Purification of synthetic RNA

RNA purification is significantly more difficult than DNA purification due to possible chain cleavage and isomerization of the internucleotide phosphate linkage. Several methodologies for deprotection and purification have been developed.

Small Quantity Purification

The first step following the synthesis of oligonucleotides involves a base treatment to remove the N-protecting and cyanoethyl groups. For base deprotection, both 28% aqueous ammonia containing ethanol (1:3) and anhydrous saturated ethanolic ammonia solutions have been used. The anhydrous saturated ethanolic ammonia solution was found to be superior in preventing the loss of any silyl groups (S. A. Scaringe, C. Francklyn, N. Usman, Nucl. Acids Res., 18, 5433, 1990). It has been reported that a certain amount of silyl groups will be lost when using the 28% aqueous ammonia containing ethanol (1:3) solution (J. Stawinski, R. Stromberg, M. Thelin, E. Westman, Nucl. Acids Res., 16, 9285, 1988).

The second step in the deprotection process involves removal of silyl protecting groups (TBDMS). This has been accomplished by 1.0 M solution of tetrabutylammoniumfluoride (TBAF) in THF. The reaction involves 50 equivalents of TBAF per TBDMS group during 24 hours. Even though initially deemed adequate, this method is slow and still leaves some TBDMS groups on the oligonucleotide. Superior results have been obtained with triethylamine-tris-hydrofluoride (TEA.3HF). In the synthesis of 76-mer tRNA the reaction with neat TEA.3HF for 14 hours resulted in complete removal of all TBDMS groups (D. Gasparutto et al., ibid.). The faster rate of silyl removal with TEA.3HF has been demonstrated by others (B. Sproat, F. Colonna, B. Mullah, D. Tsou, A. Andrus, A. Hampel, R. Vinayak, Nucleosides & Nucleotides, 14, 255, 1995).

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