Biochemical Characterization of the 2-5A Synthetase/RNase L Pathway by 2',5'-Phosphorothioate and Affinity Probes

该研究计划的重点是生物化学 核苷酸-蛋白质相互作用的表征 维持2-5A合成酶/RNase L 途径，细胞抗病毒防御的重要过程。 RNase L在核酸酶中的独特之处在于其依赖于2- 5A用于尿苷酸处的活化和切割特异性 残基 探讨了2-5A在细胞膜结合中的分子机制， 将检测RNaseL的活化。 为此，Pi 化学合成了一类新的代谢稳定的 2-5A的激动剂和拮抗剂，以研究生物化学 在体外和体内的RNA水解过程。 2 '，5'-硫代磷酸酯跨膜进入 病毒感染的细胞将通过聚（L-赖氨酸） 结合和这些探针对稳定性的影响 将测量病毒和细胞mRNA。 此外，本发明还提供了一种方法， 实验的目的是检查（i）添加的顺序 的底物和变构激活剂的RNase L所需的 生产复合体的形成，（二）要求 RNase L的2-5A与RNA之间的互补碱基配对 活化，和（iii）每个2 '，5'- 磷酸二酯键在2-5A上的结合和活化 核糖核酸酶L. 最后，单功能和双功能 核苷酸定点亲和标记已经被 合成以研究受体和2' -腺苷酸化位点， 重组40 kDa，均质纯100 kDa和110 kDa 2-5A 合成酶，蛋白激酶的ATP结合位点，以及 RNase L的2-5A结合位点。

The research proposed focusses on the biochemical characterization of nucleotide-protein interactions required for maintenance of the 2-5A synthetase/RNase L pathway, a vital process in cellular antiviral defense. RNase L is distinctive among nucleases in its dependence on 2- 5A for activation and cleavage specificity at uridylate residues. The molecular mechanism of 2-5A in the binding and activation of RNase L will be examined. To this end, The Pi chemically synthesized a new class of metabolically stable agonists and antagonists of 2-5A to study the biochemical processes for RNA hydrolysis in vitro and in vivo. Transmembrane passage of the 2', 5`-phosphorothioates into virus infected cells will be accomplished by poly(L-lysine) conjugation and the effect of these probes on the stability of viral and cellular mRNA will be measured. In addition, experiments are designed to examine (i) the order of addition of the substrate and allosteric activator to RNase L needed for productive complex formation, (ii) the requirement for complementary base pairing between 2-5A and RNA for RNase L activation, and (iii) the contribution of each 2', 5'- phosphodiester bond in 2-5A to the binding and activation processes of RNase L. Finally, mono- and bifunctional nucleotide site-directed affinity labels have been synthesized to study the acceptor and 2' -adenylation sites of recombinant 40 kDA, homogeneously pure 100 kDa and 110 kDa 2-5A synthetases, the ATP binding site of protein kinase, and the 2-5A binding site of RNase L.