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.
| Joyce, G F (2001) RNA cleavage by the 10-23 DNA enzyme. Methods Enzymol 341:503-17 |
| Sheppard, T L; Ordoukhanian, P; Joyce, G F (2000) A DNA enzyme with N-glycosylase activity. Proc Natl Acad Sci U S A 97:7802-7 |
| Santoro, S W; Joyce, G F (1998) Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37:13330-42 |
| Raillard, S A; Joyce, G F (1996) Targeting sites within HIV-1 cDNA with a DNA-cleaving ribozyme. Biochemistry 35:11693-701 |
| Tsang, J; Joyce, G F (1996) Specialization of the DNA-cleaving activity of a group I ribozyme through in vitro evolution. J Mol Biol 262:31-42 |
| Tsang, J; Joyce, G F (1996) In vitro evolution of randomized ribozymes. Methods Enzymol 267:410-26 |
| Breaker, R R; Joyce, G F (1994) Emergence of a replicating species from an in vitro RNA evolution reaction. Proc Natl Acad Sci U S A 91:6093-7 |
| Tsang, J; Joyce, G F (1994) Evolutionary optimization of the catalytic properties of a DNA-cleaving ribozyme. Biochemistry 33:5966-73 |
| Beaudry, A A; Joyce, G F (1992) Directed evolution of an RNA enzyme. Science 257:635-41 |
| Cadwell, R C; Joyce, G F (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28-33 |
