Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance

The hairpin ribozyme derived from the minus strand of the satellite RNA ass ociated with the tobacco ringspot virus is one of the small catalytic RNAs that has been shown to catalyze trans-cleavage reactions. There is much int erest in designing hairpin ribozymes with improved catalytic activity for t he development of new therapeutic agents. Extensive mutagenesis studies as well as in vitro selection experiments have been performed to define the st ructure and optimize its catalytic activity. This communication describes a comparative kinetic analysis of four structural variants, introduced, eith er alone, or in combination, into the hairpin ribozyme, We have shown that extension of the helix 2 from 4 to 6 bp resulted in a significant decrease in K-M, Furthermore, the combination of this extension with the simultaneou s stabilization of helix 4, led to a more than two-fold increase in the cat alytic efficiency. This variant showed a 15-fold reduction in the K-M value in respect to the wildtype ribozyme. This could be of great interest for t he in vivo application of this catalytic moth, The 9-bp enlargement of heli x 4 implied about a three-fold improvement in the catalytic activity, Simil arly, the U39C substitution brought up the efficiency of the ribozyme sligh tly. However, introduction of nucleotides at the hinge region between A and B domains reduced the catalytic activity. This reduction was gradually inc reased with the number of nucleotides. Results obtained with variants carry ing more than one modification always agreed with the ones obtained from ea ch single variant.