6656 Nucleic Acids Research, 2006, Vol. 34, No. 22 bonding (see below). Moreover, ﬁfteen signals in the aro- matic region due to the presence of nine guanine H8 and six thymine H6 protons were clearly observable. A combination of the analysis of 2D NOESY (700 MHz, 3T1P¼-NM25RCs),peTcOtroCsScYopysp(e2c0tr2a M(7H0z0, MTH¼z, 2T5 C¼) 2a5lloCw)e,danuds to get the almost complete assignment (see Supplementary Material) of both exchangeable and nonexchangeable protons and phosphorus resonances of 1. Particularly, the 1D proton decoupled phosphorus spectrum displays fourteen signals (see Supplementary Material). After assigning the 1H resonances within each deoxyribose by a 2D TOCSY experi- ment, the 2D proton-detected heteronuclear 1H–31P COSY (see Supplementary Material) allowed us to assign the entire backbone correlating each phosphorus resonance to the respective 50-coupled H30 proton and 30-coupled H50 and H50 protons of adjacent ribose. Further, NOEs between base protons and H10, H20 and H200 protons allowed us to assign all aromatic protons to the pertinent base. It is interesting to note that the intensities NOESY (700 MHz, T ¼ 25 C, mixing time 100 ms) cross-peaks between the H8 proton bases and sugar H10 resonances indi- cate that four (G2, G5, G10, G14) out of nine Gs of 1 adopt a syn glycosidic conformation (Figure 3), where the H8 reso- nances of syn G residues are upﬁeld shifted with respect to those of the anti ones (5,19,20). Then, four anti-Gs (G1, G6, G11 and G15) have classical H8/H20–H200 sequen- tial connectivities to 50 neighboring syn-Gs (G2, G5, G10 and G14, respectively) (Figure 4), indicating that the subunits G1–G2, G5–G6, G10–G11 and G14–G15 are involved in the formation of a four-stranded helical structure (underlined residues adopt a syn glycosidic conformation). Moreover, the entire pattern of NOEs observed for all cited Gs indicates that the backbone conformations of these tracts resemble Figure 2. Expanded region of the proton NMR spectrum of 1 (700 MHz, T ¼ 25 C). those of the unmodiﬁed TBA possessing a right-handed helix structure (Figure 4). The alternation of syn and anti G residues within each strand suggests that, as TBA, 1 folds into a monomolecular foldback quadruplex, characterized by two G tetrads. Further, a number of unusual NOE connectivities were observed between syn-Gs and Ts, indicating that 50TG30 and 50GT30 tracts do not adopt a helical winding, thus suggesting that, most probably, the TT and TGT tracts form loops. In order to assign exchangeable protons to their respective G or T residues, a 1H–15N HSQC experiment (700 MHz, T ¼ 5 C) has been acquired (see Supplementary Material). Eight exchangeable protons (the eight at lower ﬁeld) correlate with 15N in the region between 143 and 147 p.p.m. indicating that they are attributable to imino protons of G residues (21), whereas the signals at 10.65 and 11.35 p.p.m. correlate with nitrogens at 155.7 and 155.6 p.p.m., respectively, thus indi- cating that the latter belong to T residues (21). Interestingly, these two signals are characterized by strong NOE effects between each other, thus suggesting that two thymines are in close proximity and, verisimilarly, interacting by a mutual hydrogen bond, exactly as observed in the unmodiﬁed TBA. Moreover, NOEs between H10 proton of G8, methyl protons of T9 and three G imino protons (dH 12.03, 11.97 and 11.91 p.p.m.), and NOEs between other four imino protons (dH 12.37, 12.35, 12.30 and 11.95 p.p.m.) and H6, methyl, H30 and H10 protons of T4 and H30, H20, H40 and H10 of T13, led us to assign the exchangeable protons of two tetrads (see Supplementary Material). The whole of data indicates that the structure of 1 is a fold- back quadruplex characterized by two G tetrads formed by the residues G1, G6, G10, G15 and G2, G5, G11, G14, respectively, where the underlined residues possess a syn gly- cosidic conformation. Interestingly, the two tetrads assume an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the bases. It is noteworthy that even the T imino protons (dH 10.65 and 11.35 p.p.m., T ¼ 5 C) are characterized by several NOE connectivities with a set of G imino protons belonging to only one tetrad (dH 11.95, 12.80, 12.35 and 12.37 p.p.m.). Moreover, the pattern of NOEs attributable to the loop T12–T13 and T3–T4 let us to identify the T4 and T13 as the residues that are H-bonded to each other. Interestingly, T13 and T4 are mutually hydrogen bonded in TBA as well. Then, the facts that TGT loop possesses some NOEs with G imino protons of one tetrad and that the two TT loops are characterized by NOEs with G imino protons of the other tetrad indicate that no loop assumes a dog-eared Figure 3. Expanded region of the NOESY spectrum of 1 (700 MHz, T ¼ 25 C, mt ¼ 200 ms) correlating G–H8 and H1’ protons.