Differential Activity oftrans-Acting Hammerhead Ribozymes Targeted to β Amyloid Peptide Precursor mRNA by Altering the Symmetry of Helices I and III

In order to determine whether distributing the pairing bases in helices I and III of a hammerhead ribozyme asymmetrically would enhance the cleavage oftrans-acting ribozymes designed to degrade cellular mRNAs, we measured the cleavage properties of symmetric and asymmetric ribozymes targeted to the amyloid peptide precursor (βAPP) mRNA. Five ribozymes were formed from three βAPP synthetic mRNA analogs and two ribozyme RNA core sequences. Symmetric ribozymes β133/Rz133 and β125/Rz133 contained 8 bp in helix I and 7 bp in helix III. Asymmetric ribozyme β125/Rz125 had 13 bp in helix I and 4 bp in helix III, asymmetric ribozyme β123/Rz125 had 11 bp in helix I and 4 bp in helix III, while asymmetric ribozyme β133/Rz125 contained 8 bp in helix I and 4 bp in helix III. The ability of each ribozyme to cleave its substrate RNA was first assessed under single-turnover conditions at 37°C. These studies revealed that only symmetric ribozyme, β133/Rz133, and asymmetric ribozyme β123/Rz125 effectively cleaved their substrates. Further studies using a 80°C, 1-min → 37°C, 1-min temperature cycling paradigm were performed to increase the cleavage efficiency of the ribozymes. Under these conditions ribozymes β133/Rz133, β125/Rz125, and β123/Rz125 were kinetically well behaved. Therefore, the fact that the symmetric ribozyme β133/Rz133 was more active than its asymmetric counterparts indicates that symmetrically distributing the pairing bases in helices I and III around this cleavage site is preferred.