Novel Cleavage of a Hammerhead Ribozyme Targeted to β-Amyloid Peptide Precursor mRNA

A trans-acting hammerhead ribozyme (D0) was designed to cleave at the first GUC ↓ X target sequence in Alzheimer amyloid precursor (βAPP) mRNA. A synthetic 26-base substrate analog of βAPP mRNA, labeled with either [α-32P]CTP or [α-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 rihozymes. A minor 12-base product was formed by cleavage one base 5′ to this site. When the βAPP substrate analog was labeled with [α-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 ↓ 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.