CONSTRUCTION AND OPTIMIZATION OF ANTI-HIV RIBOZYMES

迫切需要有效的治疗药物来对抗人类 获得性免疫缺陷病毒1型(HIV-1) 免疫缺陷综合征(艾滋病)。这一项目将导致 选择性攻击和破坏HIV RNA的核酶的研究进展 在受感染的细胞内。核酶是催化的RNA分子；许多 催化特定部位的RNA裂解反应。基因的分子基础 对于某些核酶来说，底物选择性是被理解的。因此，它是 可以通过以下方式合理地改变核酶的底物专一性 在其底物结合部位内使用 重组/合成DNA方法。这项提议的具体目的是 为了(1)开发和鉴定选择性地攻击几个 HIV-1RNA序列，(2)组装和鉴定多价核酶 针对HIV-1RNA，(3)建立一种用于分析和 优化核酶的体内抗病毒活性，(4)使用 优化抗HIV基因的代理基因富集噬菌体系统 核酶，以及(5)评价所产生的单价和 T细胞提取物中的多价抗HIV核酶。用于本研究的核酶 将从自分裂的“发夹”减去烟草链中提取 环斑病毒卫星((-)sTRSV)。在初步工作中，我们已经产生了 一种能选择性攻击HIV-1 Poll序列的发夹核酶。优势 抗HIV核酶有几种：(I)核酶可以潜在地抑制 HIV感染细胞也抑制了HIV基因在感染体内的表达 细胞，(Ii)核酶可能是高度选择性的试剂，(Iii)核酶作用 催化作用，使单个核酶分子有可能 破坏HIV RNA的许多分子，以及(Iv)核酶不敏感 对多效性耐药。该项目将直接针对主要 开发最佳抗HIV核酶面临的挑战-实现 序列选择性、催化活性和 核酶在体内遇到条件下的稳定性。

There is an urgent need for effective therapeutic agents against human immunodeficiency virus 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). This project will result in the development of ribozymes that will selectively attack and destroy HIV RNA within an infected cell. Ribozymes are catalytic RNA molecules; many catalyze site-specific RNA cleavage reactions. The molecular basis for substrate selectivity is understood for some ribozymes. Therefore, it is possible to rationally alter the substrate specificity of a ribozyme by engineering changes within its substrate binding site using recombinant/synthetic DNA methods. The Specific Aims of this proposal are to (1) Develop and characterize ribozymes that selectively attack several HIV-1 RNA sequences, (2) Assemble and evaluate multivalent ribozymes against HIV-1 RNA, (3) Develop a bacteriophage system for analyzing and optimizing antiviral activity of ribozymes in vivo, (4) Use the bacteriophage system for surrogate genetic enrichment of optimized anti-HIV ribozymes, and (5) Evaluate the activity of resulting monovalent and multivalent anti-HIV ribozymes in T cell extracts. Ribozymes for this study will be derived from the self-cleaving "hairpin" minus strand of tobacco ringspot virus satellite ((-)sTRSV). In preliminary work, we have generated a hairpin ribozyme that selectively attacks HIV-1 pol sequences. Advantages of anti-HIV ribozymes are several: (i) Ribozymes can potentially inhibit HIV infection of cells and also inhibit HIV gene expression within infected cells, (ii) Ribozymes may be highly selective agents, (iii) Ribozymes act catalytically, so that a single molecule of ribozyme can potentially destroy many molecules of HIV RNA, and (iv) Ribozymes are not susceptible to pleiotropic drug resistance. This project will directly address major challenges in the development of optimal anti-HIV ribozymes---to achieve the optimal balance of sequence selectivity, catalytic activity and ribozyme stability under conditions encountered in vivo.