ANTIVIRAL HAIRPIN RIBOZYMES

这个项目的目标是开发一类新的有效的 基于(-)S TRSV发夹状核酶的抗病毒药物。核酶是 催化RNA分子；许多催化特定部位的RNA裂解 反应。我们将确定底物识别的机制和 切割发夹状核酶，并合理利用这一信息 通过工程序列改变改变核酶的特异性 在其底物结合域内。这项建议的具体目的 用于(1)确定发夹的底物选择规则 核酶，(2)操纵核酶的序列特异性以 产生针对多种病毒RNA序列的核酶，(3) 多变型核酶的构建及活性鉴定(4) 用于分析和优化抗病毒药物的噬菌体系统的开发 体内核酶的活性，以及(5)利用噬菌体系统 优化核酶对抗外源序列的活性。核酶 因为这项研究是基于卫星的自裂解负链 烟草环斑病毒((-)sTRSV)。在前期工作中，我们有 使核酶的底物专一性发生了重大变化， 从而选择性地攻击HIV-1轮询序列。潜力 抗病毒核酶有几个优点：(I)核酶可以 潜在地抑制病毒感染和病毒的表达 已经感染的细胞中的基因，(Ii)核酶可能高度 选择性试剂，(Iii)核酶催化作用，从而使单个核酶 核酶分子可能潜在地破坏病毒的许多分子 RNA，(Iv)核酶对多效性耐药不敏感， 以及(V)核酶技术应得到广泛应用，以便 核酶可能被迅速开发来对抗新的感染 新出现的或新发现的病毒。此外，这项工作将 提供大量关于结构的有价值的信息 和核酶的功能。

The goal of this project is to develop a novel class of effective antiviral agents based on the (-)s TRSV hairpin ribozyme. Ribozymes are catalytic RNA molecules; many catalyze site-specific RNA cleavage reactions. We will determine the mechanism for substrate recognition and cleavage of the hairpin ribozyme, and use this information to rationally alter the specificity of the ribozyme by engineering sequence changes within its substrate-binding domain. The Specific Aims of this proposal are to (1) Determine the substrate selection rules for the hairpin ribozyme, (2) Manipulate the sequence specificity of the ribozyme to generate ribozymes specific for a variety of viral RNA sequences, (3) Construct and evaluate the activity of multivariant ribozymes, (4) Develop a bacteriophage system for analyzing and optimizing antiviral activity of ribozymes in vivo, and (5) Use the bacteriophage system to optimize the activity of ribozymes against foreign sequences. Ribozymes for this study are based on the self-cleaving minus strand of satellite tobacco ringspot virus ((-)sTRSV). In preliminary work, we have engineered a major change in the substrate specificity of the ribozyme, so that it selectively attacks HIV-1 pol sequences. Potential advantages of antiviral ribozymes are several: (i) Ribozymes can potentially inhibit both viral infection and the expression of viral genes in cells that are already infected, (ii) Ribozymes may be highly selective agents, (iii) Ribozymes act catalytically, so that a single molecule of ribozyme may potentially destroy many molecules of viral RNA, (iv) Ribozymes are not susceptible to pleiotropic drug resistance, and (v) ribozyme technology should be widely applicable, so that ribozymes may be rapidly developed to combat infections by newly emerging or newly recognized viruses. In addition, this work will produce a large amount of valuable information concerning the structure and function of ribozymes.