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A ribozyme was synthesized against a conserved region in the RNA-dependent RNA-polymerase encoding cistron of the important plant pathogen potato virus Y (PVY). This ribozyme was shown to cleave PVY-specific RNA-transcripts efficiently in vitro, with up to 95% of the substrate RNA being cleaved within 2 h incubation at 37°C. A second ribozyme, designed with much shorter viral complementary arms in an attempt to optimize the efficiency of the cleavage reaction, surprisingly failed to cleave the substrates previously cleaved by the longer ribozyme. A much shorter PVY specific RNA-transcript of only 37 nucleotides (nt), however, was cleaved by this short ribozyme proving its ribozymic activity and indicating that the cleavage activity of the ribozyme is, in part, determined by the substrate involved. Analysis of cleavage reactions on non-denaturing polyacrylamide (PAA) gels indicated that incorrect basepairing, interfering with correct formation of the hammerhead structure, was likely to be responsible for the absence of detectable cleavage of the larger substrates by the short ribozyme.

We constructed a novel RNA-protein hybrid ribozyme that have the site-specific cleavage activity of the hammerhead ribozyme and the unwinding activity of the endogenous RNA helicase. This hybrid ribozyme leads to extremely efficient cleavage of any target mRNA regardless of the secondary structure of the RNA. Since the novel hybrid ribozymes can attack any site within mRNA, libraries were made of the hybrid ribozymes with randomized binding arms and introduced into cells. This procedure made it possible to readily identify the relevant genes associated with phenotype in the apoptosis pathway. This application of a randomized library of hybrid ribozymes represents a simple powerful method for identification of genes associated with specific phenotypes in the post genome era.

Background The great variety in sequence, length, complexity, and abundance of satellite DNA has made it difficult to ascribe any function to this genome component. Recent studies have shown that satellite DNA can be transcribed and be involved in regulation of chromatin structure and gene expression. Some satellite DNAs, such as the pDo500 sequence family in Dolichopoda cave crickets, have a catalytic hammerhead (HH) ribozyme structure and activity embedded within each repeat. Results We assessed the phylogenetic footprints of the HH ribozyme within the pDo500 sequences from 38 different populations representing 12 species of Dolichopoda. The HH region was significantly more conserved than the non-hammerhead (NHH) region of the pDo500 repeat. In addition, stems were more conserved than loops. In stems, several compensatory mutations were detected that maintain base pairing. The core region of the HH ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among 198 sequences. RNA folding of the HH sequences revealed that a potentially active HH ribozyme can be found in most of the Dolichopoda populations and species. Conclusions The phylogenetic footprints suggest that the HH region of the pDo500 sequence family is selected for function in Dolichopoda cave crickets. However, the functional role of HH ribozymes in eukaryotic organisms is unclear. The possible functions have been related to trans cleavage of an RNA target by a ribonucleoprotein and regulation of gene expression. Whether the HH ribozyme in Dolichopoda is involved in similar functions remains to be investigated. Future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead.

The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.

Hepatitis B virus is a DNA virus which replicates asymmetrically through reverse transcription of an RNA intermediate (pregenomic RNA). As a first step toward an antiviral strategy, a single synthetic oligodeoxynucleotide coding for 3 ribozyme motifs directed against three adjacent sites within the pregenomic RNA was synthesized, cloned and transcribed in vitro . Experiments utilizing 5′ and 3′ end labeling of RNA demonstrated that the three ribozymes accurately cleaved hepatitis B virus substrate RNA. Cleavage efficiency was similar to that of single ribozyme constructs. These results demonstrate that hepatitis B virus RNA is susceptible to ribozyme induced cleavage and illustrate that three ribozymes were simultaneously active when transcribed from a single DNA template. The expression of multiple ribozyme motifs may offer an advantage if there is high viral target sequence variability.

Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5′ and 3′ ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5′ and 3′ ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5′ and 3′ termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.

