TARGETED DEGRADATION OF MRNAS FOR SIGNALING FACTORS

针对信号因子的 MRNAS 定向降解

• 批准号: 6237445
• 负责人: ROBERT H SILVERMAN
• 金额: $ 22.23万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-16 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6237445
• 关键词: RNase protection assay; adenosine monophosphate; antisense nucleic acid; biological signal transduction; chemical cleavage; chemical kinetics; chemical structure function; computer assisted sequence analysis; double stranded RNA; enzyme activity; enzyme mechanism; gel mobility shift assay; gene expression; gene induction /repression; genetic transcription; interferons; messenger RNA; northern blottings; nuclear runoff assay; nucleic acid sequence; pancreatic ribonuclease; phospholipase A2; protein kinase; protein tyrosine kinase; transcription factor

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.

我们建议进一步开发一种耦合和放大的新方法 2',5'-寡腺苷酸 (2-5A) 和反义的抑制作用 基因表达。 目标是专门消除 mRNA 种类 干扰素和 dsRNA 信号通路中涉及的因素。 这 策略涉及 2-5A 依赖性 RNase 和核糖核酸内切酶 介导干扰素对病毒感染的抑制作用。 指挥 2-5A 依赖性 RNase 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 2-5A-反义寡核苷酸成分结合特定的RNA序列 而伴随的 2-5A 成分则激活 2-5A 依赖性 RNase 从而导致靠近RNA的区域中的RNA被切割 目标序列。 dsRNA依赖性mRNA的催化降解 蛋白激酶 (PKR) 将在含有 2-5A- 的反应中进行测量 反义和均质重组人 2-5A 依赖性 RNase。 这 反义长度和序列、化学修饰的影响，以及 周转数、kcat 和 Km 的混合不匹配 反应将被确定。 PKR 在传递 dsRNA 中的作用 生成的信号将在 PKR 耗尽的细胞中用 2-5A- 进行研究 反义。 同样，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 和胞质 磷脂酶 A2 以确定其调节干扰素的功能 受刺激的基因。 由于其特异性、多功能性和效力， 2-5A-反义是一种有前途的基因表达控制方法 通过靶向 RNA 降解。