was kept at 3 s for NOESY experiments used in the structure determination. A relaxation delay of 1.2 s was used for all other experiments. The NMR data were processed on a SGI Octane workstation using FELIX 98 software (Accelrys, San Diego, CA). Structural calculations Cross-peak volume integrations were performed with the program FELIX 98, using the NOESY experiment collected at mixing time of 100 ms. The NOE volumes were then con- verted to distance restraints after they were calibrated using known ﬁxed distances of H20/H200 of G1, G2, G5, G8, T9, G10, G11, T12, T13 and G14. Then a NOE restraint ﬁle was generated with three distance classiﬁcations as follows: strong NOEs (1.8 s < rij < 3.0 s, where 1.8 s is the van der Waals radius and rij is the interproton distance between protons i and j), medium NOEs (2.5 s < rij < 4.0 s) and weak NOEs (3.5 s < rij < 5.5 s). A total of 198 NOE derived distance restraints were used. Hydrogen bonds constraints were used: upper and lower distance limits of 2.0 s and 1.7 s for hydrogen-acceptor distance, and 3.0 s and 2.7 s for donor-acceptor distance, respectively. These constraints for H-bonds did not lead to an increase in residual constraints violation. A total of 54 backbone torsion angles were used in the calculations too. Moreover, according to NMR data, glycosidic torsion angles were kept in a range of 160 / 70 for the G anti, whereas a range of 10 /100 was used for the G syn. The calculations have been performed using a distance- dependent macroscopic dielectric constant of 4*r and an inﬁnite cut-off for nonbonded interactions to partially com- pensate for the lack of the solvent (17) have been used. Thus the 3D structures which satisfy NOE and dihedral angle constraints were constructed by simulated annealing calculations. An initial structure of the oligonucleotide was built using a completely random array of atoms. Using the steepest descent followed by quasi-Newton–Raphson method (VA09A), the conformational energy was minimized. Restrained simulations were carried out for 500 ps using the CVFF force ﬁeld as implemented in Discover software (Accelrys, San Diego, CA). The simulation started at 1000 K, and then the temperature was decreased stepwise until 273 K. The ﬁnal step was again to energy-minimize to reﬁne the obtained structures, using successively the steepest descent and the quasi-Newton–Raphson (VA09A) algo- rithms. Both dynamic and mechanic calculations were carried out by using 1 (kcal/mol)/A˚ 2 ﬂatwell distance restraints: 20 structures were generated. RMSD (root mean square deviation) values of 0.75 ± 0.27 s and 0.90 ± 0.31 s for the backbone and heavy atoms, respectively, were calcu- lated for the best seven structures. Illustrations of structures were generated using the INSIGHT II program, version 2005 (Accelrys, San Diego, CA). All the calculations were performed on a PC running Linux WS 4.0. The ﬁnal set of coordinates has been deposited in the Protein Data Bank (accession code: 2IDN). Circular dichroism CD samples of 1 and TBA were prepared at a concentration of 1 · 10 4 M, by using the buffer solution used for NMR Nucleic Acids Research, 2006, Vol. 34, No. 22 6655 experiments: 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA, pH 7.0. CD (circular dichroism) spectra and CD melt- ing curves were registered on a Jasco 715 circular dichroism spectrophotometer in a 0.1 cm pathlength cuvette. For the CD spectra, the wavelength was varied from 220 to 320 nm at 100 nm min 1. The spectra were recorded with a response of 16 s at 2.0 nm bandwidth and normalized by subtraction of the background scan with buffer. The temperature was kept constant at 20 C with a thermoelectrically controlled cell holder (Jasco PTC-348). CD melting curves were regis- tered as a function of temperature from 20 to 90 C at 294 nm with a scan rate of 10 C h 1 for both quadruplexes. The CD melting curves of TBA and modiﬁed aptamer showed sigmoidal proﬁles and were modeled by a two-state transition, using a theoretical equation for an intramolecular association, according to the van’t Hoff analysis (18). The Tm and DH values provide the best ﬁt of the experimental melting data. The reported errors for thermodynamic parame- ters are the SD values of the mean from the multiple deter- minations. The DS values were calculated by equation DS ¼ DH /Tm and free energy change values by the equation DG (T) ¼ DH TDS . Prothrombin time (PT) assay Human plasma samples were collected by venipuncture, in presence of 0.1 volumes 3.8% sodium citrate and fractionated by centrifugation at 2000 g for 5 min. PT times were mea- sured by using Koagulab MJ Coagulation System with a spe- ciﬁc kit RecombiPlas Tin HemosIL (Inst. Labs, Lexinton, USA). Brieﬂy, this method relies on the high sensitivity thromboplastin reagent based on recombinant human tissue factor. The addition of recombiplastin to the plasma in the presence of calcium ions initiates the activation of extrinsic pathway. This results ultimately in the conversion of ﬁbrino- gen to ﬁbrin, with a formation of solid gel. The procedure was performed according to the manufacturer’s instructions. TBA, 1 or vehicle (PBS) were added at different time points in a volume of 2 ml at a ﬁnal concentration of 20 mM. Data are expressed as mean ± SEM and are representative of at least three different measurements. RESULTS AND DISCUSSION The NMR sample of 1 was heated for 10 min at 80 C and slowly cooled down to room temperature, then its 1H-NMR spectrum was recorded by using pulsed-ﬁeld gradient WATERGATE (12) for H2O suppression (Figure 2). With the exclusion of some weak resonances due to very minor conformations also present in solution (whose relative inten- sities turned out to be insensitive to temperature changes), the simple appearance of 1D spectra of 1 indicates that, in the conditions used here, the modiﬁed oligomer forms mainly a single well-deﬁned hydrogen-bonded conformation. In fact, the 1H-NMR spectrum of 1 (700 MHz, T ¼ 25 C) shows the presence of eight well-deﬁned signals in the region 11.5–12.5 p.p.m., attributable to imino protons involved in Hoogsteen hydrogen bonds of at least two G-quartets, and of two broader and less intense singlets at 10.6 and 11.29 p.p.m., respectively, probably belonging to two imino protons of two thymines involved in mutual hydrogen