KINETIC FOOTPRINTING OF DNA TRIPLEX FORMATION

Kinetic parameters of tripler-forming reaction between 22-base-pair du plex oligonucleotide (5'-d[AAAGGAGGAGAAGAAGAAAA], sequence of purine s trand) and the third strand 5'-d[TTTCCTCCTCTTCTTCTTTTTT] were determin ed by quantitative footprinting using DNase I as the cleavage reagent. When the third strand oligonucleotide is present in 10-fold excess ov er its duplex target, the binding reaction kinetics is pseudo first or der in oligonucleotide concentration, Under the conditions of these me asurements (10 mM sodium cacodylate, pH 6.9, 2 mM MBCl(2)), the reacti ons are slow with relaxation times on the order of minutes (4 to 28 mi n). As is generally found for helix-formation reactions, the forward r ate constant (helix formation) decreased with temperature, the bimolec ular association rate constants ranged from 237 M(-1) s(-1) at 10 degr ees C to 13 M(-1) s(-1) at 30 degrees C. These data are consistent wit h an activation energy of -25 kcal/mol (strands), The dissociation rat e constant apparently is temperature independent under these condition s; the changes observed were within the error that this parameter coul d be determined. Advantages and limitations of this technique for obta ining 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. (C) 1996 Acade mic Press, Inc.