Kinetic footprinting of DNA triplex formation.

Kinetic parameters of triplex-forming reaction between 22-base-pair duplex oligonucleotide (5'-d[AAAGGAGGAGAAGAAGAAAAAA], sequence of purine strand) and the third strand 5'-d[TTTCCTCCTCTTCTTCTTTTTT] were determined by quantitative footprinting using DNase I as the cleavage reagent. When the third strand oligonucleotide is present in 10-fold excess over its duplex target, the binding reaction kinetics is pseudo first order in oligonucleotide concentration. Under the conditions of these measurements (10 mM sodium cacodylate, pH 6.9, 2 mM MgCl2), the reactions are slow with relaxation times on the order of minutes (4 to 28 min). As is generally found for helix-formation reactions, the forward rate constant (helix formation) decreased with temperature, the bimolecular association rate constants ranged from 237 M-1 s-1 at 10 degrees C to 13 M-1 s-1 at 30 degrees C. These data are consistent with an activation energy of -25 kcal/mol (strands). The dissociation rate constant apparently is temperature independent under these conditions; the changes observed were within the error that this parameter could be determined. Advantages and limitations of this technique for obtaining the kinetic parameters of reactions involving sequence-specific DNA complexes are discussed. The technique can be readily implemented in most biochemistry or molecular biology laboratories.