HAIRPIN RIBOZYME-MEDIATED CLEAVAGE OF THE FULL-LENGTH BETA-GLUCURONIDASE (GUS) MESSENGER-RNA

We designed three hairpin ribozymes to cleave Escherichia coil beta-gl ucuronidase (GUS) mRNA and tested those activities ill vitro, One of t he ribozymes was designed to form 9 base pairs in total with the targe t GUS mRNA, and the other two ribozymes had longer substrate binding s ites. All ribozymes cleaved the model substrate (100 bases long) at th e predicted target site, Two ribozymes containing longer substrate bin ding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site, Also, the ribozyme s with long substrate binding sites had high activity against the full -length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant ce lls. Effects of the substrate binding site length of the ribozyme on c leavage activity are discussed.