TARGETING SITES WITHIN HIV-1 CDNA WITH A DNA-CLEAVING RIBOZYME

A variant Of the Tetrahymena ribozyme that efficiently cleaves single- stranded DNA under simulated physiological conditions [Tsang, J., & Jo yce, G.F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potent ial therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites wit hin HIV-1 cDNA. In order to increase the sequence selectivity of the r ibozyme, its substrate recognition domain was extended from 6 to 12 nu cleotides, allowing base pairing with substrate nucleotides that lie b oth upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a va riety of different DNA targets. The ribozyme exhibited a high degree o f sequence specificity, discriminating by a factor of 10(2) to more th an 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the c leavage site led to a greater decrease in activity compared to those t hat occurred farther away.