shortening of residence lifetimes to 1.5 and 2.0 s for O1I and O2I processes at 313 K, respectively. Volumes of cross-peaks are further tuned by longitudinal relaxation. Analysis showed that O1I and O2I processes are char- acterized by T1 relaxation times of 0.77 and 0.57 s at 283 K, which are reduced to 0.46 and 0.30 s at 313 K, respectively. The third resolved cross-peak (IOx) corre- sponding to 15NH4þ ion movement within d[G4(T4G4)3] G-quadruplex has been observed for the reverse move- ment, but did not oﬀer suﬃcient chemical shift diﬀerence to analyze IO1 and IO2 cross-peaks individually. However, the asymmetry of IOx cross-peak has shown that more ions move from the inner binding site to the site O1 in comparison to O2. Interpretation of kinetic data on movement of 15NHþ 4 ions between the inside of the G-quadruplex and bulk solution is more challenging due to proton exchange with solvent. The loss in intensity of cross-peaks due to exchange with bulk proved to be considerable and highly pH dependent. The same goes for any cross-peaks for exchange with bulk. Three cross-peaks (O1B, O2B and IB) correspond to movement of 15NH4þ ions from the binding sites within G-quadruplex to bulk. The fourth resolved cross-peak, BO2 corresponds to the movement of bulk ions into G-quadruplex. The O2B cross-peak is much more intense than O1B suggesting that most of the 15NHþ 4 ions leave G-quadruplex through O2-binding site. Similarly, the vast majority of 15NH4þ ions enter G-quadruplex through the O2-binding site. The BO2 cross-peak, however, exhibits broad line which prevents its quantitative analysis. We were able to calculate the individual exchange rate constants for O1B and O2B processes. Although they are not directly comparable to the exchange rate constants within d[G4(T4G4)3] due to loss by proton exchange, the O2B exchange rate constant of 0.75 s 1 at 293 K demonstrates that 15NH4þ ion exchange with bulk is faster than exchange within the G-quadruplex. The comparison of O2B and O1B exchange rate constants shows that O2B process is roughly ﬁve times faster at 293 K. Volumes of O1, I and O2 autocorrelation peaks are decreasing with mixing time ( m). The starting values (extrapolated to m ¼ 0 s) of the three peaks are not the same due to diﬀerent 15NH4þ ion occupancies of the binding sites. Qualitatively, the volumes for autocorrela- tion peaks for the two outer binding sites decrease at roughly the same rate, while decrease for the inner binding site is noticeably slower. The decrease of autocorrelation peak volumes has been described with a biexponential function of mixing time. All three autocorrelation peaks initially decay quickly with characteristic d1 values between 7.5 and 14.1 s 1 at 293 K. This fast relaxation process makes up from 26 to 38% of the total signal decay of autocorrelation peaks in the temperature range from 283 to 303 K. It is interesting to note that this fast process exhibits small if any temperature dependence. The second rate constant, d2, represents the sum of the rate constants for 15NH4þ ion movements from a speciﬁc binding site and relaxation (1/T1) at that binding site. It increases with the rise in temperature. Its values are the lowest for the binding site I (e.g. 0.57 s 1 at 293 K) and the highest for O2 Nucleic Acids Research, 2007, Vol. 35, No. 8 2561 (e.g. 1.82 s 1 at 293 K). 15NH4þ ions occupying binding site I experience longer T1 relaxation times than ions occupy- ing the two outer binding sites. The ionic radius of 15NH4þ ion is too large to allow it a free passage through G-quartets. In comparison, the smaller Naþ ion can ﬁt in the plane of a G-quartet which intimately facilitates its movement. It is noteworthy however, that the consideration of cation interactions of Kþ and Naþ with respect to 15NH4þ ions cannot be based solely on cation size. 15NH4þ ions are potential hydrogen- bond donors, which is a feature that distinguishes them strongly from alkali cations. G-quartet has to open slightly for 15NH4þ ion movement to occur. In this way, the rigidity of individual G-quartet is directly related to exchange rate constant. Movement within d[G4(T4G4)3] G-quadruplex requires partial opening of at least one of the inner G-quartets. The inner core of the G-quadruplex appears to be relatively rigid resulting in slow exchange rates of inner 15NH4þ ions. On the other hand, the exchange rate constants through the outer G-quartets diﬀer substantially which can be attributed to the diﬀerent structure of T4 loops. One of the T4 loops near the binding site O1 spans the diagonal of the outer G-quartet. The diagonal loop makes the neighboring G-quartet tighter which is reﬂected in slower rates for movement of 15NHþ 4 ions from O1-binding site. In addition, this loop represents steric barrier for the 15NH4þ ion movement from bulk into the interior of G-quadruplex and vice versa. Two T4 loops on the other side of the G-quadruplex that span along the opposite edges of the outer G-quartet allow for the two halves of this G-quartet to open more easily upon the passage of 15NH4þ ion(s). The two T4 loops can swing to the sides and allow easier passage of ions. The stiﬀness and steric hindrance contribute to the 5-fold diﬀerence in the exchange rate constants through the outer G-quartets in d[G4(T4G4)3] G-quadruplex. The related sequence d[G4T4G4] consisting of 1.5 telomeric repeat of O. nova has been shown to exhibit three 15NH4þ-ion-binding sites within its bimolecular G-quadruplex structure (49). 15NHþ ions move along 4 the central axis of the G-quadruplex. The exchange rate constant of the central 15NH4þ ion is 4 s 1 at 283 K (49,61), which is 80 times faster than in the case of unimolecular G-quadruplex studied here. Bimolecular d[G4T4G4]2 quadruplex is a symmetric system and there is a single cross-peak that experimentally demonstrates movement from the inner to the outer binding sites. In comparison, d[G4(T4G4)3] quadruplex exhibits two separate cross- peaks for the two outer binding sites (O1 and O2). Our analysis has shown that O1I and O2I exchange rate constants are comparable within the 283–303 K tempera- ture range. At 313 K, however, O1I process becomes more eﬃcient which suggests local thermal lability of d[G4(T4G4)3] G-quadruplex. A recent study based on quantitative analysis of h2JN2N7 coupling constants across hydrogen bonds revealed that the 50 strand end is the most thermolabile region of bimolecular quadruplex adopted by d[G4T4G4]2 (63). The slower movement of 15NH4þ ions within d[G4(T4G4)3] quadruplex results in slower decrease in intensity of autocorrelation peaks in comparison to d[G4T4G4]2. The actual volumes are the result of 15N