EVOLUTIONARY ENGINEERING OF ANTI-HIV-1 RIBOZYMES

反义寡核苷酸在普通核酶和反义核酶中的应用 尤其提供了一种很有前途的方法来开发 抗HIV-1治疗药物。虽然大部分工作都涉及 反义核酶利用“锤头”核酶，第一组 众所周知，核酶还能以序列特异性的方式切割RNA。 最近，我们报道了一组I核酶可以特异性地切割 单链DNA也是如此。因此，I族核酶提供了一种方法 遗传物质在分子水平上的核内切割 不是RNA就是DNA。 我们已经开发了一个实验室系统，用于控制进化 核糖核酸酶。该系统已被用作一种工具来帮助 具有增强DNA切割活性的新型核酶的开发。我们 建议使用这一系统来开发专门切割的核酶 特定的遗传靶点，在以下条件下优化活性 与蜂窝环境相似。最优化程序 是在体外进行的，使用了10(9)-10(12)个突变体 核酶受到重复的几轮选择性 放大。每轮选择性扩增需要一小时 这样我们就可以调查大量的核酶 在短时间内产生变种。 这项拟议的研究的目标是开发一种反义家族。 使用体外进化技术的核酶。那些拥有 最理想的催化性能将被引入到两个 细菌宿主和真核宿主，并测试其携带 排除体内的RNA或DNA裂解反应。我们特别是 有兴趣开发一种能特异性切割的核酶 双链DNA。

The use of antisense oligonucleotides in general and antisense ribozymes in particular offers a promising approach to the development of anti-HIV-1 therapeutic agents. While most of the work involving antisense ribozymes has made use of "hammerhead" ribozymes, group I ribozymes are also known to cleave RNA in a sequence-specific manner. Recently we reported that a group I ribozyme can specifically cleave singlestranded DNA as well. Thus group I ribozymes offer an approach to the endonucleolytic cleavage of the genetic material at the level of either RNA or DNA. We have developed a laboratory system for the controlled evolution of RNA enzymes. The system has been used as a tool to aid in the development of novel ribozymes with enhanced DNA cleavage activity. We propose to use this system to develop ribozymes that specifically cleave specific genetic targets, optimizing for activity under condidons that resemble those of the cellular environment. The optimization procedure is carried out in vitro, using a population of 10(9) - 10(12) mutant ribozymes that are subjectcd to repeated rounds of selective amplification. Each round of selective amplification requires one hour to perform, so that we can survey a very large number of ribozyme variants in a short period of time. The goal of the proposed research is to develop a family of antisense ribozymes using in vitro evolution techniques. Those individuals with the most desirable catalytic properties will be introduced into both a bacterial host and a eukaryotic host and tested for the ability to carry out the RNA or DNA cleavage reaction in vivo. We are especially interested in developing a ribozyme that specifically cleaves double-stranded DNA.