PROBING ACCESSIBLE SITES FOR RIBOZYMES ON HUMAN ACETYLCHOLINESTERASE RNA

In order to design ribozymes for the efficient cleavage of a human ace tylcholinesterase (AChE) in vitro transcript, a completely randomized decadeoxyribonucleotide (dN(10)) was used in conjunction with RNase H to identify suitable sites for annealing. Based on the observed cleava ge pattern, ribozymes were designed to cleave the transcript at these positions. Five ribozymes so designed proved to be efficient in the tr anscript cleavage (k(react)/K-m ranged from 0.9 x 10(4) to 68.2 x 10(4 ) M(-1) min(-1)). The best was 150-fold more active than the best desi gned on the basis of the MFold program. Thus, the RNase H mapping demo nstrated a high predictive power for favorable ribozyme cleavage sites . The digestion pattern with RNase H differed dramatically from that o bserved with the single-strand-specific mung bean nuclease.