Radioimmune, radiobinding and HPLC analysis of 2-5A and related oligonucleotides from intact cells

The enzyme (2-5 A synthetase) which synthesizes ppp(A2′p)_n A where n = 2 to 4 (collectively referred to as 2-5A)¹ is widely distributed in a variety of cells and tissues in amounts which increase in response to interferon^2-5 and vary with growth and hormone status⁶. 2-5A activates a nuclease which inhibits protein synthesis^7-10. The non-phosphorylated 'core' of 2-5A ((A2′p)_nA, n = 2 to 4) can inhibit DNA synthesis and cell growth^11,12. Here we describe convenient and sensitive radioimmune (RI) and radiobinding (RB) assays for core and 2-5A. In combination with more satisfactory high performance liquid chromatography (HPLC) methods using reverse-phase C18 columns, these assays have been used to detect core and 2-5A in crude extracts from interferon-treated cells. The novel 2-5A synthetase products NAD2′p5′A2′p5′A and A5′p ₄5′A2′p5′A2′p5′A (ref. 13), which can also be detected using the RB assay, were not found in significant amounts. The natural occurrence of core has not been described previously.