EQUILIBRIUM ASSOCIATION CONSTANTS FOR OLIGONUCLEOTIDE-DIRECTED TRIPLE-HELIX FORMATION AT SINGLE DNA SITES - LINKAGE TO CATION VALENCE AND CONCENTRATION

The linkage between the energetics of oligonucleotide-directed triple helix formation and the cationic solution environment has been investi gated in mixed-valence salt solutions. Equilibrium constants for forma tion of the local pyrimidine.purine.pyrimidine structure afforded by b inding of the oligonucleotide 5'-d(TTTTTCTCTCTCTCT)-3' to a single si te within a 339-bp plasmid fragment were measured using quantitative a ffinity cleavage titrations at pH 7.0 and 22-degrees-C in the presence of various concentrations of KCl, MgCl2, and spermine tetrahydrochlor ide (SpmCl4). In a solution containing 10 mM NaCl, 140 mM KCl, 1.0 mM MgCl2, and 1.0 mM SpmCl4, the measured binding constant was 3.3 (+/- 1 .4) X 10(5) M-1. The equilibrium constant previously reported for the same association reaction in 100 mM NaCl and 1 mM SpmCl4 at the same t emperature and pH was 10-fold higher [Singleton, S. F., & Dervan, P. B . (1992) J. Am. Chem. Soc. 114, 6957-6965]. Further study demonstrated that varying the potassium ion concentration between 5.0 and 140 mM ( in the presence of 10 mM NaCl, 1.0 mM MgCl2, and 1.0 mM SpmCl4) result ed in an overall 100-fold decrease in the binding affinity from the lo west to the highest concentration. In contrast, measured binding const ants increased 500-fold as the spermine concentration was increased fr om 0.40 to 4.0 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM MgCl2). There was a modest effect on the binding constant (a 3-fold decrease) upon varying the magnesium ion concentration from 0.10 to 1 0 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM SpmCl4). T he results are consistent with a valence-specific cationic stabilizati on of the local triple-helical complex decreasing in order from the mo st stabilizing to the least: Spm4+ > Mg2+ > K+. The observed trends ar e in good qualitative agreement with the expected effects of competiti on among the cations on changes in the thermodynamic binding fraction and the differential phosphate charge screening potential of each cati on.