Enhanced folding of hairpin ribozymes with replaced domains

Reversely joined ribozymes (Komatsu et al., 1995) have been proven to be active. Here we describe the construction of hairpin ribozymes with separated domains, but containing complementary arms for association of the two domains. Linker nucleotides were inserted between the arms and domains. These ribozymes were active under the standard conditions (12 mM MgCl2), depending on the length of the linker. When the-complementary arms were covalently joined through a stable loop, these ribozymes showed cleavage activities. However, the K-m value of the stem-loop ribozymes was found to be larger than that of the parent ribozyme, which can adopt both linear and bent conformations. Kinetic analyses of these modified hairpin ribozymes suggest a higher turnover of the hairpin ribozyme as compared to other small ribozymes. The present ribozymes provide insight into the nature of the domain interaction and are suitable for physicochemical studies on the tertiary structure of the hairpin ribozyme.