Synthesis and Purification of Telomeric DNA Oligonucleotides

DNA oligonucleotides were chemically synthesized on an ABI 394 DNA/RNA synthesizer (Applied Biosystem) at 5 or 1 μmol scale, using the standard β-cyanoethylphosphoramidite solid phase chemistry as described elsewhere (27 (link)). In particular, the following oligonucleotides were synthesized: d[(TTAGGG)₄TT] and d(TTAGGGT), corresponding to two different truncations of human telomeric DNA sequence. After synthesis, the oligomers were detached from the support and deprotected by treatment with concentrated aqueous ammonia at 55°C for 17 h. The combined filtrates and washings were concentrated under reduced pressure, dissolved in H₂O, and purified by high-performance liquid chromatography (HPLC) employing standard protocols. The isolated oligomers were proved to be >98% pure by NMR. The concentration of oligonucleotides was determined by UV adsorption measurements at 90°C using appropriate molar extinction coefficient values ϵ (λ = 260 nm), calculated by the nearest-neighbor model (28 (link)). All G4s were prepared in 10 mM KH₂PO₄ buffer containing 70 mM KCl, pH 7.0. Samples were then heated at 90°C for 5 min, gradually cooled to room temperature overnight, and finally incubated at 4°C for 24 h, before data acquisition. The G4 parallel arrangement of the 26-mer telomeric sequence (TelG4-up) was prepared and checked as previously described (29 (link),30 (link)).