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(+) ions into the solution of d(G(3)T(4)G(4))(2) folded in the presence of [Formula: see text] ions uncovered a mixed mono-K(+)-mono- [Formula: see text] form that represents intermediate in the conversion of [Formula: see text] into di-K(+) form. Analogously, [Formula: see text] ions were found to replace Na(+) ions inside d(G(3)T(4)G(4))(2) quadruplex. The preference of [Formula: see text] over Na(+) ions for the two binding sites is considerably smaller than the preference of K(+) over [Formula: see text] ions. The two cation binding sites within the G-quadruplex core differ to such a degree that [Formula: see text] 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 [Formula: see text] ions by Gibbs free energies of approximately −24 and −18 kJ mol(−1) which includes differences in cation dehydration energies, respectively.