Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity.
The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.
Hammann
C
C
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion Crete, Greece.
Martinez
E
E
Moosbauer
J
J
Hormes
R
R
Tabler
M
M
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D016372:RNA, Antisense / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D016372:RNA, Antisense; D016337:RNA, Catalytic