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 fo rm 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 t ested for in vitro cleavage and for inhibition of HIV-1 replication in huma n cells, Truncation of helix III to 8 bp did not affect the in vitro cleava ge potential of the parental catalytic antisense RNA 2as-Rz12. Further trun cation of helix III led to decreased cleavage rates, with no measurable cle avage 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 s low rates or not at all. Gel purification of preformed ribozyme-substrate c omplexes led to a significant increase in cleavage rates. However, the comp lex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the compar able antisense RNA on HIV-1 replication.