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

本研究的重点是维持2-5A合成酶/RNase L通路所需的核苷酸-蛋白相互作用的生化特性，这是细胞抗病毒防御的重要过程。在核酸酶中，RNase L的独特之处在于它依赖于2- 5A来激活和特异性切割尿苷酸残基。2-5A在RNase L的结合和活化中的分子机制将被研究。为此，The Pi化学合成了一类新的代谢稳定的2-5A激动剂和拮抗剂，在体外和体内研究RNA水解的生化过程。2', 5 ' -硫代磷酸酯通过聚l -赖氨酸偶联进入病毒感染细胞，并测量这些探针对病毒和细胞mRNA稳定性的影响。此外，实验旨在检查(i)底物和变构激活剂添加到RNase L所需的生产复合物形成的顺序，（ii） 2- 5a和RNA之间互补碱基配对对RNase L激活的要求，以及（iii）每个2'的贡献。最后，合成了单功能和双功能核苷酸位点亲和标记，研究了重组40 kDA、均质纯100 kDA和110 kDA 2- 5a合成酶的受体和2' -腺苷化位点、蛋白激酶的ATP结合位点、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.