2 o pivaloyloxymethyl phosphoramidite monomers

RNA sequences were chemically synthesized on a solid support using an ABI 394 synthesizer. After RNA elongation with 2′O-pivaloyloxymethyl phosphoramidite monomers^{34 (link),35} (Chemgenes, USA), the 5′-hydroxyl group was phosphorylated and the resulting H-phosphonate derivative^{36 (link)} oxidized and activated into a phosphoroimidazolidate derivative to react with either phosphoric acid (for ppRNA synthesis), pyrophosphate (pppRNA)^{36 (link)} or guanosine diphosphate (GpppRNA)^{37 (link),38 (link)}. N7-methylation of the purified GpppRNA was performed enzymatically using N7-hMTase^{37 (link),38 (link)}. To prepare monophosphate RNA (pRNA), the 5′-H-phosphonate RNA was treated with a mixture of N,O-bis-trimethylacetamide (0.4 ml), CH₃CN (0.8 ml) and triethylamine (0.1 ml) at 35 °C for 15 min, and then oxidized with a tert-butyl hydroperoxide solution (5–6 M in decane, 0.4 ml; 35 °C, 15 min). After deprotection and release from the solid support, RNA sequences were purified by IEX-HPLC (>95% pure) and their identity were confirmed by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Fight) spectrometry.