2556 Nucleic Acids Research, 2007, Vol. 35, No. 8 The magnetization of 15NH4þ during the mixing time is in Nz state. A series of NzExHSQC spectra at mixing times of 13, 50, 100, 200, 300, 400, 500 and 600 ms were acquired at 283, 293, 303 and 313 K. Quantitative analysis of cross- peak volumes as a function of mixing time was used to determine rate constants for 15NH4þ site exchange. Data analysis Volumes of cross-peaks were integrated using Varian VNMRJ 2.1A software. All volumes at a given tempera- ture were integrated relative to the most intense auto- correlation peak (O1 at m of 13 ms). The arbitrary volume of 1.00 in the ﬁgures was assigned to volume of autocorrelation peak O1 at m of zero. Iterative least- square ﬁtting was done with Origin 7.5 software (www. originlab.com). Errors in variables are reported as calculated by the Origin program and are estimates of standard deviation. Quality of the ﬁts is expressed as root mean square deviation (RMSD). RESULTS d[G4(T4G4)3] folds into a unimolecular quadruplex in the presence of 15NH4þ ions The oligonucleotide d[G4(T4G4)3] has been folded into a G-quadruplex structure by overnight dialysis against a 40 mM 15NH4Cl solution, which resulted in well- resolved imino (and other) resonances in 1H NMR spectra (Figure 2A and B). The chemical shifts of 16 imino protons were diﬀerent from those reported for the same oligonucleotide in the presence of Naþ ions (1). This is not surprising because the diﬀerent ionic radii of the two cations can induce diﬀerences in the local structure of the G-quadruplex, which lead to diﬀerent shielding of imino protons by the nearby aromatic ring currents. In addition, 15NH4þ and Naþ ions can themselves exert distinct (de)shielding due to their diﬀerent electrostatic densities and diﬀerent preferred positions between neighboring G-quartets. Our initial assumption that the general fold of Figure 2. Aromatic (A) and imino (B) regions of 1H NMR spectrum of 15NH4þ form of d[G4(T4G4)3] G-quadruplex at 298 K. Plot of 2D 15N–1H HSQC spectrum (C). The cross-peak corresponding to 15NHþ 4 ions in bulk is labeled as B, while those residing at the binding sites within the G-quadruplex are labeled as O1, O2 and I. the G-quadruplex is the same in the presence of Naþ and 15NH4þ cations was substantiated by acquiring a series of NOESY spectra, by measuring translational diﬀusion coeﬃcients and by other NMR experiments. The fold- back nature of the unimolecular d[G4(T4G4)3] quadruplex was conﬁrmed by the presence of eight guanines in syn conformation, which is in full accordance with the NMR structure of the Naþ form (Figure 1). Additionally, if both Naþ and 15NH4þ forms of G-quadruplex exhibit the same folding topology their hydrodynamic properties should be the same. Pulsed-ﬁeld gradient spin-echo diﬀusion experiments were used to determine translational diﬀusion coeﬃcients of 1.2 10 6 and 1.1 10 6 cm2/s for the Naþ and NH4þ forms, respectively. The equivalence of these diﬀusion coeﬃcients, within the experimental error, also supports equivalent folding topologies. Cation localization The 15NH4þ-ion-binding sites within the d[G4(T4G4)3] G-quadruplex were identiﬁed with the use of 2D 15N– 1 H HSQC NMR spectra. Analysis of these spectra revealed four peaks that correspond to 15NH4þ ions in four distinct chemical environments (Figure 2C). The most intense peak at d(1H) of 7.10 p.p.m. corre- sponds to 15NH4þ ions in bulk solution, while the three smaller peaks correspond to 15NH4þ ions occupying diﬀerent binding sites within the G-quadruplex. The 15NH4þ-ion-binding sites are located between pairs of adjacent G-quartets. The cross-peaks with the similar 1H NMR chemical shifts of 7.38 and 7.36 p.p.m. were Tashseigirnecdhetmoitchael osuhtieftrsbainlodninggtshietes15ON1 danimdeOns2i,ornespareect3iv8e.l1y8. and 38.35 p.p.m., respectively (Figure 2C). The cross-peak at d(1H) of 7.28 p.p.m. was assigned to the inner binding site (I). The assignment of bound 15NH4þ ions and their localization sites were conﬁrmed by 2D NOESY data. The outer binding site designated as O1 was assigned to the intra-quadruplex binding site near the outer G-quartet that is spanned by the diagonal T4 loop. The outer binding site designated as O2 was assigned to site composed of the outer G-quartet spanned by the two lateral loops (vide infra). It has been previously suggested that cations may reside between the outer G-quartets and T4 loops with greater residence times than delocalized cations. Our NMR data does not oﬀer support for 15NH4þ ions residing at loop-binding sites, or at least not cations with relatively long (e.g. millisecond) resonance times, as we only observe a single NMR resonance corresponding to bulk ions. It is noteworthy that volume integrals of these three cross-peaks in HSQC spectrum are not the same. The ratios of volume integrals of cross-peaks O1, I and O2 at 130 mM 15NH4þ ion concentration are 1.0:0.7:0.7, respec- tively. The occupancies of binding sites O1, I and O2 calculated on the basis of equilibrium binding constants at 1.8 mM G-quadruplex and 130 mM 15NH4þ ion concen- trations are 90, 75 and 80%, respectively. 15NH4þ ion movement Cations inside the d[G4(T4G4)3] quadruplex are not static, as demonstrated by several cross-peaks in NzExHSQC