MECHANISM OF SYNTHETIC RIBOZYME ACTION

植物核糖核酸病原体中自裂解结构的发现开启了 建立一种新的方法来开发新的治疗药物 控制由艾滋病毒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拥有 无论是“锤头”图案还是“发夹”图案，都有能力识别 靶RNA链内的特定序列，然后切割特定的 每个序列中的磷酸二酯键。建筑结构的合理设计 利用这些特性的高效抗艾滋病毒药物将极大地 通过对结构和机制的详细了解来促进 被这两个核酶基序所使用。为此，有几个小核酶 每个基序的结构域将被大量化学合成 并且处于高纯度的状态。属性域将由形成的双链组成 从寡核苷酸对，其中一个成员对应于 催化部位和底物部位各有一处。对于后者， 在可裂解的磷酸二酯附近引入化学修饰 在每种情况下，为了防止自毁活动 分析。这些修改旨在防止卵裂，同时避免 负责结构的大的构象变化 乳沟。修饰将包括2‘-羟基的取代 参与裂解反应的基团，可裂解的异构化 从3‘-5’构型到2‘-5’构型的磷二酯，以及取代 位于磷原子之间的氧的亚甲基的 3‘-5’双酯和相邻核苷酸的5‘碳。技术 将用于确定这些合成核酶的结构 包括热力学测量、双晶X射线衍射、 和双链溶液的核磁共振分析。

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.