EVOLUTIONARY ENGINEERING OF ANTI-HIV-1 RIBOZYMES

反义寡核苷酸的一般用途和反义核酶 特别是提供了一个有希望的方法， 抗HIV-1治疗剂。 虽然大多数工作涉及 反义核酶利用了“锤头”核酶，I组 还已知核酶以序列特异性方式切割RNA。 最近我们报道了I组核酶可以特异性切割 单链DNA也一样。 因此，I组核酶提供了一种方法， 遗传物质的核酸内切酶切， RNA或DNA。 我们已经开发了一个实验室系统， RNA酶。 该系统已被用作一种工具， 开发具有增强的DNA切割活性的新型核酶。 我们 我建议使用这个系统来开发特异性切割的核酶 特异性遗传靶点，在 类似于细胞环境中的那些。 优化过程 使用10（9）-10（12）个突变体的群体在体外进行 核酶经过重复的选择性循环， 放大 每轮选择性扩增需要一小时 这样我们就可以测量大量的核酶 在短时间内的变化。 这项研究的目的是开发一个反义寡核苷酸家族， 使用体外进化技术的核酶。 这些人与 最理想的催化性能将被引入到 细菌宿主和真核宿主，并测试携带 体内RNA或DNA裂解反应。 我们特别 对开发一种能特异性切割 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.