NMR evaluation of ammonium ion movement within a unimolecular G-quadruplex in solution

d[G(4)(T(4)G(4))(3)] has been folded into a unimolecular G-quadruplex in the presence of (15) [Formula: see text] ions. NMR spectroscopy confirmed that its topology is the same as the solution state structure determined earlier by Wang and Patel (J. Mol. Biol., 1995; 251: 76–94) in the presence of Na(+) ions. The d[G(4)(T(4)G(4))(3)] G-quadruplex exhibits four G-quartets with three (15) [Formula: see text]-ion-binding sites (O(1), I and O(2)). Quantitative analysis utilizing (15) [Formula: see text] ions as a NMR probe clearly demonstrates that there is no unidirectional (15) [Formula: see text] ion movement through the central cavity of the G-quadruplex. (15) [Formula: see text] ions move back and forth between the binding sites within the G-quadruplex and exchange with ions in bulk solution. (15) [Formula: see text] ion movement is controlled by the thermodynamic preferences of individual binding sites, steric restraints of the G-quartets for (15) [Formula: see text] ion passage and diagonal versus edge-type arrangement of the T(4) loops. The movement of (15) [Formula: see text] ions from the interior of the G-quadruplex to bulk solution is faster than exchange within the G-quadruplex. The structural details of the G-quadruplex define stiffness of individual G-quartets that intimately affects (15) [Formula: see text] ion movement. The stiffness of G-quartets and steric hindrance imposed by thymine residues in the loops contribute to the 5-fold difference in the exchange rate constants through the outer G-quartets.