spectra, which are observed in addition to the four autocorrelation peaks in standard 1H–15N HSQC spectra (Figures 2C and 3). These cross-peaks clearly show that during mixing time ( m) a fraction of 15NH4þ ions move from their initial binding site to another binding site within the G-quadruplex, or out into bulk solution. A two-letter code is used in Figure 3 to denote the initial and ﬁnal locations of certain 15NH4þ ions over the course of an NMR pulse sequence. A series of NzExHSQC spectra were acquired with diﬀerent mixing times in the range from 13 ms to 3 s, and at several temperatures. Two initial observations are noteworthy. First, at short mixing times (up to 50 ms) the volumes of 15NH4þ ion site-exchange cross-peaks grow more slowly than previously demon- strated for the bimolecular G-quadruplex of d(G4T4G4) (49,61). Thus, 15NH4þ ion movement is slow within the unimolecular d[G4(T4G4)3] quadruplex in comparison to the dimeric d[G4T4G4]2 quadruplex (49). Second, certain autocorrelation peaks (e.g. O1 and I) persist for mixing times as long as 3 s and their signal intensities reduce more slowly than expected for cations participating in site exchange. Inspection of Figure 3 clearly demonstrates 15NH4þ ion movement between the three binding sites within the d[G4(T4G4)3] quadruplex. A portion of ions from the inner binding site moved to two outer binding sites. Considerable overlap due to nearly identical 1H chemical shifts for 15NH4þ ions bound at sites O1 and O2 resulted in Figure 3. Plot of 2D 15N–1H NzExHSQC spectrum ( m ¼ 500 ms) of d[G4(T4G4)3] quadruplex at 293 K. The autocorrelation peaks are labeled with single letter, while the cross-peaks, which conﬁrm movement of 15NH4þ ions are labeled with two letters where the ﬁrst indicates the initial and the second the ﬁnal location. Asterisk denotes cross-peaks of deuterium isotopomers of ammonium ions. Nucleic Acids Research, 2007, Vol. 35, No. 8 2557 the observation of only a single combined cross-peak for the movement of 15NH4þ ions from the inner binding site, I, to both outer binding sites (i.e. IOx in Figure 3). However, upon close examination it becomes evident that the IOx cross-peak is not symmetric. This asymmetry reveals that more ions moved from the inner binding site to the site O1 in comparison to O2. 15NH4þ ions are also observed to move from the outer binding sites to the inner binding site. We observed two resolved cross-peaks (i.e. O1I and O2I in Figure 3). A comparison of the O1I and O2I cross-peaks reveals that more ions move to the inner binding site from the site O1 than from O2. The cross-peaks corresponding to exchange from the interior of the G-quadruplex into bulk solution were also observed (Figure 3). The cross-peak O2B is signiﬁcantly more intense than O1B which suggests that more 15NHþ 4 ions move into bulk through binding site O2. 15NH4þ ions from bulk solution enter the interior of G-quadruplex almost exclusively through the O2 site, which leads to cross-peak BO2. The cross-peaks anticipated for move- ment of cations from bulk to O1 and I (i.e. BO1 and BI) are not observed above the noise level, presumably due to their low intensity and broad nature. Some NzExHSQC cross-peaks in Figure 3 denote a two-step 15NH4þ ion movement during the mixing time of the NMR experiment. The IB cross-peak corresponds to a two-step movement from binding site I into bulk via one of the outer binding sites. Exchange between the outer sites (O1O2 and O2O1) may also take place. However, the movement of 15NHþ between binding sites O1 and O2 4 cannot be experimentally substantiated by existing NzExHSQC spectra due to spectral overlap and low intensities of the relevant cross-peaks. Taken together, our qualitative analysis of cross-peak volumes reveals that there is no ion-channel-like net traﬃc of 15NHþ ions 4 through the G-quadruplex. 15NH4þ ions move back and forth between the binding sites within the G-quadruplex and exchange with ions in bulk solution. Exchange rate constants for 15NH4þ ion movement within d[G4(T4G4)3] A quantitative analysis of 15NH4þ ion movement was conducted based upon a series of NzExHSQC spectra where the mixing time ( m) was systematically increased from the lowest possible value of 13 ms, which is determined by the inherent delays of the pulse sequence and spectrometer hardware, to values over 1 s, where signal intensities are reduced signiﬁcantly by nuclear spin relaxation. We attempted to completely analyze the relationship between autocorrelation and cross-peaks’ volume integrals as a function of mixing time in terms of 15NH4þ ion movements. There are eight possible single-step ion movements taking place within the G-quadruplex and between the quadruplex and bulk solution. These exchange events are designated in our nomenclature as IO1, IO2, O1I, O2I, BO1, BO2, O1B and O2B. Only the cross-peaks corresponding to O1I and O2I could be quantitatively evaluated due to the above- mentioned spectral overlap and low signal intensity of cross-peaks corresponding to exchange with bulk solution.