The kinetics of DNA triple helices

Paes, Hazel Margaret (1997) The kinetics of DNA triple helices. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

A detailed examination of triplex formation at the target sites (GGA)₅GG and (GGA)₂GGG(GGA)₂GG, showed that GT-containing oligonucleotides bind much faster than GA- or CT-containing third strands. The reaction is faster in the presence of manganese than magnesium. In several instances the observed rate constants are faster at the centre than at the edges of the target site. This is consistent with a model in which triplex formation at this repetitive site is achieved via intermediate complexes in which the third strand is not properly aligned with its target, and which subsequently migrate to the correct position. Single triplet mismatches had little effect on the rate of association, though the reaction was slowed by addition of an excess of non-specific DNA. The association reaction was also faster in the presence of a naphthylquinoline triplex-binding ligand, consistent with the suggestion that this ligand stabilises the transient nucleation intermediates which are formed before zippering up of the complex.

The kinetics of formation of triplexes containing different arrangements of 3XC^+.GC and 9x ^. TAT triplets was also investigated. In general the third strands bound faster when the C^+.GC triplets were furthest away from each other.

Studies on the dissociation of these intermolecular triplexes confirmed that the reaction is slow with t_1/2 values of 12-500 minutes. The dissociation of parallel triplexes was much slower than that of antiparallel triplexes. However the dissociation rate was dependent on the length and concentration of the competing oligonucleotide, suggesting that we are not observing a simple dissociation process.

The final chapter examines the kinetic properties of triplexes formed at the sequence (GA)₆T₁₁, using the third strands T₁₁(AG)₆ and T₁₁(TG)₆, designed to form blocks of parallel and antiparallel triplets. We find that this structure forms at a rate intermediate between that of the two halves.

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