6654 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 1. Schematic illustration of the structures adopted by TBA, modified TBA (1), the three human telomeric repeats sequence into a dimeric quadruplex (22) (2), the four-repeat Tetrahymena d(T2G4)4 sequence (23) (3), and the four-repeat human telomeric d[TAGGG(TTAGGG)3] sequence in K+ solution (24,25). Black arrowheads indicate 50!30 polarity of the strands. Black circle in 1 represents the 50–50 inversion of polarity site. Anti and syn guanines are depicted as yellow and blue solids, respectively. Red arrows indicate the direction of the proton donors and acceptors in Hoogsteen hydrogen bonds. analysis and a structure–activity relationship of this new modiﬁed TBA. MATERIALS AND METHODS The oligonucleotide 30GGT50-50TGGTGTGGTTGG30 (1) and the natural counterpart 50GGTTGGTGTGGTTGG30 (TBA) were synthesized on a Millipore Cyclon Plus DNA synthe- sizer, using solid phase b-cyanoethyl phosphoramidite chem- istry at 15 mmol scale. The synthesis of 1 was performed by standard methods for the 50TGGTGTGGTTGG30 tract, and using 50-phosphoramidites for the 30GGT50 tract. The oli- gomers were detached from the support and deprotected by treatment with concentrated aqueous ammonia at 55 C for 12 h. The combined ﬁltrates and washings were concentrated under reduced pressure, redissolved in H2O and analyzed and puriﬁed by HPLC on a Nucleogel SAX column (Macherey- Nagel, 1000-8/46); using buffer A: 20 mM KH2PO4 aqueous solution, pH 7.0, containing 20% (v/v) CH3CN; buffer B: 1 M KCl, 20 mM KH2PO4 aqueous solution, pH 7.0, containing 20% (v/v) CH3CN; a linear gradient from 0% to 100% B in 30 min and ﬂow rate 1 ml/min were used. The isolated oligo- mers have the following retention times: 1 ¼ 19.0 min; TBA ¼ 20.5 min. The oligomers were collected and successively desalted by Sep-Pak cartridges (C18). The isolated oligomers resulted to be >99% pure (NMR). Nuclear magnetic resonances NMR samples were prepared at a concentration of 2 mM, in 0.6 ml (H2O/D2O 9:1) buffer solution having 10 mM KH2PO4, 70 mM KCl, 0.2 mM EDTA, pH 7.0. For D2O experiments, the H2O was replaced with D2O by drying down the sample, lyophilization and redissolution in D2O alone. NMR spectra were recorded with Varian UnityINOVA 500 MHz and 700 MHz spectrometers. 1H chemical shifts were referenced relative to external sodium 2,2-dimethyl- 2-silapentane-5-sulfonate (DSS), whereas 31P chemical shifts were referenced to external phosphoric acid (H3PO4 85% v/v). 1D proton spectra of samples in H2O were recorded using pulsed-ﬁeld gradient WATERGATE (12) for H2O suppression. A proton-detected 1H–31P heteronuclear COSY was recorded in D2O in the hypercomplex mode with 2048 t2 points and 96 t1 increments, and a spectral width of 500 Hz along the 31P dimension. Phase-sensitive NOESY spectra (13) were recorded with mixing times of 100 and 200 ms (T ¼ 25 C and 5 C). Pulsed-ﬁeld gradient WATERGATE was used for NOESY and 1H–15N HSQC (14) spectra in H2O. TOCSY spectra (15) with mixing times of 100 ms were recorded with D2O solutions. All experiments were recorded using STATES-TPPI (16) procedure for quadrature detection. In all 2D experiments, the time domain data consisted of 2048 complex points in t2 and 400–512 ﬁds in t1 dimension. The relaxation delay