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 investigated in mixed-valence salt solutions. Equilibrium constants for formation of the local pyrimidine.purine.pyrimidine structure afforded by binding of the oligonucleotide 5'-d(T*TTTTCTCTCTCTCT)-3' to a single site within a 339-bp plasmid fragment were measured using quantitative affinity cleavage titrations at pH 7.0 and 22 degrees C in the presence of various concentrations of KCl, MgCl_2, and spermine tetrahydrochloride (SpmCl_4). In a solution containing 10 mM NaCl, 140 mM KCl, 1.0 mM MgCl_2, and 1.0 mM SpmCl_4, 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 SpmCl_4 at the same temperature 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 MgCl_2, and 1.0 mM SpmCl_4) resulted in an overall 100-fold decrease in the binding affinity from the lowest to the highest concentration. In contrast, measured binding constants increased 500-fold as the spermine concentration was increased from 0.40 to 4.0 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM MgCl_2). There was a modest effect on the binding constant (a 3-fold decrease) upon varying the magnesium ion concentration from 0.10 to 10 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM SpmCl_4). The results are consistent with a valence-specific cationic stabilization of the local triple-helical complex decreasing in order from the most stabilizing to the least: Spm^(4+) > Mg^(2+) > K^+. The observed trends are in good qualitative agreement with the expected effects of competition among the cations on changes in the thermodynamic binding fraction and the differential phosphate charge screening potential of each cation