MECHANISM OF SYNTHETIC RIBOZYME ACTION

合成核酶的作用机制

• 批准号: 3304452
• 负责人: PETER T GILHAM
• 金额: $ 13.06万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-09 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304452
• 关键词: AIDS; RNA; X ray crystallography; enzyme mechanism; enzyme structure; enzymes; human immunodeficiency virus 1; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; ribozymes; thermodynamics

The discovery of self-cleaving structures in plant RNA pathogens has opened up a novel approach to the development of new therapeutic agents for the control of diseases caused by the HIV virus and by other retroviral infections. These ribozyme structures, which are classified as possessing either a "hammerhead" or a "hairpin" motif, have the capacity to recognize specific sequences within a target RNA strand and then cleave a particular phosphodiester linkage within each sequence. The rational design of efficient anti-HIV agents that exploit these properties would be greatly facilitated by a detailed understanding of the structures and mechanisms used by these two ribozyme motifs. To this end, a few small ribozyme domains of each motif will be chemically synthesized in large quantities and in states of high purity. The domains will consist of duplexes formed from pairs of oligoribonucleotides in which one member corresponds to the catalytic site and the other to the substrate site. For the latter, chemical modifications will be introduced near the cleavable phosphodiester linkage in each case, in order to prevent self-destructing activity during analysis. The modifications are designed to prevent cleavage while avoiding large conformational changes in the structure that is responsible for cleavage. Modifications will include substitutions for the 2'-hydroxyl group involved in the cleavage reaction, isomerization of the cleavable phosphodiester from the 3'-5' to the 2'-5' configuration, and substitution of a methylene group for the oxygen located between the phosphorus atom of the 3'-5' diester and the 5' carbon of the adjacent nucleotide. Techniques to be used in determining the structures of these synthetic ribozymes will include thermodynamic measurements, X-ray diffraction of duplex crystals, and NMR analysis of duplex solutions.

植物RNA病原体中自切割结构的发现开启了 为开发新的治疗药物提供了一种新的方法， 艾滋病毒和其他逆转录病毒引起的疾病 感染.这些核酶结构，被归类为具有 无论是“锤头”还是“发夹”图案， 靶RNA链内的特定序列，然后切割特定的 每个序列内的磷酸二酯键。的合理设计 利用这些特性的有效抗艾滋病毒药物将大大 通过对结构和机制的详细了解， 这两个核酶基序所使用的。为此，几个小核酶 每个基序的结构域将被大量化学合成 并且处于高纯度状态。结构域将由形成的双链体组成 来自寡核糖核苷酸对，其中一个成员对应于 催化位点，另一个到底物位点。对于后者， 在可裂解的磷酸二酯附近引入化学修饰 在每一种情况下，为了防止自毁活动， 分析.这些修饰旨在防止切割，同时避免 结构中的巨大构象变化， 乳沟修饰将包括2 '-羟基取代。 基团参与裂解反应，可裂解的异构化 磷酸二酯从3 '-5'到2 '-5'构型，和取代 的亚甲基基团的氧位于磷原子之间的 3 ′-5 ′二酯和相邻核苷酸的5 ′碳。技术 用于确定这些合成核酶的结构， 包括热力学测量、双相晶体X射线衍射 和双链体溶液的NMR分析。