1 m triethylamine trihydrofluoride

RNA oligonucleotides were synthesized using the solid-phase phosphoramidite chemistry implemented on an ABI-394 DNA/RNA synthesizer. The ribonucleotide phosphoramidites, Pac-A-CE, Ac-C-CE, iPr-Pac-G-CE, and U-CE, with a t-BDMS protecting group on the 2′O, were obtained from Link Technologies. Fluorescein (Link Technologies) and Cy3 (GE Healthcare) were attached to the 5′ termini of the oligonucleotides as phosphoramidites in the final cycle of the synthesis, as required.
RNA oligonucleotides were deprotected in 25% ethanol/ammonia solution for 3 h at room temperature and evaporated to dryness. They were redissolved in 115 µL DMSO (Sigma-Aldrich) to which was added 60 µL triethylamine (Sigma-Aldrich) and 75 µL 1 M triethylamine trihydrofluoride (Sigma-Aldrich), and incubated at 65°C for 2.5 h to remove the t-BDMS protecting groups. Thereafter, samples were cooled on ice for 10 min and 250 µL RNA quenching buffer (Glen Research) was added to stop the reaction. The oligonucleotides were then desalted by application to NAP-10 columns (GE Healthcare).

Ribooligonucleotides were synthesized using tert-butyldimethylsilyl phosphoramidite chemistry (26 ), as described in Wilson et al. (27 (link)). Oligoribonucleotides were deprotected in 25% ethanol/ammonia solution at 55°C for 2 h, and evaporated to dryness. Oligoribonucleotides were redissolved in 100-µl dimethyl sulfoxide to which 125 µl of 1 M triethylamine trihydrofluoride (Sigma-Aldrich) was added and incubated at 65°C for 2.5 h to remove tert-butyldimethylsilyl-protecting groups. All oligonucleotides were purified by gel electrophoresis in polyacrylamide in the presence of 7 M urea, and the full-length RNA product was visualized by ultraviolet shadowing. The band was excised and electroeluted using an Elutrap (Whatman) into 45 mM Tris borate (pH 8.5) and 5 mM ethylenediaminetetraacetic acid buffer for 8 h at 200 V at 4°C. The RNA was precipitated with ethanol, washed once with 70% ethanol and dissolved in water. The concentration of RNA was determined by measuring the absorbance at 260 nm using an extinction coefficient of 319.4 mM⁻¹ cm⁻¹.