Identification of mixed di-cation forms of G-quadruplex in solution

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d( G(3)T(4)G(4)) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K 1 ions into the solution of d( G(3)T(4)G(4))(2) folded in the presence of (NH4+)-N-15 ions uncovered a mixed mono-K+-mono-(NH4+)-N-15 form that represents intermediate in the conversion of di-(NH4+)-N-15 into di-K+ form. Analogously, (NH4+)-N-15 ions were found to replace Na+ ions inside d( G(3)T(4)G(4))(2) quadruplex. The preference of (NH4+)-N-15 over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over (NH4+)-N-15 ions. The two cation binding sites within the G-quadruplex core differ to such a degree that (NH4+)-N-15 ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d( G(3)T(4)G(4))(2) is more favorable with respect to (NH4+)-N-15 ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.