The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes

Abstract

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 determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.

Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

“…The rate for the AUCt motif, which was cleaved best (kcleav = 2.4 x 10-3/min = 4 x 10-5/s), was about four times less than that described for another asymmetric hammerhead ribozyme (18). This reflects the different sequence context which was found to have a great influence on the detectable cleavability, even with the same cleavable motif (17). Analysis of the ribozyme/substrate sequences with the MFOLD programme (19) revealed that helices III and II are quite stable, but alternatives to the base pairing in helix I can occur, which may contribute to an overall reduced cleavage rate.…”

“…As described previously (14,17,18,21), under these conditions the ribozyme reaction follows pseudo-first order kinetics which allowed to determine the detectable cleavage rate constants that are summarized in Figure 3. It was found that the cleavage rates were highest if the target nucleotide 17 was C, which resulted in -2-3-fold stronger detectable cleavage compared to A in position 17. An U residue in position 17 resulted in a further reduction of the cleavage rates.…”

“…In general, the cleavage rates are reduced -100-500-fold compared with hammerhead ribozymes that can catalyze multiple cleavage (4), possibly because the cleavage reaction requires an additional conformational change (21). It has also been observed, that the sequence context ofthe target RNA influences the cleavage rate (17). Regardless of the stoichiometric cleavage at relatively low rates, it was shown that the catalytic activity enhanced the antisense effect (14,18).…”

“…Complex formation was analyzed on a non-denaturating polyacrylamide gel as described by Zoumadakis et al (17).…”

“…The GUCl motif found in nature represents nucleotides 16.2, 16.1 and the unpaired nucleotide 17 according to the numbering system for hammerhead ribozymes by Hertel et al (8). This motif has been converted to AUC4, CUC4, UUC4, GUA4 or GUUt each by Koizumi et al and Ruffner et al (9,10), without loss of activity, even though the cleavage efficiencies varied among the motifs and were generally lower compared with the GUC4 motif.…”