In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues.

The conventional hammerhead ribozyme cleaves RNA 3' to nucleotide triplets with the general formula NUH, where N is any nucleotide, U is uridine and H is any nucleotide except guanosine. In order to isolate hammerhead ribozyme sequences capable of cleaving 3' to the GUG triplet, we performed a mutagenic selection protocol starting with the conventional sequence of an NUH-cleaving ribozyme. The 22 nucleotides in the core and the stem-loop II region were subjected to mutagenic PCR using the two nucleotide analogues 6-(2-deoxy-beta-d-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C)][1, 2] oxazin-7-one and of 8-oxo-2'-deoxyguanosine. After five repetitions of the selection cycle, several clones showed cleavage activity. One sequence, having one deletion, showed at least a 90 times higher in trans cleavage rate than the starting ribozyme. It cleaved 3' to GUG and GUA. The sequence of this ribozyme is essentially identical with that obtained previously by selection for AUG cleavage starting with a randomised core and stem-loop II region. This identical result of two independent selection procedures supports the notion that sequences for NUR cleavage, where R is a purine nucleotide, are not compatible with the classical hammerhead structure, and that the sequence space for this cleavage specificity is very limited. The cleavage of NUR triplets is not restricted to the sequence of the substrate that was used for selection but is sequence-independent for in trans cleavage, although the sequence context influences the value for the cleavage rate somewhat. Analysis of cleavage activities indicates the importance of A at position L2.5 in loop II.

Expanding the structural and functional diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids.

Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH(2)) as a uridine substitute was a more effective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine triphosphate (USH). However, both functioned in transcription assays of 100 nt templates to generate RNA transcripts in quantities sufficient to initiate RNA selection procedures. Transcription of RNA pools with T7 RNA polymerase and UNH(2) or USH occurred with efficiencies of 43 and 29%, respectively, of the values obtained for native UTP transcription. In addition, the transcribed RNA containing roughly 25% UNH(2) residues exhibited better substrate properties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing approximately 25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.

Recent developments in the hammerhead ribozyme field.

Developments in the hammerhead ribozyme field during the last two years are reviewed here. New results on the specificity of this ribozyme, the mechanism of its action and on the question of metal ion involvement in the cleavage reaction are discussed. To demonstrate the potential of ribozyme technology examples of the application of this ribozyme for the inhibition of gene expression in cell culture, in animals, as well as in plant models are presented. Particular emphasis is given to critical steps in the approach, including RNA site selection, delivery, vector development and cassette construction.

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH.

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

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.

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 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.

Structure-function studies of the hammerhead ribozyme.

Elucidation of the catalytic mechanism and structure-function relationship studies of the hammerhead ribozyme continue to be an area of intensive research. A combination of diverse approaches, such as X ray crystallography, spectral studies, chemical modifications, sequence variations and kinetic analyses, have provided valuable insight into the cleavage mechanism of this ribozyme. The hammerhead ribozyme crystal structures have provided valuable insight into conformational deformations needed to attain the catalytically active structure. Similarly, determination of ribozyme solution structure by spectroscopic analyses and the effect of divalent metal ions on RNA folding has further aided in the construction of a model for hammermead catalysis.

Conformation of the oxalamide group in retro-bispeptides. Three crystal structures.

We have determined the structures of a range of peptides having the oxalamide (-NH-CO-CO-NH-) unit located at their center. The oxalamide group has the trans conformation in two retropeptides and an approximately orthogonal conformation in the peptide with Pro residues. Torsional angles about the CO-CO bond are 180 degrees in MeO-Aib-CO-CO-Aib-OMe (1), 175 degrees in MeO-L-Leu-CO-CO-L-Leu-OMe (2), and -108 and -106 degrees for the two independent molecules in the crystal of MeO-L-Pro-CO-CO-L-Pro-OMe (3), owing to steric hindrance between CO and the pyrolidine ring. Crystal data are: (1) C12H20N2O6, triclinic, space group P1, a = 6.190(1), b = 10.044(2), c = 11.989(2) A, alpha = 86.13(12), beta = 83.13(2), gamma = 80.16(2) degrees, R = 0.057 for 1646 observed reflections [magnitude of Fo > 3 sigma (magnitude of Fo)]; (2) C16H28N2O6, tetragonal, space group P4(1), a = b = 11.121(3), c = 15.775(6) A, R = 0.058 for 1216 observed reflections [magnitude of Fo > 3 sigma (magnitude of Fo)]; (3) C14H20N2O6, monoclinic space group P2(1), a = 9.556(2), b = 17.864(3), c = 9.618(2) A, beta = 104.35(1) degree; R = 0.051 for 2100 observed reflections [magnitude of Fo > 3 sigma (magnitude of Fo)].