THE INFLUENCE OF IMPERFECTLY PAIRED HELIX-I AND HELIX-II ON THE CATALYTIC ACTIVITY OF HAMMERHEAD RIBOZYMES

Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determ ine the rates of the cleavage process for the different ribozymes unde r single-turnover conditions. It was found that the sequence context s urrounding the cleavable motif influenced the cleavage efficiencies. D eletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. (1) de stroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not dest ruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions wi thin helices I and III resulted in alternative cleavage sites. The pot ential consequences for the specificity of the ribozyme reaction are d iscussed.