EVOLUTIONARY ENGINEERING OF ANTI-HIV-1 RIBOZYMES

一般使用反义寡核苷酸和反义核酶

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.