DIFFERENTIAL ACTIVITY OF TRANS-ACTING HAMMERHEAD RIBOZYMES TARGETED TO BETA-AMYLOID PEPTIDE PRECURSOR MESSENGER-RNA BY ALTERING THE SYMMETRY OF HELIX-I AND HELIX-III

In order to determine whether distributing the pairing bases in helices I and III of a hammerhead ribozyme asymmetrically would enhance the cleavage of transacting ribozymes designed to degrade cellular mRNAs, we measured the cleavage properties of symmetric and asymmetric ribozymes targeted to the amyloid peptide precursor (beta APP) mRNA. Five ribozymes were formed from three beta APP synthetic mRNA analogs and two ribozyme RNA core sequences. Symmetric ribozymes beta 133/Rz133 and beta 125/Rz133 contained 8 bp in helix I and 7 bp in helix III. Asymmetric ribozyme beta 125/Rz125 had 13 bp in helix I and 4 bp in helix III, asymmetric ribozyme beta 123/Rz125 had 11 bp in helix I and 4 bp in helix III, while asymmetric ribozyme beta 133/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 degrees C. These studies revealed that only symmetric ribozyme, beta 133/Rz133, and asymmetric ribozyme beta 123/Rz125 effectively cleaved their substrates. Further studies using a 80 degrees C, 1-min --> 37 degrees C, 1-min temperature cycling paradigm were performed to increase the cleavage efficiency of the ribozymes. Under these conditions ribozymes beta 133/Rz133, beta 125/Rz125, and beta 123/Rz125 were Identically well behaved. Therefore, the fact that the symmetric ribozyme beta 133/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. (C) 1995 Academic Press, Inc