Self-association of adenine-dependent hairpin ribozymes

Hairpin ribozymes are flexible molecules that catalyse reversible self-cleavage after the docking of two independently folded internal loops, A and B. The activities, self-association and structures in solution of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) were studied by native gel electrophoresis, analytical centrifugation, and small angle neutron scattering. Bi-molecular RNA interactions such as linear–linear, loop–loop, loop–linear or kissing interactions have been found to be important in the control of various biological functions, and hairpin loops present rich potential for establishing both intra- and intermolecular interactions through standard Watson-Crick base pairing or non-canonical interactions. Similar results were obtained for ADHR1 and ADHR2. At room temperature, they indicated end-to-end self-association of the ribozymes in rod-like structures with a cross-section corresponding to two double strands side-by-side. Dimers, which predominate at low concentration (∼0.1 mg/ml), associate into longer rods, with increasing concentration (∼1 mg/ml). Above 65°C, the dimers and rods dissociated into compact monomers, with a radius of gyration similar to that of tRNA (about 70 bases). The dimers were non-active for catalysis, which suggests that dimer formation, probably by preventing the correct docking of loops A and B, could act as an inhibition mechanism for the regulation of hairpin ribozyme catalysis.