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., & Joyce, G. F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potential therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites within HIV-I cDNA. In order to increase the sequence selectivity of the ribozyme, its substrate recognition domain was extended from 6 to 12 nucleotides, allowing base pairing with substrate nucleotides that lie both upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a variety of different DNA targets. The ribozyme exhibited a high degree of sequence specificity, discriminating by a factor of 10(2) to more than 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the cleavage site led to a greater decrease in activity compared to those that occurred farther away.
Raillard
S A
SA
Department of Chemistry, Scripps Research Institute, La Jolla, California 92037, USA.
Joyce
G F
GF
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D019020:Directed Molecular Evolution; D015497:HIV-1 / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism* / Q000627:therapeutic use; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic