6658 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 5. Stereoview representation of the superimposed seven best structures of 1. Backbones and bases are depicted in colored ‘stick’ (carbons, green; nitrogens, blue; oxygens, red; hydrogens, white). Table 2. Rise, tilt, roll and twist of TBA and of the best-minimized structure of 1 Table 3. Shear, stretch, stagger, buckle, propeller, opening of TBA and of the best-minimized structure of 1 TBA G1/G2 G5/G6 G10/G11 G14/G15 Rise 3.44 3.25 2.67 3.64 2.80 3.92 4.67 3.24 10.60 7.84 6.22 1.25 10.45 4.04 17.19 12.25 179.75 160.55 161.77 178.12 178.83 151.25 177.35 156.98 50.93 45.79 48.90 49.88 48.50 52.77 55.65 49.22 G1/G2 G5/G6 G10/G11 G14/G15 Tilt Roll Twist 1 Shear Stretch Stagger Buckle Propeller Opening 1 TBA G1/G6 G1/G10 G1/G15 G2/G5 G2/G11 G2/G14 (Figure 5). These data, along with the lack of signiﬁcant vio- lations of the experimental restraints (see Table 1), suggest that the obtained structures are representative of the structure actually adopted in solution by 1. As expected, 1 shows a right-handed helical backbone geometry and three edge-wise connecting TT, TGT and TT loops. As the unmodiﬁed TBA, 1 involves two stacked G-tetrads, but differs in guanine syn/anti distribution around the GGGG tetrads, being characterized by the unusual anti-anti-anti-syn and syn-syn-syn-anti arrangement of the two tetrads respectively, and in the relative strand orienta- tions, with three strands parallel to each other and with only one oriented in opposite manner. Interestingly, a similar strand orientation and glycosidic angle arrangement of the bases of the tetrads have already been observed for the assembly of three human telomeric repeats into a dimeric G-quadruplex (23) (2), the four-repeat Tetrahymena d(T2G4)4 sequence (24) (3), and the four-repeat human telomeric d(TAGGG(TTAGGG)3] sequence in K+ solution (25,26) (4) (Figure 1). Even though sequences 2–4 differ each other in both molecularity and folding topology, when their structures are represented in such a way that the four strands possess the same orientation (Figure 1), a certain resemblance among the three models is clearly apparent. Particularly, all the corresponding guanines are characterized by the same glycosidic angle arrangements. It is noteworthy that the only two tetrads in the modiﬁed TBA (1) show the same glycosidic angle arrangements as those in the lower two planes in 2–4. A further structural element joining the four complexes is the presence in all of them of one narrow, one wide and two medium width grooves. 6.15 6.67 4.82 6.59 6.02 6.95 6.42 6.23 6.04 7.00 5.41 6.59 1.72 2.57 2.32 2.09 1.87 2.01 2.77 2.08 1.75 2.51 2.07 1.88 0.88 0.38 2.69 0.71 0.59 0.37 0.11 0.02 2.05 0.04 1.82 0.37 11.32 3.51 3.47 38.43 5.12 28.42 5.50 5.58 24.52 26.55 5.12 8.34 176.45 174.13 176.25 164.49 162.35 172.46 157.97 168.29 177.68 147.31 162.35 176.04 89.48 91.17 0.95 6.02 86.32 86.37 91.51 87.08 3.39 0.96 86.32 89.79 G1/G6 G1/G10 G1/G15 G2/G5 G2/G11 G2/G14 Furthermore, a comparison of the most representative structures of 1 (the one with lowest energy after minimiza- tion), and the already reported NMR structure of the unmodi- ﬁed TBA (PDB code: 148D) has been accomplished by analyzing the helix parameters by CURVES (27,28) (Tables 2 and 3). These data clearly suggest that the overall structure adopted by 1 is similar to that of TBA. The base stacking of the two quadruplexes is quite similar, being the ﬁve- membered rings of the top guanines overlapped to the ﬁve- membered rings of the underneath guanine bases (Figure 6). Furthermore, as suggested by Tilt and Stagger values, the stacking between G pairs is slightly better in 1 than in TBA. The groove widths for 1 are just a little bit different from that of unmodiﬁed TBA. Particularly, TBA is character- ized by two wide grooves ( 19 s) between the strands G5–G6 and G10–G11 and between the strands G1–G2 and G14–G15 respectively, and other two narrow grooves that are calculated to be 13 s. On the other hand, 1 possesses the groove between the strands G1–G2 and G5–G6 just a little bit wider ( 16 s) and the groove between the strands G1–G2 and G14–G15 just a little bit narrower ( 15 s) (Figure 6). As for the sugar puckering, the two molecules possess the same sugar ring conformations, being in S-type conformation. It is interesting to note that the main structural difference rely on the orientation of T3. In fact, as indicated by the