Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.     

Activity of hammerhead ribozymes containing non-nucleotidic linkers.

Hammerhead ribozymes were synthesized in which the tetranucleotide loop II was replaced by non-nucleotidic linkers of 7, 13, 17 and 19 atoms length. Ribozymes with 17 and 19 atom linkers, in combination with a 4 base pair stem II, had catalytic efficiencies which were 2 fold increased to that of the parent ribozyme with a tetranucleotide loop. Ribozymes with these linkers, but in combination with a 2 base pair stem II, showed a 2 fold decrease in catalytic efficiency when compared to the parent ribozyme. Prolonged preincubation in the presence of MgCl2 was required for hexaethylene glycol linker-modified ribozymes to obtain maximum activity and reproducible kinetic data.     

In vitro activity of minimised hammerhead ribozymes.

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37 degrees C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.3 above which the cleavage rate constants decline rapidly. The reactions show a biphasic dependence on magnesium-ion concentration; one of the interactions has an apparent dissociation constant of around 20 mM while the other appears to be very weak, showing no sign of saturation at 200 mM MgCl2. The minizymes are significantly less active than comparable, full-size ribozymes when cleaving short substrates. However, at a particular site in a transcribed TAT gene from HIV-1, minizymes are more effective than ribozymes.     

Generation and application of asymmetric hammerhead ribozymes.

Most researchers who intend to suppress a particular gene are interested primarily in the application of ribozyme technology rather than its mechanistic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribozyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribozyme carries at its 5' end the catalytic domain and at its 3' end a relatively long antisense flank that is complementary to the target RNA. Asymmetric hammerhead ribozymes can be constructed via polymerase chain reaction amplification, and rules are provided on how to select the DNA oligonucleotides required for this reaction. In addition to details on construction, we describe how to test asymmetric hammerhead ribozymes for association with the target RNA in vitro, so that RNA constructs can be selected and optimized for fast hybridization with their target RNA. This test can allow one to minimize association problems caused by the secondary structure of the target RNA. Additionally, we describe the in vitro cleavage assay and the determination of the cleavage rate constant. Testing for efficient cleavage is also a prerequisite for reliable and successful application of the technology. A carefully selected RNA will be more promising when eventually used for target suppression in living cells.     

Kinetics of intermolecular cleavage by hammerhead ribozymes.

The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.     

Design, characterization and testing of tRNA3Lys-based hammerhead ribozymes

A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3Lys without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3Lys-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy.     

In vitro selection of hammerhead ribozymes containing a bulged nucleotide in stem II.

Hammerhead ribozymes were transcribed from a dsDNA template containing four random nucleotides between stems II and III, which replace the naturally occurring GAA nucleotides. In vitro selection was used to select hammerhead ribozymes capable of in cis cleavage using denaturing polyacrylamide gels for the isolation of cleaving sequences. Self-cleaving ribozymes were cloned after the first and second rounds of selection, sequenced and characterised. Only sequences containing 5'-HGAA-3', where H is A, C or U, between stems II and III were active; G was clearly not tolerated at this position. Thus, only three sequences out of the starting pool of 256 (4(4)) were active. The Michaelis-Menten parameters were determined for the in trans cleaving versions of these ribozymes and indicate that selected ribozymes are less efficient than the native sequence. We propose that the selected ribozymes accommodate the extra nucleotide as a bulge in stem II.     

Catalytic diversity of extended hammerhead ribozymes

Chimeras of the well-characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full-length hammerhead at equilibrium and thereby deduce the elemental cleavage ( k 2) and ligation ( k -2) rate constants. The nine chimeras were all more active than minimal hammerheads and exhibited a very broad range of catalytic properties, with values of k 2 varying by 750-fold and k -2 by 100-fold. At least two of the hammerheads exhibited an altered dependence of k obs on magnesium concentration. Since much less catalytic diversity is observed among minimal hammerheads that lack the tertiary interactions, a possible role for the different tertiary interaction is to modulate the hammerhead cleavage properties in viroids. For example, differing hammerhead cleavage and ligation rates could affect the steady state concentrations of linear, circular, and polymeric genomes in infected cells.     

Design and specificity of hammerhead ribozymes against calretinin mRNA.

We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.     

The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes.

Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.     

1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes.

The incorporation of 1-deazaadenosine (c1A, 1b) into a hammerhead ribozyme and the resulting catalytic activity is described. For this purpose the phosphoramidite 2a and the 3'-phosphonate 2b as well as Fractosil-linked 1-deazaadenosine (3b) were prepared. The methoxyacetyl group was used for the 6-amino group protection and the triisopropylsilyl residue was introduced as the 2'-OH protecting group. Replacement of residues A14and A15.1 of the hammerhead ribozyme by 1-deazaadenosine resulted in a significantly reduced catalytic activity. Substitution of the A6, A9 and A13 residues has only a minor influence. The findings observed on ribozymes modified with 1-deazaadenosine were compared with those containing other adenosine analogues.     

Functional analysis of the pro-apoptotic factor Bax using hammerhead ribozymes.

A pro-apoptotic protein Bax is a Bcl-2 family member and forms homodimers and also heterodimerizes with death antagonists, Bcl-2 and Bcl-XL. To elucidate the detail of function of Bax in cells, we constructed a hammerhead ribozyme targeted to the Bax mRNA. The level of Bax protein in Hela-K cells expressing Bax-ribozyme was decreased compared with that of wild type Hela-K cells. Therefore, the Bax-ribozyme should be useful for the future investigations of the details of apoptosis pathway.     

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

BACKGROUND: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. RESULTS: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. CONCLUSIONS: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.