A HELIX 1-EXTENDED HAIRPIN RIBOZYME EXHIBITS ALTERED CLEAVAGE BEHAVIOR IN-VITRO

The catalytic domain of a hairpin ribozyme was incorporated at the 3'- end of a 254-base antisense RNA directed against the RNA of human immu nodeficiency virus type 1 (HIV-1), generating a hairpin ribozyme with a largely extended helix 1. In parallel, a catalytic antisense RNA bas ed on a hammerhead ribozyme was directed toward the same cleavage moti f in the HIV-1 target. Both ribozymes were expected to create identica l cleavage products, Cleavage analysis in vitro confirmed that the ham merhead ribozyme delivered the expected cleavage products. However, th e helix 1-extended hairpin ribozyme catalyzed additional RNA cleavage at several unexpected sites, which were mapped. Some of the 3' cleavag e products had other nucleotides than G at their 5'-terminus, indicati ng that the helix 1-extended hairpin ribozyme was able to cleave bonds other than NpG(+1). Inspection of the sequence context of the differe nt cleavage sites suggested that unconventional helices 2 in combinati on with an asymmetric loop A consisting of up to 32 unpaired nucleotid es in the substrate strand were formed, A second variant of a helix 1- extended hairpin ribozyme that differed in two nucleotides gave consis tent results.