TARGETED DEGRADATION OF MRNAS FOR SIGNALING FACTORS

我们建议进一步开发一种新的方法，将其耦合和放大 2‘，5’-寡腺苷(2-5A)及其反义寡核苷酸的抑制作用 基因表达。我们的目标是特异性地去除mRNAs。 干扰素和dsRNA信号通路中涉及的因素。这个 策略涉及依赖2-5A的核糖核酸酶和内切核糖核酸酶 介导干扰素对病毒感染的抑制作用。导演，导演 2-5A依赖的核糖核酸酶切割独特的RNA序列，2-5A是共价的 连接到反义寡核苷酸(2-5A-反义)。反义词 2-5A反义寡核苷酸成分结合特定的RNA序列 而伴随的2-5A组分激活2-5A依赖的核糖核酸酶 从而导致RNA在邻近区域的RNA被切割 目标序列。依赖dsRNA的信使核糖核酸的催化降解 蛋白激酶(PKR)将在含有2-5A-的反应中被测定。 反义和同源的重组人2-5A依赖的核糖核酸酶。这个 反义长度和序列、化学修饰和 在成交量、kcat和里程上的混合不匹配 反应将是确定的。PKR在dsRNA传递中的作用 产生的信号将在耗尽PKR的细胞中用2-5A- 不合常理。同样，我们也会耗尽细胞中的酪氨酸蛋白 激酶、JAK-1和JAK-2、ISRE结合蛋白、IBF-1和胞浆 磷脂酶A2以确定其在调节干扰素- 受刺激的基因。由于其特异性、多功能性和效力， 2-5A反义基因是一种很有前途的基因表达调控方法 通过有针对性的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.