RNASE P RIBOZYMES FOR ANTIVIRAL APPLICATIONS

描述：这项建议的目标是研究催化 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 由来自大肠杆菌的核糖核酸酶P的催化RNA亚单位改造而成。这是一项新的 核酶切割包括病毒mRNAs在内的RNA底物，使该碱基对 它的引导序列。为了提高卵裂效率， 申请者将进行实验以阐明催化的机理。 和底物识别。他还将测试病毒复制是否可以 通过靶向重要的病毒基因ICP4而被废除。核酶 靶基因的识别将通过体外动力学和 物理映射分析。高效的核酶将由以下任一种方式产生 定点突变或体外选择。对以下各项的要求 将定义组织培养中的有效核酶活性，并 确定了序列特异性。最后，基因工程核酶是否 可以通过取消ICP4的表达来抑制HSV-1病毒的复制 调查过了。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和基因打靶工具的应用 临床应用。

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.