A model for the secondary structure of the self-cleaving RNA from hepatitis delta virus was tested. Specific base changes were introduced in each of four regions with the potential for base-pairing (stems I-IV), and for each variant sequence, a rate constant for cleavage was determined. In each stem, mutations that would interfere with Watson-Crick base-pairing also reduced the first-order rate constants by 10-10(4)-fold relative to the unmodified version. Within stems I and II and a shortened form of stem IV, compensatory changes resulted in rates of cleavage equal to or greater than the unaltered ribozyme sequence. Stem III compensatory mutants cleaved faster than the uncompensated mutants although they were not as active as the natural sequence, suggesting additional sequence-dependent requirements within this region. Structure probing of RNA containing the stem II mutations provided an independent confirmation of stem II in the ribozyme. The predictive value of the model was tested by designing two trans-acting ribozymes which were circularly permuted composites of genomic, antigenomic, and unique sequences. The core of these two catalytic RNAs was the same, but they otherwise differed in that, in one of them, a constraining tetraloop sequence was added to stem II. Both ribozymes catalyzed the trans cleavage of a substrate oligoribonucleotide, thus providing additional evidence for stem II and the proposed structure in general.

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

A ribozyme was constructed that specifically cleaves RNA that contains the first coding exon of the tat gene of HIV-1. This anti-tat ribozyme was incorporated into a Moloney murine leukemia virus vector. A sequence containing only the 48-nucleotide antisense region of the ribozyme was also inserted into the retroviral vector. Human T-cell lines constitutively producing the tat-antisense and the anti-tat ribozyme RNA were created by transduction into Jurkat cells. When challenged with HIV-1, both the tat-antisense and anti-tat ribozyme-producing cells inhibited the replication of HIV-1. The antisense vector conferred a greater resistance to HIV-1 replication than did the anti-tat ribozyme vector.

The cleavage properties of a trans-acting hammerhead ribozyme targeted 51 bases upstream of the putative splicing branch point in the hamster prion pre-mRNA intron were investigated in cell-free model systems in vitro. The specificity of cleavage was demonstrated by the inability of this ribozyme to cleave a non-homologous synthetic message encoding part of the β amyloid peptide precursor, βAPP, and by the inability of the prion pre-mRNA to be cleaved by a ribozyme targeted to β amyloid peptide precursor mRNA. Also, the addition of total RNA isolated from rat brain had only a minimal effect on the cleavage of the prion substrate pre-mRNA by the ribozyme. Finally neither the presence of 100 ng of nuclear or cytoplasmic proteins were found to affect the rate of cleavage in vitro.

We analyzed the cleavage properties and the transcription regulation of the newt (Triturus vulgaris meridionalis) self-cleaving RNA. In vitro self-cleavage of model oligoribonucleotides occurs within a double hammerhead structure. In addition, an entire ribozyme molecule, as well as its catalytic domain, "trans-cleaves" in vitro appropriate oligoribonucleotide substrates. Signals encoded within the ribozyme DNA sequences regulate the ribozyme transcription, which is RNA polymerase II dependent. Finally, the deduced secondary structure of the self-cleaving RNA appears to be conserved in evolutionarily distant newt species. These features suggest that the newt ribozyme could play some role in the cell, possibly related to its cleavage properties.

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Maintaining RNA stability is a major problem in the delivery of preformed inhibitory RNA to target cells. In this study, we delivered a hammerhead ribozyme directed against tumour necrosis factor α into human promyelocytic leukaemia cells by cationic liposome-mediated transfection. Delivering a ribozyme in this manner reduced by 90% and 85% tumour necrosis factor α mRNA and protein, respectively. A modified ribozyme with a bacteriophage T7 transcription terminator at its 3′ end was more stable than one lacking this sequence. This indicates that ribozyme stability can be improved by the addition of terminal sequences expected to protect against cellular nucleases.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.

