TARGETED DEGRADATION OF MRNAS FOR SIGNALING FACTORS

针对信号因子的 MRNAS 定向降解

• 批准号: 6269633
• 负责人: ROBERT H SILVERMAN
• 金额: $ 23.08万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6269633
• 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