Synthesis, Characterization and Properties of ppp(A2′p)nApCp and Related High-Specific-Activity 32P-Labelled Derivatives of ppp(A2′p)nA

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Abstract
T4 RNA ligase (EC 6.5.1.3) was used to link cytidine 3'',5''-[5''-32P]bisphosphate or unlabeled cytidine 3'',5''-bisphosphate (pCp) covalently to the 3''-OH of individual components of 5''-triphospho-oligo[(2''-5'')adenylyl]adenosine [ppp(A2''p)nA with n = 2 or 3] and adenylyl(2''-5'')adenyly(2''-5'')adenosine [(A2''p)2A] to yield 5''-triphospho-oligo[(2''-5'')adenylyl]adenylyl(3''-5'')cytidine 3''-phosphate [ppp(A2''p)nApCp with n = 2 or 3] and adenylyl(2''-5'')adenylyl(2''-5'')adenylyl(3''-5'')cytidine 3''-phosphate [A2''p)2ApCp], respectively. The radioactive products isolated by high-performance liquid chromatography had specific activities > 106 Ci/mol. These products were effective probes for use in radiobinding and radioimmune assays for ppp(A2''p)nA and (A2''p)nA. ppp(A2''p)nA is unstable in cell-free systems and in intact cells. ppp(A2''p)nApCp was much more stable than ppp(A2''p)nA in extracts of rabbit reticulocytes or [mouse] Ehrlich ascites tumor cells. (A2''p)2Ap, (A2''p)2ApC (reticulocyte only) or (A2''p)2ApCp were also more stable than unmodified (A2''p)2A in these systems. The results are consistent with a specific degradation pathway for ppp(A2''p)nA which proceeds from the 3'' terminus. ppp(A2''p)3ApCp activated the ppp(A2''p)nA-dependent RNase and inhibited protein synthesis in a reticulocyte cell-free system at least as well as unmodified tetramer ppp(A2''p)3A, suggesting that an unmodified 3'' terminus is not required for full activity in this system. In extracts from Ehrlich ascites tumor or L-cells, ppp(A2''p)nApCp was .gtoreq. 30-fold less active (if directly active at all).