In our previous studies, it was demonstrated that the activity of a ribozyme in vivo was governed by several parameters, which include a high level-expression of ribozyme, the intracellular stability of the ribozyme and colocalization of the ribozyme with its target RNA in the same cellular compartment. To generate ribozymes with significant activity in vivo, we have developed a ribozyme-expression system based on a human tRNA(Val) promoter. Our tRNA-embedded ribozymes produced by our ribozyme-expression system remain relatively stable in cultured cells with half-lives longer than 30 min. Moreover, tRNA-ribozymes with a cloverleaf structure were efficiently exported from the nucleus to the cytoplasm, where they would effectively cleave target RNAs. In the present study, we investigated the relationship between the secondary structure of the tRNA-ribozymes and the transport efficacy of them in mammalian cells by using a screening system in vivo. Furthermore, we also investigated the mechanism of the export of tRNA-embedded ribozymes both in mammalian cells and in Xenopus oocytes.

Self-cleavage efficiency of ribozymes derived from hepatitis delta virus (HDV) has been shown to depend on the RNA structure, which in turn may be determined by the length of the considered sequences. Here we describe the construction and functional analysis of a 71 nucleotide-long RNA genomic fragment, Rz71, which carries an 18 nucleotide deletion in a very stable GC-rich stem-loop (stem IV), predicted to be present in several computer-derived secondary structure models. Rz71 is able to undergo self-cleavage under non-denaturing conditions (the t1/2 of the reaction at 37 degrees C is 3 min). The deletion, however, is not neutral, since under the same conditions the non-deleted ribozyme cleaves to 50% in less than 15 sec. Therefore, stem-loop IV seems to play a structural role, not being directly involved in the catalytic reaction, but contributing to the correct positioning of the catalytic core of the HDV ribozyme. Rz71 is the smallest self-cleaving sub-fragment of HDV genomic RNA reported so far.

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

Kinetic and thermodynamic factors that determine specificity of RNA cleavage by ribozymes are illustrated with examples from recent work with a ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA. The conclusions also apply to other ribozymes, to antisense oligonucleotide experiments, and to RNA and DNA cleavage agents that can recognize a single-stranded or double-stranded region of variable length. At first, adding bases to a ribozyme's recognition sequence is expected to increase cleavage of the target RNA relative to cleavage of other RNAs. However, adding more bases ultimately reduces this discrimination, as cleavage occurs essentially every time the target RNA or a mismatched RNA binds the ribozyme. This occurs despite the weaker binding of the mismatched RNA because dissociation becomes too slow (binding is too strong) to allow the ribozyme to "choose" between cleavage of the target RNA and a mismatched RNA. In summary, more (base pairing) isn't always better, because maximal discrimination requires equilibrium binding prior to cleavage. The maximum discrimination that can be obtained is expected to be greater with an A + U-rich recognition sequence than with a G + C-rich recognition sequence. This is because the weaker A.U base pairs (relative to G-C base pairs) allow recognition to be spread over a larger number of bases while preventing binding that is too strong. Finally, creating an A-rich ribozyme rather than a U-rich ribozyme avoids the loss in discrimination expected with U-rich ribozymes from the formation of U.G wobble pairs in addition to the "targeted" Watson-Crick U.A pair.

The ribozymes derived from Hepatitis delta virus (HDV) RNA appear unique in their sequence requirements for self-cleavage. While truncating the 1679 nucleotide antigenomic HDV RNA, we have characterized the cleavage requirements of a number of ribozymes of intermediate length. Two of these, containing 186 and 106 HDV nucleotides respectively, cleaved to completion in the presence of 18 M formamide. The 186 nucleotide ribozyme also cleaved to completion in 10 M urea. Removal of an additional 10 nts from the 3' terminus of the 106 nt ribozyme resulted in a loss of the ability to cleave in high concentrations of the denaturants. The interaction of nucleotides near the cleavage site with a sequence within this 10 base region may confer unusual stability on these ribozymes.

Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus (HDV) has revealed that certain co-transcribed vector sequences significantly affect the activity of the ribozyme. Specifically, the t1/2 of self-cleavage for a 135 nucleotide HDV RNA varied, at 42 degrees C, from 5 min to 88 min, depending on the vector-derived sequences flanking the 5' end of the ribozyme. Further analysis suggested that this phenomenon was most likely due to the interaction of vector-derived sequences with a 16 nucleotide region found at the 3' end of the ribozyme. These findings have implications for studies of ribozymes transcribed from cDNA templates, and may provide information regarding the catalytic structure of the HDV ribozyme.

Human hepatitis delta virus (HDV) ribozyme can catalyze self-cleavage reaction in the presence of Mg2+ ions, yielding products with 2',3'-cyclic phosphate and 5'-OH termini as do hammerhead and hairpin ribozymes. Recently, the tertiary structure of 3'-cleaved product of genomic HDV ribozyme was solved by X-ray crystallographic analysis. In this structure three single-stranded regions (SSrA, -B and -C) interacts intricately with hydrogen bonds between bases, phosphate oxygens and 2'-OHs to form nested double pseudoknot structure. Especially two Watson-Crick base pairs, 726G-710C and 727G-709C, between SSrA and SSrC, seems to be important for compact folding. To characterize the necessity of the two base pairs, we performed in vitro selection of active ribozymes using random RNA pool which mutated at 709, 710, 726 and 727. The result indicates that basically one G-C base pair is necessary for the activity.

A new type of hammerhead ribozyme, with cleavage activity enhanced by oligonucleotides, was constructed. Stem II of the ribozyme was substituted with a non complementary loop (loop II). The modified ribozyme exhibited negligible cleavage of a target RNA; however, it was converted to an active molecule in the presence of oligonucleotides which were complementary to loop II. The oligonucleotide compensated for the disabled stem II by binding with the ribozyme. The induction of the cleavage activity was sequence-specific and the oligonucleotides containing a purine base as the 3'-dangling end were able to induce the cleavage activity of the ribozyme most efficiently. A photo-crosslinking experiment proved that a pseudo-half-knot structure was formed in the active molecule. The cleavage of two kinds of substrate RNAs with different sequences was controlled by the corresponding ribozymes activated by specific oligonucleotides.

We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures., Author Summary Enzymes use variations of a few standard approaches to catalyze reactions. One of these approaches, acid–base catalysis, is of such fundamental importance that it is common to both protein enzymes and RNA-based enzymes, or ribozymes. The hammerhead ribozyme is one such ribozyme that uses an invariant guanine residue as a general base in its catalytic reaction. By changing this to an adenine, we can slow the reaction rate 100,000-fold, permitting us to capture both active, precatalytic, and postcatalytic forms of the ribozyme. We have exploited this approach to obtain near-atomic–resolution three-dimensional structures of the hammerhead ribozyme both before and after catalytic self-cleavage. These structures provide complementary views of the chemical step of hammerhead ribozyme catalysis., The hammerhead ribozyme exploits acid/base catalysis as an enzymatic mechanism. Weakening the general base, G12, by mutating it into an A, greatly slows the reaction. As a result, the before and after cleavage structures could be captured in a crystal lattice.

