNase I footprinting is used to examine a number of strategies aimed at enhancing the stability of intermolecular DNA triple helices. In particular, the effect of a naphthoquinoline triplex-binding ligand on the interaction of both unmodified and acridine-linked oligonucleotides with DNA fragments containing the target sites A6G6·C6T6 and G6A6·T6C6 is examined. The unmodified oligonucleotides T5C5 and C5T5 bind weakly to their respective target sites, even at pH 5.5, however 10μM of the ligand can reduce the concentration of oligonucleotide required to generate a DNase I footprint by over 100-fold. Acridine-linked complexes are stabilised to a lesser extent. Further examination reveals that the ligand selectively stabilises T·AT rather than C+·GC triplets.At physiological pH, antiparallel triplexes with GT-containing oligonucleotides are also stabilised, but require higher ligand concentrations. The binding of GA-containing oligonucleotides is not induced by the ligand. The ligand has a greater stabilising effect on these complexes at lower pH, suggesting that the active species is protonated. Coralyne displays similar triplex-stabilising activity, although it also shows some interaction with duplex DNA. Ethidium bromide does not promote the formation of any of these triplexes and destabilises the interaction of Acr-T5C5 and Acr-C5T5 with their respective target sites.These triplex-stabilising ligands do not shift the formation of C+·GC-containing triplexes to physiological pH, however replacement of cytosine with β-aminopyridine permits the recognition of GC base pairs at neutral pH. Substitution with nuclease-resistant aminopyridine bases in the α-configuration weakens this interaction, although binding can be enhanced in the presence of the triplex-binding ligand.Minor-groove bind ligands can dramatically affect the binding of triplex-forming oligonucleotides within the DNA major groove. Both mithramycin (GC-selective) and distamycin (AT-selective) abolish parallel triplex formation with acridine-linked oligonucleotides. Antiparallel complexes are also destabliised ny mithramycin, however distamycin-boun DNA appears compatible with this motif.