Novel cleavage of a hammerhead ribozyme targeted to beta-amyloid peptide precursor mRNA

A trans-acting hammerhead ribozyme (D0) was designed to cleave at the first GUC decreases X target sequence in Alzheimer amyloid precursor (beta APP) mRNA. A synthetic 26-base substrate analog of beta APP mRNA, labeled with either [alpha-32P]CTP or [alpha-35S]CTP underwent Mg(+2)-dependent, site-specific cleavage in vitro. Cleavage was found to occur at two sites. The major product of the reaction, a 13-base oligonucleotide, resulted from cleavage at the consensus cut site of hammerhead ribozymes. A minor 12-base product was formed by cleavage one base 5' to this site. When the beta APP substrate analog was labeled with [alpha-35S]ATP (Rp isomer) another cleavage site, amounting to 25-30% of the total product formed, was also activated. A series of ribozyme deletion mutants delimited the new cleavage site to the phosphate linkage 5' to the G residue of the hammerhead consensus cut site, GUC decreases X. We conclude that small changes in RNA conformation can dramatically alter ribozyme specificity in vitro. This may have implications for targeting hammerhead ribozymes in vivo.