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.     

Targeting mortalin using conventional and RNA-helicase-coupled hammerhead ribozymes

Mortalin, also known as mot2/mthsp70/GRP75/PBP74, is a member of the heat-shock protein 70 family that is heat-uninducible. It is differentially distributed in cells that have normal and immortal phenotypes, has been localized to various subcellular sites, and has several binding partners and functions. Here, we describe the construction and use of mortalin-specific conventional and hybrid ribozymes to elucidate its crucial role in cell proliferation. Whereas conventional hammerhead ribozymes did not cause any repression of endogenous mortalin expression, RNA-helicase-linked hybrid ribozymes successfully suppressed the expression of mortalin, which resulted in the growth arrest of transformed human cells. We show that, first, RNA helicase-coupled hybrid ribozymes that have a linked unwinding activity can be used to target genes for which conventional hammerhead ribozymes are ineffective; second, the targeting of mortalin by RNA-helicase-coupled hybrid ribozymes causes growth suppression of transformed human cells and could be used as a treatment for cancer.     

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.     

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.     

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.     

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.     

mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes.

Subcellular localization signals for several mRNAs are positioned in their 3' untranslated regions (UTR). We have utilized the human alpha- and beta-actin 3' UTRs as signals for colocalizing hammerhead ribozymes with a lacZtarget mRNA. Ribozyme and target genes containing matched or unmatched 3' UTRs were cotransfected into 12-day-old chicken embryonic myoblast and fibroblast (CEMF) cultures and assayed by in situ hybridization (ISH) using a dual label, antibody sandwich procedure, and dual fluorescence microscopy to monitor intracellular colocalization. Beta-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl beta-D-galactopyranoside (ONPG) assay. We found that the percentage of colocalization using the matched alpha- or beta-actin 3' UTR (alpha-alpha or beta-beta) was enhanced approximately threefold relative to unmatched 3' UTRs. The increase in ribozyme-mediated inhibition of beta-galactosidase activity observed when matched 3' UTRs were used was consistent with the observed percentage of colocalization. These results represent the first direct demonstration that mRNA localization signals (zipcodes) can be utilized to enhance intracellular ribozyme efficacy.     

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.     

Functional gene-discovery systems based on libraries of hammerhead and hairpin ribozymes and short hairpin RNAs

Abundant information about the nucleotide sequence of the human genome has become readily available and it is now necessary to develop methods for the identification of genes that are involved in important cellular, developmental and disease-related processes. Identification methods based on the activities of hammerhead and hairpin ribozymes and of short hairpin RNAs (shRNAs), whose target specificities are coupled with loss-of-function phenotypes, have received increasing attention as possible tools for the rapid identification of key genes involved in such processes. We describe here recent advances that have been made with libraries of ribozymes and shRNAs and compare the advantages of the different types of library. The use of such libraries has already revealed new details of several important physiological phenomena.     

Intronic hammerhead ribozymes are ultraconserved in the human genome

Small ribozymes have been regarded as living fossils of a prebiotic RNA world that would have remained in the genomes of modern organisms. In this study, we report the ultraconserved occurrence of hammerhead ribozymes in Amniota genomes (reptiles, birds and mammals, including humans), similar to those described previously in amphibians and platyhelminth parasites. The ribozymes mapped to intronic regions of different genes, such as the tumour suppressor RECK in birds and mammals, a mammalian tumour antigen and the dystrobrevin beta in lizards and birds. In vitro characterization confirmed a high self-cleavage activity, whereas analysis of RECK-expressed sequence tags revealed fusion events between the in vivo self-cleaved intron and U5 or U6 small nuclear RNA fragments. Together, these results suggest a conserved role for these ribozymes in messenger RNA biogenesis.     

Kinetic characterization of two I/II format hammerhead ribozymes.

Five new hammerhead ribozymes were designed that assemble through the formation of helices I and II (I/II format) instead of the more standard assembly through helices I and III (I/III format). The substrate binding and cleavage properties of such hammerheads could potentially be different due to the absence of loop II and the requirement for the entire catalytic core to assemble. Two I/II format hammerheads, HHalpha1 and HHalpha5, which show structural homogeneity on native gels, were characterized kinetically. The association rate constants of both I/II hammerheads are unusually slow compared to the rate of RNA duplex formation. The dissociation rate constants indicate that the hammerhead core destabilizes an uninterrupted RNA helix somewhat less than was observed for I/III hammerheads. Whereas the cleavage rate constant of HHalpha5 is similar to that observed for I/III hammerheads, HHalpha1 cleaves 10-fold faster than any hammerhead previously reported. The temperature and pH dependence of the cleavage rate constant of HHalpha1 are similar to those reported for I/III hammerheads, suggesting a similar mechanism of cleavage.     

Structure-based search reveals hammerhead ribozymes in the human microbiome

Deep sequencing of viral or bacterial nucleic acids monitors the presence and diversity of microbes in select populations and locations. Metagenomic study of mammalian viromes can help trace paths of viral transmissions within or between species. High throughput sequencing of patient and untreated sewage microbiomes showed many sequences with no similarity to genomic sequences of known function or origin. To estimate the distribution of functional RNAs in these microbiomes, we used the hammerhead ribozyme (HHR) motif to search for sequences capable of assuming its three-way junction fold. Although only two of the three possible natural HHR topologies had been known, our analysis revealed highly active ribozymes that terminated in any of the three stems. The most abundant of these are type II HHRs, one of which is the fastest natural cis-acting HHR yet discovered. Altogether, 13 ribozymes were confirmed in vitro, but only one showed sequence similarity to previously described HHRs. Sequences surrounding the ribozymes do not generally show similarity to known genes, except in one case, where a ribozyme is immediately preceded by a bacterial RadC gene. We demonstrate that a structure-based search for a known functional RNA is a powerful tool for analysis of metagenomic datasets, complementing sequence alignments.     

Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag

A new type of 5'-lipophilic capture tag is described, enabling the facile reverse phase HPLC purification of chemically modified hammerhead ribozymes (oligozymes) whilst still carrying the 2'-O-tert.-butyldimethylsilyl protection of the essential riboses. In its most convenient form, the capture tag consists of a simple diol, such as hexan-1,6-diol, which at one end is attached via a silyl residue to a highly lipophilic entity such as tocopherol (vitamin E) or cholesterol, and the other end is functionalized as a phosphoramidite. This lipophilic capture tag is added as the last residue in the solid-phase synthesis of chemically modified hammerhead ribozymes. Cleavage from the support and release of all protecting groups except for the silyl groups is achieved with ethanolamine/ethanol. The crude product is then loaded directly on to a reverse phase HPLC column. Separation of failure peaks from full length product is achieved easily using a short run time. The retarded product peak is collected, lyophilized, desilylated in the normal way and then desalted. This method removes the lipophilic capture tag yet leaves behind the hexanediol entity which helps protect the compound against degradation by 5'-exonucleases. The purity of the product as judged by analytical anion-exchange HPLC and capillary gel electrophoresis is generally better than 95% full-length, and yields of 2-4 mg from a 1 micromol scale synthesis are routine. In addition, the method can be readily scaled up, an important feature for the development of such chemically modified ribozymes as potential therapeutics.