Triethylamine trihydrofluoride

RNA oligonucleotides were synthesized using t-BDMS phosphoramidite chemistry (Beaucage and Caruthers, 1981 ) as described in Wilson et al. (Wilson et al., 2001 (link)), implemented on an Applied Biosystems 394DNA/RNA synthesizer. Oligoribonucleotides containing 5-bromocytidine (ChemGenes) were deprotected in a 25% ethanol/ammonia solution for 36 h at 20°C. All oligoribonucleotides were redissolved in 115 μL of anhydrous DMSO, 60 μl triethylamine (Aldrich) and 75 μL triethylamine trihydrofluoride (Aldrich) to remove t-BDMS groups, and agitated at 65°C in the dark for 2.5 h. After cooling on ice for 10 min, 250 μL RNA quenching buffer (Glen Research) was added to stop the reaction and the oligonucleotides were desalted using NAP-10 columns (GE Healthcare).
RNA was further purified by gel electrophoresis in polyacrylamide under denaturing conditions in the presence of 7 M urea. The full-length RNA product was visualized by UV shadowing. The band was excised and electroeluted using an Elutrap Electroelution System (GE Healthcare) into 45 mM Tris-borate (pH 8.5), 5 mM EDTA buffer for 8 h. at 200 V at 4°C. The RNA was precipitated with ethanol, washed once with 70 % ethanol and dissolved in double-distilled water.

Oligoribonucleotides were synthesized using UltraMILD ribonucleotide phosphoramidites (Link Technologies) with 2′-O-tert-butyldimethylsilyl (TBDMS) protection implemented on an Applied Biosystems 394 synthesizer^{1 (link)}. Oligoribonucleotides were cleaved from the support and base deprotected in 25% ethanol/ammonia solution at 20 °C for 3 h, and evaporated to dryness. Removal of TBDMS protecting groups was achieved by redissolving oligoribonucleotides in 115 μL dimethyl sulfoxide to which was added 125 μL 1 M triethylamine trihydrofluoride (Sigma-Aldrich) and incubated at 65 °C for 2.5 h prior to butanol precipitation. All oligonucleotides were purified by gel electrophoresis in 20% polyacrylamide under denaturing conditions (7 M urea) in 90 mM Tris-borate (pH 8.3), 10 mM EDTA (TBE buffer). The full-length RNA product was visualized by brief ultraviolet shadowing. The band was excised and electroeluted using an Elutrap (Whatman) into 45 mM Tris-borate (pH 8.5), 5 mM EDTA buffer, 8 M ammonium chloride at 200 V. The RNA was precipitated with ethanol, washed with 70% ethanol, dried and resuspended in water. Oligoribonucleotides were subjected to further purification by reversed-phase HPLC (ACE C18-AR, Advanced Chromatography Technologies), using an acetonitrile gradient with an aqueous phase of 100 mM triethylammonium acetate (pH 7.0).

RNA oligonucleotides were synthesized using t-BDMS phosphoramidite chemistry (24 ) as described in Wilson et al. (25 (link)), implemented on an Applied Biosystems 394DNA/RNA synthesizer. RNA was synthesized using ribonucleotide phosphoramidites with 2′O-tert-butyldimethyl-silyl (t-BDMS) protection (26 ,27 (link)) (Link Technologies). All oligoribonucleotides were redissolved in 100 μl of anhydrous DMSO and 125 μl triethylamine trihydrofluoride (Sigma-Aldrich) to remove t-BDMS groups, and agitated at 65°C in the dark for 2.5 h. After cooling on ice for 10 min, the RNA was precipitated with 1 ml of butanol, washed once with 70% ethanol and suspended in double-distilled water. 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 synthesized using phosphoramidite chemistry (Beaucage and Caruthers 1981 (link)) implemented on an Applied Biosystems 394DNA/RNA synthesizer as described in Wilson et al. (2001) (link). Ribonucleotide phosphoramidites were protected by 2′O-tert-butyldimethyl-silyl (t-BDMS) (Hakimelahi et al. 1981 (link); Perreault et al. 1990 (link)) (Link Technologies). All oligoribonucleotides were redissolved in 100 µL of anhydrous DMSO and 125 µL triethylamine trihydrofluoride (Sigma-Aldrich) to remove t-BDMS groups, and agitated at 65°C in the dark for 2.5 h. After cooling on ice for 10 min, the RNA was precipitated with 1 mL of butanol, washed once with 70% ethanol and suspended in double-distilled water. Fluorescein (Link Technologies) and Cy3 (GE Healthcare) were attached to the 5′ termini of the oligonucleotides as phosphoramidites in the final synthesis cycle as required.

RNA oligonucleotides were synthesized using t-BDMS phosphoramidite chemistry (14 ) as described in Wilson et al. (15 (link)), implemented on an Applied Biosystems 394DNA/RNA synthesizer. RNA was synthesized using ribonucleotide phosphoramidites with 2′O-tert-butyldimethyl-silyl (t-BDMS) protection (16 ,17 (link)) (Link Technologies). Oligonucleotides containing 5-bromocytidine (ChemGenes) were deprotected in a 25% ethanol/ammonia solution for 36 h at 20°C. All oligoribonucleotides were redissolved in 100 μl of anhydrous DMSO and 125 μl triethylamine trihydrofluoride (Sigma-Aldrich) to remove t-BDMS groups, and agitated at 65°C in the dark for 2.5 h. After cooling on ice for 10 min, the RNA was precipitated with 1 mL of butanol, washed once with 70% ethanol and suspended in double-distilled water. RNA without bromine modification was purchased from Accurate Biotechnology (Hunan, China) Co., Ltd.