TARGETED DEGRADATION OF MRNAS FOR SIGNALING FACTORS

我们建议进一步开发一种新的方法， 2 '，5'-寡腺苷酸（2-5A）和反义寡腺苷酸（2-5A）对 基因表达。 目标是特异性地消融mRNA种类， 干扰素和双链RNA信号通路中涉及的因子。 的 该策略涉及2- 5A依赖性RNA酶和核糖核酸内切酶， 介导干扰素对病毒感染的抑制作用。 以引导 2- 5A依赖性RNA酶切割独特的RNA序列，2-5A共价结合 与反义寡核苷酸连接（2- 5A-反义）。 反义 2- 5A-反义寡核苷酸组分结合特异性RNA序列 而伴随的2-5A组分激活2- 5A依赖性RNase 从而引起RNA在邻近所述RNA的区域中的切割， 目标序列。 dsRNA依赖的mRNA的催化降解 蛋白激酶（PKR）将在含有2-5A- 反义和同源的重组人2- 5A依赖性RNA酶。 的 反义长度和序列的影响，化学修饰， 混合不匹配的周转数，kcat，和Km的 反应将被确定。 PKR在介导dsRNA中作用 产生的信号将在PKR耗尽的细胞中用2-5A- 反义 同样地，我们将耗尽细胞中的蛋白质酪氨酸， 激酶，JAK-1和JAK-2，ISRE结合蛋白，IBF-1和细胞溶质 磷脂酶A2，以确定它们在调节干扰素- 刺激基因 由于其特异性、多功能性和效力， 2- 5A反义核酸是一种很有前途的基因表达调控方法 通过靶向RNA降解。

We propose to further develop a novel method which couples and amplifies the inhibitory effects of 2',5'-oligoadenylates (2-5A) and antisense on gene expression. The goal is to specifically ablate mRNA species for factors involved in interferon- and dsRNA-signaling pathways. The strategy involves the 2-5A-dependent RNase, and endoribonuclease which mediates inhibitory effects of interferon on virus infection. To direct 2-5A-dependent RNase to cleave unique RNA sequences, 2-5A is covalently linked to antisense oligonucleotide (2-5A-antisense). The antisense oligonucleotide component of 2-5A-antisense binds a specific RNA sequence while the accompanying 2-5A component activates 2-5A-dependent RNase thereby causing the cleavage of the RNA in a region proximal to the targeted sequence. The catalytic degradation on mRNA for dsRNA-dependent protein kinase (PKR) will be measured in reactions containing 2-5A- antisense and homogeneous, recombinant human 2-5A-dependent RNase. The effects of antisense length and sequence, chemical modifications, and hybrid mismatches on the turnover number, kcat, and the Km of the reactions will be determined. the role of PKR in relaying dsRNA generated signals will be studied in cells depleted of PKR with 2-5A- antisense. Similarly, we will deplete cells of the protein tyrosine kinases, JAK-1 and JAK-2, the ISRE-binding protein, IBF-1 and cytosolic phospholipase A2 to determine their functions in regulating interferon- stimulated genes. Because of its specificity, versatility and potency, 2-5A-antisense is a promising approach to the control of gene expression through targeted RNA degradation.