The hammerhead ribozyme is one of the smallest ribozymes known and catalyses the site-specific hydrolysis of a phosphodiester bond. This small ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression. The first part of the review summarizes the sequence requirements of the hammerhead, its three-dimensional structure and the proposed mechanism, in addition to ribozyme specificity and turnover. The second part of the review focuses on the in vivo application of the ribozyme. The processes involved in designing ribozymes for efficient cleavage in vivo are described, together with possible delivery strategies.

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 A9U mutation was observed while in the other two mutations at A9U and U7A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 microM(-1) min(-1), respectively, as compared to 163 microM(-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.

The diastereomers of adenosine and uridine 2',3'-cyclic phosphorothioates were tested as substrates for 2',3'-cyclic nucleotide 3'-phosphodiesterase from bovine brain. The enzyme cleaves the Sp (or exo) diastereomers efficiently, whereas the Rp (or endo) diastereomers are resistant to hydrolysis, even after long incubation. As the enzyme exhibits strong substrate inhibition the precise determination of kinetic parameters posed problems, particularly with phosphorothioates. The stereoselectivity of this enzyme is opposite to that of RNase T1 and RNase A and thus could be a useful complement in determination of the configuration of nucleoside 2',3'-cyclic phosphorothioates resulting from hydrolysis reactions of unknown stereochemical course.

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.The hammerhead ribozyme is one of the smallest ribozymes known and has thus attracted much attention for the study of structure-function relationships in catalytic RNAs as well as for its potential for the sequence-specific inhibition of gene expression (1-3). The hammerhead cleaves RNA sequencespecifically adjacent to the general triplet sequence NUH, where N is any nucleotide and H can be A, U, or C. Cleavage 3Ј to a guanosine such as in GUG is very slow (4.3 ϫ 10 Ϫ5 min Ϫ1

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