Synthesis, characterization and properties of ppp(A2'p)nApCp and related high-specific-activity 32P-labelled derivatives of ppp(A2'p)nA.

Abstract

T4 RNA ligase (EC 6.5.1.3) has been used to link cytidine 3',5'-[5'-32P]bisphosphate or unlabelled 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')adenylyl(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 greater than 10(6) Ci/mol. These products were found to be effective probes for use in radiobinding and radioimmune assays for ppp(A2'p)nA and (A2'p)nA [M. Knight et al. (1980) Nature 288, 189-192]. ppp(A2'p)nA is unstable in cell-free systems and in intact cells. ppp(A2'p)nApCp, however, was found to be much more stable than ppp(A2'p)nA in extracts of rabbit reticulocytes or Ehrlich ascites tumour 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 mouse Ehrlich ascites tumour or L-cells, however, ppp(A2'p)nApCp was greater than or equal to 30 fold less active (if directly active at all).