Isolation of hammerhead ribozymes with altered core sequences by in vitro selection

The hammerhead ribozyme has an invariant nucleotide sequence in the core region. In order to search for alternative sequences which can support the cleavage after the triplet GUC, the core region of 10 nucleotides was randomized and subjected to in vitro selection by repeated cycles of transcription, reverse transcription, and PCR. Active sequences were isolated after each transcription by denaturing PAGE, and after nine cycles of selection, two sequences dominated the pool. Both sequences conformed broadly to the consensus core region except that in one sequence a single A(9)U mutation was observed while in the ether two mutations at A(9)U and U(7)A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 mu M-1 min(-1), respectively, as compared to 163 mu M-1 min(-1) for the consensus sequence. Interestingly, the consensus was not found in any of the selected sequences. This discrimination against the consensus sequence was attributed to the specificity of the enzymes used in the selection procedure.