Background Hepatitis delta virus (HDV) is a satellite virus of hepatitis B. During viral replication the 1700-nucleotide-long genomic RNA and its complement, the antigenomic RNA, undergo self-cleavage catalyzed by internal ribozyme motifs that are essential for propagation of the virus in vivo. These self-cleavage activities are provided by 85-nucleotide-long sequence elements, the genomic and antigenomic forms of HDV ribozyme. Recently four permuted variants of the antigenomic HDV cis-ribozyme with a self-cleavage site located at the 5' proximity, in the middle, or nearby the 3' end of the molecule were constructed and synthesized. These constructs exhibit equal activity, a bi-phasic kinetics of self-cleavage reaction and reaction products with low and high stability. We have used ribonuclease probing to footprint the structures of uncleaved and post-cleaved forms of the antigenomic HDV ribozymes in solution. Uncleaved ribozymes, associated and individual products of the self-cleavage reaction were analyzed using ribonuclease and Fe(II)-EDTA protection assays to reveal the differences in the structure of pre- and post-cleaved antigenomic HDV ribozyme in solution. Findings Our findings demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, in contrast to minor conformational switch observed in crystals of the genomic form. This study indicates that changes in the structure of stem P1 and stem P4 are minor, those of the region ascribed to stem P2, stem P3 and loop l3 are dramatic, while stem P1.1 results from the self-cleavage reaction. Conclusion Our data agree with the structure of post-cleaved and disagree with that of pre-cleaved forms of HDV ribozyme published elsewhere.

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.

Two ribozymes were synthesized which were designed to cleave potato leafroll virus (PLRV) positive strand RNA within the regions known to encode the viral coat protein and the predicted RNA polymerase gene. DNA sequences encoding the ribozymes were inserted into the Escherichia coli plasmids pTz18R and pTz19R under the control of the bacteriophage T7 promoter and enzymically active RNA molecules generated by transcription by T7 RNA polymerase in vitro. Each ribozyme cleaved its cognate site in RNA derived from either cDNA or PLRV particles. Ribozyme cleavage sites within the polymerase gene and coat protein gene were determined and shown to be at the predicted sequence immediately downstream from a GUC motif. An altered version of the ribozyme which recognized the sequence in the coat protein gene was isolated in which a single adenosine residue in the enzymic loop of the ribozyme was deleted. This mutated ribozyme was unable to cleave RNA molecules containing the coat protein ribozyme target site.

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.

A highly-modified DNA building block, lacking both sugar and base moieties, is synthesized and incorporated into oligonucleotides to form functional mimics of ribozymes.

RAS genes encode guanine nucleotide binding proteins and are ubiquitously expressed although different level of expression in each tissue. RAS proteins play essential roles in transducing several crucial signaling pathways that regulate normal cellular proliferation. However, RAS genes are frequently mutated in human cancers, particularly in pancreatic and colon carcinoma. Mutant RAS oncogenes often harbor point mutations, leading the oncogenic proteins in a persistently activated GTP-bound state. Especially, point mutations in codon 12, 13, and 61 of the K-RAS gene are necessary for an early event in lung cancer. Such K-RAS point mutations are found in 30% to 56% of pulmonary adenocarcinomas. Therefore, K-RAS is a good target for cancer therapy. Several approaches specifically targeting mutant RAS genes have been performed for cancer therapy. However, they have some limitations in target specificity and/or therapeutic efficacy. Here, we describe a novel approach to human cancer gene therapy that is based on targeted replacement of cancer-specific mutant K-RAS (K-RASV12) transcripts using Tetrahymena group I intron-based trans-splicing ribozyme. RNA replacement approach with trans-splicing ribozyme will have the advantage of cumulative effects such as mutant K- RAS level reduction and simultaneous anti-cancer gene induction specifically in the mutant RNA-expressing cancer cells. These ribozymes were designed to specifically recognize mutated codon 12 site of K-RAS RNA, cleave the downstream sequence of the targeted site, and ligate with and hence induce the specific expression of reporter gene which was tagged at 3′ exon of the ribozymes in human cells as well as in vitro. Importantly, expression of the specific ribozyme with HSV-tk gene as 3′ exon caused efficient and specific cell regression with GCV treatment only in cancer cells expressing K-RASV12 transcript. Taken together, K-RASV12-targeting trans- splicing ribozyme could be a specific and potent agent for the treatment of tumors which express the mutant K-RAS transcript.

