Procedures for the Assay of (2′-5′)-Oligoadenylic Acids and Their Derivatives in Intact Cells

This chapter describes two methods by which penetration of the cell membrane by the oligonucleotide triphosphates is achieved. An alternative approach is to remove the triphosphate groups, which renders the oligonucleotides inactive in inhibiting protein synthesis in cell-free systems, but allows them to penetrate cells, and be processed by the cell to some active form. A procedure for the treatment of intact cells with the non-triphosphorylated 2'-5'(A) is also described. (2'-5')-Oligoadenylic acids are extremely potent inhibitors of protein synthesis, being effective at the subnanomolar level in cell-free systems. However, the setting-up of cell-free system assays, involving the preparation and testing of cell extracts and messenger RNAs, is not to be undertaken lightly. Removal of the 5'-triphosphate group from ppp5'A(2'p5'A), radically reduces its polarity, thus permitting its penetration of the cell membrane. Methods using intact cells have distinct advantages including they require less skill, are less costly, and are probably as reproducible, although this may depend on the relative experience of the operator with cell extracts versus whole cells.