Attempts to obtain more efficient GAC-cleaving hammerhead ribozymes by in vitro selection

An in vitro selection was carried out to identify hammerhead ribozymes cleaving 3' to GAC triplets more efficiently than the wild type ribozyme. A double-stranded DNA containing the sequence for the hammerhead ribozyme with 10 randomizations in the catalytic core, designed for in cis cleavage, was transcribed and the cleavage product amplified by reverse transcription and PCR. After seven selection cycles, the DNA was cloned and 50 colonies sequenced. One sequence, appearing six times, was active for in cis cleavage of GAC. It was identical to the consensus sequence except for a mutation at position 7. Another cleaved GUC and two more, cleaved GUA. The cleavage rates of these ribozymes for in trans cleavage were slower than the rate of the consensus ribozyme. Interestingly, the consensus sequence was not found in the selection. This strongly suggests that the consensus hammerhead ribozyme has evolved to an optimal sequence.