Nucleic Acids Research, 2006, Vol. 34, No. 22 6657 Figure 4. Expanded region of NOESY spectrum of 1 (700 MHz, T ¼ 25 C, mt ¼ 100 ms) correlating G–H8 and H20–H200 protons. Classical G–H8/H20–H200 NOEs (nuclear Overhauser effect) correlations for the four-stranded part of the complex are reported. Pattern of NOEs for the Gs in syn glycosidic conformation are highlighted by vertical dashed lines. Table 1. Experimental constraints and structure statistics of the best 20 structures Experimental constraints Total NOEs NOEs from non-exchangeable protons NOEs from exchangeable protons Hydrogen bonds constraints Dihedral angle constraints CVFF energy (kcal mol 1) of the minimized structures Total nonbond restraint NOEs violations Number > 0.2 s Maximum (A˚ ) Sum (A˚ ) Average violation (A˚ ) r.m.s. deviations from the mean structure (A˚ ) All backbone heavy atoms All heavy atoms 198 185 13 36 62 370.158 ± 4.529 40.783 ± 5.145 13.213 ± 1.417 3.40 ± 1.63 0.560 ± 0.263 3.821 ± 0.693 0.016 0.75 ± 0.27 0.90 ± 0.31 conformation. All this means that the 1 folds into a chair-like quadruplex structure possessing three strands parallel to each other and only one strand oriented in reverse manner (Figure 1). In order to get the 3D structure of 1 at atomic level, an estimation of proton–proton distances has been retrieved from cross-peak intensities in 2D NOESY experiments acquired at 700 MHz, both at T ¼ 25 C and T ¼ 5 C (mixing time 100 ms for the experiment acquired in D2O and 200 ms for the experiments acquired in H2O). Pseudo-atoms were introduced where needed. A total of 198 distances were used for the calculations and, as suggested by the presence of 8 G and 2 T imino protons in the 1D 1H-NMR spectrum, 32 supplementary distance restraints (HN1–O6, N1–O6, HN2–N7, N2–N7) for 16 hydrogen bonds corresponding to the 2 G-quartets, and 4 distance restraints (HN3–O4, N3– O4) for 2 hydrogen bonds between T13 and T4 were also incorporated during the computations (Table 1). The back- bone torsion angles b and e were estimated from the scalar coupling measured in the 2D proton-detected heteronuclear 1H–31P COSY by using the semi-empirical Karplus equation (22). Thus, since for all sugars in 1 all JP,H50 and JP,H500 were <8 Hz, b backbone torsion angles were restricted to 180 ± 30 (22). For residues 3, 4, 5, 6, 7, 8, 9 and 11, JP,H30 was >8 Hz, and the corresponding e angles were restricted to 120 ± 30 (21). Sugar ring conformations were obtained from the anal- ysis of the PE-COSY spectrum. From the SJH40 estimation (<10 Hz), no dihedral restraints for g were applied to the four G nucleotides in the syn glycosidic conformation, whereas g backbone torsion angles for residues G6 and G11 (possessing an anti conformation) were restricted to 60 ± 30 (21). All measurable JH10,H20 were reasonably large, indicat- ing a predominant S-type nature of sugar ring conformations: d and n2 angle constraints have been added consistently. Further, in agreement with NMR data, glycosidic torsion angles for four out of eight guanines involved in the forma- tion of the two G-tetrads were ﬁxed in the anti-domain ( 160 / 70 ), whereas the c angle was kept in a range of 10 /100 (syn-conformation) for the remaining four G residues. Therefore, 3D structures which satisfy NOEs were con- structed by simulated annealing (SA) calculations. An initial structure of the oligonucleotide was constructed possessing a random conformation and minimized, in order to eliminate any possible source of initial bias in the folding pathway. Restrained simulations were carried out for 500 ps using the CVFF (consistent valence force ﬁeld). The restrained SA (simulated annealing) calculations started at 1000 K, and, thereafter, the temperature was decreased stepwise down to 273 K. The aim was to energy-minimize and reﬁne the structures. A total of 20 structures were generated. Average RMSD values of 0.75 ± 0.27 s and 0.90 ± 0.31 s for the backbone and all heavy atoms, respectively, were obtained from the superimposition of the best seven structures