Nucleic Acids Research, 2006, Vol. 34, No. 22 6659 Figure 6. Top view of the two G-tetrads of TBA (A) and 1 (B). Backbones and bases are depicted in colored ‘stick’ (carbons, green; nitrogens, blue; oxygens, red; hydrogens, white). White arrows indicate groove widths. Figure 7. Side view of TBA (A) and 1 (B). All residues are depicted in magenta, except T3 and T7 (yellow). The different orientations of T3 and T7 are clearly observable. diagnostic NOE connectivities between T3–Me and G2–H8, and between T3–H6 and G2–H8, the base of T3 folds back into the groove between the strands G1–G2 and G5–G6 in 1, and faces closely the G2–H8, thus justifying the very low chemical shift value of T3–H6 (dH 6.84) (Figure 7). Fur- thermore, another slight structural difference is to be ascribed to the orientation of T7. In fact, T7 folds back into the groove formed by the strands G5–G6 and G10–G11. Actually, this latter structural change seems due to a different folding of the backbone of this part of the molecule, where the sugar of T7 is closer to the bases G8 and G6 than the unmodiﬁed TBA (Figure 7). In order to determine the effects of the inversion of polarity site on the CD proﬁle and the thermal stability of the resulting quadruplex structure, CD spectra and CD melting and anneal- ing experiments were acquired for 1 and its natural counter- part (Figure 8). In particular, the CD spectra of 1 and TBA, performed at 20 C, are almost superimposable, both exhibit- ing two positive bands at 248 and 294 nm and a negative one at 267 nm. As expected, the TBA spectrum is typical of anti-parallel quadruplex structures (29–31). The spectrum of 1 is also typical of antiparallel quadruplex structures because 1 possesses two G-tetrads with an alternating anti and syn glycosidic conformation along each strand. However, this spectrum differs from CD spectra previously reported for the same ‘3+1’ strand arrangements (26), where the presence of an additional peak at 260 nm was attributed to the presence of third G-tetrad that implies a non-alternating guanine glyco- sidic conformation between the middle and bottom G-tetrads. As for melting and annealing CD experiments, taking into account that the rates of quadruplex formation/dissociation are very slow, we collected the data at 10 C/h. No signiﬁcant hysteresis emerged for each oligonucleotide comparing annealing and melting curves thereby indicating that, at the scan rate used, both systems were at equilibrium. The melting curves were analyzed using van’t Hoff analysis, and thermo- dynamic parameters are shown in Table 4. The values for TBA are in good agreement with those recently obtained by