A Helix 1-Extended Hairpin Ribozyme Exhibits Altered Cleavage BehaviorIn Vitro

Abstract

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 immunodeficiency virus type 1 (HIV-1), generating a hairpin ribozyme with a largely extended helix 1. In parallel, a catalytic antisense RNA based on a hammerhead ribozyme was directed toward the same cleavage motif in the HIV-1 target. Both ribozymes were expected to create identical cleavage products. Cleavage analysis in vitro confirmed that the hammerhead ribozyme delivered the expected cleavage products. However, the helix 1-extended hairpin ribozyme catalyzed additional RNA cleavage at several unexpected sites, which were mapped. Some of the 3' cleavage products had other nucleotides than G at their 5'-terminus, indicating that the helix 1-extended hairpin ribozyme was able to cleave bonds other than NpG+1. Inspection of the sequence context of the different cleavage sites suggested that unconventional helices 2 in combination with an asymmetric loop A consisting of up to 32 unpaired nucleotides in the substrate strand were formed. A second variant of a helix 1-extended hairpin ribozyme that differed in two nucleotides gave consistent results.