diﬀerent cations than others (35–39). Oligonucleotide with four human telomeric repeats, d(T2AG3), forms a unim- olecular G-quadruplex with diagonal- and edge-type loops in the presence of Naþ ions (40), whereas completely diﬀerent structure with four parallel G-rich strands has been determined by X-ray crystallography in the presence of Kþ ions (41). A mixture of parallel–anti-parallel strands has also been observed for sequences containing d(T2AG3) repeats in solution (42–45). Kþ and Naþ are biologically important cations, as they are present in and around living cells at the highest concentrations. Naþ, a relatively small cation can be coordinated in the plane of a G-quartet, while larger cations (e.g. Kþ, NHþ) have to be coordinated between 4 two adjacent G-quartets (3,46–49). At present, there are limited detailed studies of the binding and dynamic properties of cations within G-quadruplex structures. The use of heteronuclear (metal ion) NMR to directly monitor Naþ, Tlþ, Kþ and Rbþ cations has conﬁrmed the direct coordination of these cations inside G-quadruplex structures (50–59). The introduction of 15NH4þ ions as a probe for cation localization within G-quadruplex struc- tures has opened a whole range of new opportunities for studies with NMR (49,60–62). In the current study, we have focused on d[G4(T4G4)3], which consists of 3.5 units of the telomeric repeat sequence d(G4T4)n of the protozoan Oxytricha nova. The unim- olecular G-quadruplex adopted by this oligonucleotide in solution with Naþ ions consists of four G-quartets with alternating parallel and anti-parallel strands (Figure 1) (1). The conformation of guanine nucleosides along the strands alternates between syn and anti. One of the T4 loops spans diagonally across the outer G-quartet, while the two T4 loops on the other side of the G-quadruplex core run along the opposite edges of the outer G-quartet. d[G4(T4G4)3] was folded into a G-quadruplex in the presence of 15NH4þ ions and conﬁrmed by NMR to adopt the same solution state structure. As expected, three binding sites for 15NH4þ ions were identiﬁed within the G-quadruplex, whereas no 15NH4þ ions could be localized between the outer G-quartets and T4 loops. Cations bound inside the d[G4(T4G4)3] quadruplex are not static. These cations exchange amongst the three binding sites within the G-quadruplex and with bulk solution. We have studied exchange using a 2D 15N–1H NzExHSQC NMR experiment and assessed the exchange processes of 15NHþ 4 ions in a qualitative and quantitative manner. The comparison of our results for 15NH4þ ion movement with cation movement measurements previously obtained for a bimolecular G-quadruplex adopted by the related DNA sequence d(G4T4G4) reveals that 15NH4þ ion move- ment is 80 times slower in the case of the unimolecular G-quadruplex studied here. MATERIALS AND METHODS Sample preparation The oligonucleotide d[G4(T4G4)3] was synthesized on an Expedite 8909 synthesizer using standard phosphor- amidite chemistry and deprotected with concentrated Nucleic Acids Research, 2007, Vol. 35, No. 8 2555 Figure 1. (A) Topology of unimolecular G-quadruplex adopted by d[G4(T4G4)3] and 15NH4þ-ion-binding sites. The three binding sites are labeled as O1, I and O2. The guanine bases are shown as numbered rectangles, where cyan and magenta rectangles represent nucleobases in anti and syn conformation, respectively. (B) Birds-eye view of a 15NH4þ ion above an individual G-quartet. aqueous ammonia. DNA was then puriﬁed by passage over a 1.0 m Sephadex G15 column. Fractions containing only full-length oligonucleotide were pooled, lyophilized, redissolved in 1 ml H2O and dialyzed against 40 mM 15NH4Cl overnight. Samples were lyophilized and subse- quently redissolved in 0.3 ml of 95% H2O–5% 2H2O. LiOH or HCl were added to adjust pH of NMR samples to 6.0. Oligonucleotide concentration was 1.8 mM. NMR spectroscopy NMR data were collected on a Varian Unity Inova 600 MHz NMR spectrometer. Standard 1D 1H spectra were acquired with 16 k complex points, a spectral width of 10 kHz and 128 scans using WATERGATE solvent suppression. 2D NOESY spectra were acquired at mixing times of 80 and 300 ms with 4 k complex points in F2 and 320 increments in F1 dimensions, 16 scans for each increment and spectral width of 10 kHz in both dimen- sions at 298 K. Twenty diﬀerent gradient strengths (0.53–20.55 G/cm) were used in diﬀusion experiments (BPPSTE) with 16 k complex points, a spectral width of 10 kHz, 64 scans and WATERGATE solvent suppression at 298 K. 15N– 1 H HSQC spectra were acquired at 293 K with 1024 complex points in the F2 dimension and 256 increments in the F1 dimension, 16 scans for each increment and a spectral width of 4 kHz in F2 and 1 kHz in F1. 15N–1H NzExHSQC spectra were acquired with 1024 complex points in the F2 and 256 increments in the F1 dimensions, 16 scans for each increment and a spectral width of 4 kHz in F2 and 1 kHz in F1. Cross-peaks in this experiment appear due to the movement of 15NH4þ ions from an initial to a diﬀerent chemical environment during the mixing time ( m).