Nolan-Sorden N L, Lesiak K, Bayard B, Torrence P F, Silverman R H
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Anal Biochem. 1990 Feb 1;184(2):298-304. doi: 10.1016/0003-2697(90)90684-2.
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.
2',5'-寡腺苷酸，即2-5A [px(A2'p)nA；χ = 2或3，n≥2]，是在干扰素处理的细胞中由双链RNA诱导产生的。2-5A以高亲和力与一种依赖2-5A的核糖核酸酶结合，导致单链RNA的切割。为了便于检测依赖2-5A的核糖核酸酶，开发了一种高效、快速且极其灵敏的光亲和标记方法。合成了一种2-5A的溴代放射性衍生物，即5'-单磷酸，p(A2'p)2(br8A2'p)2A3'-[32P]Cp，作为依赖2-5A的核糖核酸酶的探针。尽管这种2-5A的溴代类似物没有5'-末端三磷酸或二磷酸，但它与依赖2-5A的核糖核酸酶结合的亲和力与2-5A本身相同，不过在当前的检测条件下，它作为核糖核酸酶的激活剂效果较差。2-5A类似物中的溴原子在紫外灯下使与依赖2-5A的核糖核酸酶的交联增强了200多倍；许多其他多肽也被标记，但水平要低得多。此外，使用高强度紫外激光照射（308 nm），在10^(-6)秒内即可轻松实现溴代2-5A类似物与依赖2-5A的核糖核酸酶的共价连接。
