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.