MODIFICATION OF PRIMARY STRUCTURES OF HAIRPIN RIBOZYMES FOR PROBING ACTIVE CONFORMATIONS

Hairpin ribozymes consist of two stem-loop domains, and these domains are assumed to interact with each other to produce the self-cleavage a ctivity We have studied the relationship of the tertiary structure of the hairpin ribozyme and the cleavage activity by dividing and re-join ing the domains. A hairpin ribozyme (E50) was divided at the hinge reg ion, and the main part was joined to a substrate (S1) using tri-or pen ta-cytidylates. These ribozymes retained the cleavage activity in the presence of the rest of the molecule, indicating that the active confo rmation could be maintained if the two domains interacted with each ot her. Based on the these results, we designed a new type of hairpin rib ozyme by replacing one of the domains. To maintain the interaction of the domains, oligocytidylates were inserted at a junction. These rever sely joined ribozyme complexes showed cleavage activity that was depen dent on the linker lengths. These modifications in the primary structu re of the hairpin ribozyme confirm the structural requirement for the catalytic reaction and provide information for the correlation of the tertiary structure with the cleavage of the hairpin ribozyme. (C) 1995 Academic Press Limited