Synthesis, Characterization and Properties of ppp(A2′p)_nApCp and Related High‐Specific‐Activity ³²P‐Labelled Derivatives of ppp(A2′p)_nA

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 > 10⁶ 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 30 fold less active (if directly active at all).