RNASE P RIBOZYMES FOR ANTIVIRAL APPLICATIONS

用于抗病毒应用的核糖核酸酶核酶

• 批准号: 6017097
• 负责人: Fenyong Liu
• 金额: $ 17.49万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6017097
• 关键词: RNase protection assay; antiviral agents; chemical cleavage; chemical kinetics; crosslink; enzyme mechanism; enzyme substrate; gene expression; gene targeting; genetic transcription; genetic transduction; herpes simplex virus 1; messenger RNA; nucleic acid sequence; nucleic acid structure; pancreatic ribonuclease; ribozymes; site directed mutagenesis; synthetic nucleotide; tissue /cell culture; virus RNA; virus genetics; virus replication

DESCRIPTION: The objective of this proposal is to study the catalytic mechanisms of ribonuclease P ribozymes and their applications for inhibition of viral gene expression. The applicant has constructed a ribozyme engineered from the catalytic RNA subunit of RNase P from E. coli. This new ribozyme cleaves RNA substrates, including viral mRNAs, that base pair to its guide sequence. It order to increase the cleavage efficiency, the applicant will conduct experiments to elucidate the mechanism of catalysis and substrate recognition. He will also test whether viral replication can be abolished by targeting an essential viral gene, ICP4. Ribozyme recognition of target mRNA will be studied by both in vitro kinetic and physical mapping analyses. Efficient ribozymes will be generated by either site-directed mutagenesis or in vitro selection. The requirements for efficient ribozyme activity in tissue culture will be defined and the sequence specificity determined. Finally, whether the engineered ribozyme can inhibit HSV-1 viral replication by abolishing ICP4 expression will be investigated. The long term goal is to develop a system to study RNA catalysis and its use as a gene-targeting tool in basic research and clinical applications.

描述：本提案的目的是研究催化 核糖核酸酶P核酶的作用机制及其在抑制中的应用 病毒基因的表达。 申请人构建了一种核酶， 由E.杆菌 这个新 核酶切割RNA底物，包括病毒mRNA，其碱基配对以 其指导序列。 为了提高裂解效率， 申请人将进行实验以阐明催化机理 和底物识别。 他还将测试病毒复制是否能 通过靶向一个重要的病毒基因ICP 4来消除。 核酶 靶mRNA的识别将通过体外动力学和 物理映射分析。 高效的核酶将通过以下方式产生： 定点诱变或体外选择。 的要求 有效的核酶在组织培养中的活性将被定义， 确定序列特异性。 最后，是否工程核酶 可以通过消除ICP 4表达来抑制HSV-1病毒复制， 研究了 长期目标是开发一个研究RNA的系统 催化及其作为基础研究中的基因靶向工具的用途， 临床应用。