In vitro selection of a purine nucleotide-specific hammerheadlike ribozyme.

Abstract

The in vitro selection for an intramolecular AUG-cleaving hammerhead-like ribozyme is described. One of the ribozymes selected was found to cleave after this triplet, both intramolecularly and intermolecularly, with rates comparable to the rate of the native GUC-cleaving hammerhead ribozyme. Although the selection was designed for cleavage 3' of the AUG triplet, the ribozyme also cleaves 3' of the AUA triplet. AUU and AUC triplets are, however, not cleaved, and thus the selected ribozyme is purine-specific for the third position in the triplet. In addition, cleavage 3' of the AAG triplet has been observed, thus the central U is not essential. Nuclease digestion indicates that the selected ribozyme has a secondary structure similar to that of the native hammerhead ribozyme, although with an altered core and stem-loop II sequence. All nucleotides in the core, except one, are essential for activity. The nucleotides in loop II are sensitive to changes and cannot, as in the hammerhead ribozyme, be replaced by other sequences or a nonnucleotide linker. Thus there are differences between these two ribozymes even though they have similar two-dimensional structures. The new ribozyme enlarges the application of hammerhead ribozymes for the inhibition of gene expression by extending the range of cleavable triplets.