After confirming that the SoM protecting group and SoA linker can function as planned for RNA synthesis, we completed the synthesis of the remaining required phosphoramidites 1b-c, and went ahead to synthesize ac4C-containing RNAs. The 26-mer RNA 19b (Figure ) was chosen as the target. This RNA was designed for the study of the effects of site-specific ac4C modification on mRNA translational efficiency, results of which will be published when they become available. The RNA was synthesized under the same conditions as described for 19a. The ac4C modification was introduced using standard commercially available phosphoramidite 1e (Figure ). Details are provided in the Supporting Information. The fully protected RNA can be represented with 2 (Scheme 1). Deprotection and cleavage were also carried out under the same conditions for 19a. However, purification was conducted differently due to the difficulty of removal of unknown impurities using the Glen-Pak TM method. The solution of 5′-DMTr-on otherwise fully deprotected crude RNA resulted from Et3N-3HF treatment was quenched with excess Me3SiOMe. After removing volatiles, the RNA was purified with trityl-on RP HPLC. The Me3SiOMe quenching is important for preventing potential damage to HPLC column by fluoride. The crude trityl-on HPLC profile is given in Figure . As can be seen, the full-length sequence, which has a retention time of 35.7 minutes, is separated well from failure sequences at 12-25 minutes and other impurities at 39-55 minutes. The fulllength sequence was collected, and detritylated with 80% acetic acid. After removing volatiles, the purified RNA was analyzed with RP HPLC. A single peak with a retention time of 17.3 minutes was given indicating that pure RNA could be obtained (Figure ). The RNA was analyzed with MALDI MS. As shown in Figure , the molecular peak at m/z 8246 indicates that the acetyl group of the ac4C modification in RNA 19b survived all the RNA deprotection and cleavage conditions, which include treating with 10% DBU in ACN at room temperature and Et3N-3HF in DMSO at 65 °C (Scheme 1). To further confirm that RNA 19b contains ac4C, the same batch of solid support carrying the un-deprotected RNA 19b was subjected to the same deprotection, cleavage and purification conditions except that the DBU treatment was omitted and nucleobase deprotection was caried out under nucleophilic conditions using AMA (saturated NH4OH and MeNH2, v/v 1:1) at 65 °C. Under these conditions, the acetyl group of ac4C as well as the SoM groups and the SoA linker were all cleaved. After removal of 2′-OTBS groups under standard RNA deprotecting conditions, RNA 19c (Figure ) was given. MALDI MS analysis gave the molecular peak at m/z 8202, which corresponds to 19c (Figure ). A MALDI MS sample containing both 19b and 19c was then analyzed. As expected, two peaks were observed with m/z 8247 for 19b and m/z 8206 for 19c (Figure ). The 41 mass difference between the two peaks matched closely with the mass difference between ac4C and C. This further confirmed that the SoM method is suitable for the synthesis of ac4C-RNAs.