Mechanism of DNA triplex formation and its specificity as studied by filter binding assay

The filter binding method was found to be a very useful method for triple-helical formation of oligo DNA duplexes with homoprymidine single-strands. Using this method, we have obtained dissociation constants of triplexes and association rates of triple-helical formation of a variety of combinations of double-strands (23-mer) and pyrimidine single-strands as functions of pH and temperature. pH dependences of dissociation constants and association rates are theoretically discussed in terms of acid-base equilibrium of pyrimidine strands. Temperature-dependence of dissociation constants and association rates were considerably different between acidic and neutral pH range, suggesting that mechanism of triple-helical formation differs between two pH ranges. The results were best interpreted in terms of a three-state model for the triple-helical formation. Furthermore, the effect of mismatched sequences on the stability of the triplexes were also discussed.