3.4. Solid-phase Synthesis of RNA Oligonucleotides

RNA oligonucleotides were synthesized using an ABI model 394 DNA/RNA synthesizer on a 1 µmol scale using 2′-O-PivOM and 2′-O-AcSM phosphoramidites (Chemgenes) and a long chain alkylamine (LCAA) controlled-pore glass (CPG) as solid support (Link technologies).

Oligonucleotides (ON) were assembled in TWIST^TM synthesis columns (Glen Research). Phosphoramidites were vacuum dried prior to their dissolution in extra dry acetonitrile (Biosolve) at 0.1 M concentration. Coupling for 180 s was performed with 5-benzylmercaptotetrazole (BMT, 0.3 M) as the activator. The oxidizing solution was 0.1 M iodine in THF/pyridine/H₂O (78:20:2; v/v/v; Link Technologies). The capping step was performed for 160 s with a mixture of 5% phenoxyacetic anhydride (Pac₂O) in THF and 10% N-methyl imidazole in THF (Link Technologies). Detritylation was performed with 3% TCA in CH₂Cl₂. After RNA assembly completion, the column was removed from the synthesizer and dried under a stream of argon.