Photochemical crosslinking in oligonucleotide-protein complexes between a bromine-substituted 2-5A analog and...

2',5'-oligoadenylates known as 2-5A [px(A2'p)nA; chi = 2 or 3, n greater than or equal to 2] are produced in interferon-treated cells in response to double-stranded RNA. 2-5A binds with high affinity to a 2-5A-dependent RNase resulting in the cleavage of single-stranded RNA. An efficient, rapid, and extremely sensitive photoaffinity labeling method was developed to facilitate detection of 2-5A-dependent RNase. A bromine-substituted and radioactive derivative of 2-5A, the 5'-monophosphate, p(A2'p)2(br8A2'p)2A3'-[32P]Cp, was synthesized as probe for 2-5A-dependent RNase. Even though this bromine-substituted analog of 2-5A bore no 5'-terminal triphosphate or diphosphate, it bound to 2-5A-dependent RNase with the same high affinity as did 2-5A per se but it was a less effective activator of the RNase under the present assay conditions. The presence of bromine atoms in the 2-5A analog enhanced by more than 200-fold crosslinking to 2-5A-dependent RNase under a uv lamp; many additional polypeptides were also labeled but at much lower levels. Furthermore, using high-intensity uv laser irradiation (308 nm) covalent attachment of the bromine-substituted 2-5A analog to 2-5A-dependent RNase was readily achieved within 10(-6) s.