AUA-CLEAVING HAMMERHEAD RIBOZYMES - ATTEMPTED SELECTION FOR IMPROVED CLEAVAGE

In order to find a ribozyme which can cleave the AUA triplet efficiently, the specificities and rates of intermolecular cleavage by the ribozyme of the satellite RNA of the barley yellow dwarf virus have been determined. Although it cleaves the AUA triplet in the plus strand of the viroid RNA, cleavage of AUC and AUU is more efficient, and AUG is essentially not cleaved. Attempts were made to increase cleavage efficiency by in vitro selection with randomization at positions 7, 10.1, and 11.1 in the core region. Fifteen clones were analyzed, two of which showed increased AUA cleavage efficiency. They have a G(10.1).C-11.1 base pair and a pyrimidine at position 7. This corresponds to the sequence of the consensus hammerhead ribozyme. Attempts to further increase cleavage efficiency by in vitro selection of the consensus hammerhead ribozyme with randomization of the 10 nucleotides in the core region or of the sBYDV ribozyme with 12 core nucleotides randomized were not successful.