Kinetic Footprinting of DNA Triplex Formation

Abstract

Kinetic parameters of triplex-forming reaction between 22-base-pair duplex oligonucleotide (5′-d[AAA-GGAGGAGAAGAAGAAAAAA], 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 mMsodium cacodylate, pH 6.9, 2 mMMgCl2), 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 237M−1s−1at 10°C to 13M−1s−1at 30°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.