6660 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 8. CD spectra of TBA (A) and 1 (C) at 20 C (continuous lines) and 90 C (dashed lines). Melting (continuous lines) and annealing (dashed lines) CD experiments of TBA (B) and 1 (D). Table 4. Thermodynamic parameters for the unfolding of 1 and TBA Tm ( C) DH (kJ mol 1) DS (kJ mol 1 K 1) DG (298 K) (±1) (±20) (±0.05) (kJ mol 1) (±1) 1 57 TBA 52 180 160 0.54 0.49 17 13 UV technique (32,33). The transition for the modiﬁed aptamer occurs 5 C above the transition of TBA and the enthalpy change values are consistent with the opening of two G-tetrads (34,35). The entropy values suggest that the modiﬁed aptamer possesses a more rigid structure with respect to TBA, according to a better stacking between G pairs found in the NMR study. The whole set of thermodyn- amic parameters shows that the quadruplex formed by 1 is more stable than its unmodiﬁed counterpart. In order to study the effects of the structural changes on the biological activity of 1, if any, we performed a PT assay. PT assay on 1 was performed on human plasma in strict compari- son with TBA. In order to eliminate the variation caused by the measurements performed on different days, the samples of 1 and TBA were prepared at the same time and folded together by heating the samples for 10 min at 80 C and slowly cooling them down to room temperature. The assays have been conducted after one week from the preparation procedure. As shown in Figure 9, overall, TBA showed a sta- tistically signiﬁcant and a more marked effect over 1, both in terms of the onset of activity and endurance. Indeed, incuba- tion studies demonstrated that TBA effect is already present after 10 s of incubation and lasts up to 15 min of incubation. On the other hand, 1 showed a slower onset of the effect, indeed PT time started to be signiﬁcantly prolonged follow- ing 5 min of incubation. Also the magnitude of the activity of 1 is reduced compared with TBA. The biological data reported here imply that the modiﬁcations of the structure adopted by 1 affect sensibly the biological activity, and there- fore its interaction with the thrombin. Looking at the overall folding of the modiﬁed aptamer, such a reduction in the bio- logical activity was not expected. However, it is interesting to note that the TBA inhibits the thrombin activity when inter- acting with its ﬁbrinogen exosite (36). In particular, it has been proposed that TBA could associate with thrombin in two different ways: through the T7-G8-T9 loop (as suggested by X-ray data) or through the T3 and T12 residues (as sug- gested by NMR models) (36). As far as the X-ray data are concerned, among all the observed interactions, T7 is buried in a pocket of hydrophobic residues of the ﬁbrinogen exosite of thrombin made by Ile24, His71, Ile79 and Tyr117, whereas, according to NMR data, the T3 residue interacts with the thrombin residues Tyr76 and Ile82. Therefore, the reduced biological activity of 1 could be ascribed to the dif- ferent orientation adopted by T3, and, to a less extent, by T7 in 1 with respect to the unmodiﬁed TBA (Figure 7). The longer onset displayed by 1 could be tentatively explained considering that 1 needs to properly orientate T3 and T7 upon its binding to thrombin. Nevertheless, the anticoagulant activity of 1 could not be simply ascribed to these changes, but could even be due to a combination of all structural changes observed such as: (i) relative strand orientation; (ii) glycosidic arrangements of the bases (iii) groove widths. The present data give a further insight into the understand- ing of the variables involved in the formation and the stability of quadruplex structures. In fact, 1 clearly prefers to adopt a ‘3+1’ strand orientation instead of a classical ‘2+2’ typical of this kind of structure (see TBA, Figure 1), in spite of the fact that both arrangements are equally possible. Thus, these ﬁnd- ings further enforce the idea that the ‘3+1’ core topology