The self-splicing group I intron from Tetrahymena thermophila has been shown to mediate trans-splicing of an exon ligated to its 3' end onto a targeted and separate 5' exon RNA. In human cells, the trans-splicing ribozymes were demonstrated to be useful for therapeutic repair of defective transcripts which cause genetic or malignant diseases. In addition, we have previously presented that this ribozyme could be also utilized for the selective induction of new gene activities in target cells by recognizing the cell-specific RNA and catalyzing the in-frame fusion of desired RNA sequences. Especially, we developed hTERT-targeting specific trans-splicing ribozyme to efficiently and specifically direct tumor-specific cytotoxicity by transcriptional targeting and RNA replacement because human telomerase reverse transcriptase (hTERT) is highly and selectively expressed in 90% of malignant tumors and tumor cell lines. Delivery of adenoviral vector encoding the ribozyme into a peritoneal carcinomatosis nude mouse model induced highly selective expression of a reporter gene in the human tumors but not in normal mouse organs, and moreover, caused the efficient regression of hTERT-positive tumors. This trans-splicing ribozyme will have the advantage of cumulative effects of hTERT level reduction and cytotoxin gene induction in hTERT positive cancer cells with tissue specificity. In this study, in order to further investigate therapeutic feasibility of the ribozyme, we studied trans-splicing efficacy and the limiting factor of trans-splicing ribozyme's activity not only in cells but in vivo. We observed the expression level of hTERT RNA which is ribozyme's target, the ribozyme, and the trans-splicing RNA product in various cancer cell lines using real-time RT-PCR analysis, and then found that the activity and efficacy of the trans-splicing ribozyme was dependent on the amount of ribozyme in cells which have limited level of target RNA. Moreover, we could observe that the efficacy of the trans-splicing ribozyme in tumor tissues in which ribozyme-encoding adenoviral vector has been delivered is enhanced in a virus dose-dependent manner. However, reduction level of target RNA has reached to plateau even at highest amount of the ribozyme, indicating that inhibition alone of telomerase level by ribozyme might be insufficient to reduce caner cell proliferation. In contrast, trans-splicing-mediated delivery of the HSVtk suicide gene with the corresponding prodrug (GCV) treatment hampered tumor progression in an hTERT-specific manner with almost the same efficacy as the strong constitutional CMV promoter-driven adenoviral vector. Taken together, this study indicate that RNA replacement by trans-splicing ribozyme could be a potent and specific anti-cancer agent by delivery of therapeutic gene activities only into cancer cells expressing the chosen target RNA.

Severe forms of osteogenesis imperfecta are produced by dominant negative mutations in type I collagen. These mutations allow the inclusion of aberrant protein in type I collagen molecules. Therefore, therapy for this disorder must eliminate mutant transcripts prior to translation of mutant protein. This can be achieved by hammerhead ribozymes: small catalytic RNA molecules that can be targeted to any RNA molecule containing a putative nucleotide triplet cleavage site. Osteogenesis imperfecta was modeled in cellulo by stable expression of a COL1A1 minigene (pMG155) in MC3T3-E1 osteoblasts. This minigene expresses a truncated COL1A1 in which exons 5 and 47 are fused, creating a ribozyme cleavage site. A monomeric ribozyme containing a 3' self-cleaving site (Rz547) was designed to target this cleavage site. A multimeric ribozyme (M8Rz547) containing 8 tandemly arranged copies of the monomer was then produced. The self-cleaving multimeric ribozyme was designed to increase the titer and stability, and therefore the effective activity, of the ribozyme under physiological conditions. Expression of both Rz547 and M8Rz547 are driven by the COL1A1 promoter. Rz547 and M8Rz547 have previously been shown to cleave the COL1A1 target sequence, and to show self cleavage, when tested in vitro. The activities of the monomeric and multimeric ribozymes were compared in cellulo by stable co-expression of either Rz547 or M8Rz547 and the COL1A1 minigene target in MC3T3-E1 osteoblasts. Both Rz547 and M8Rz547 exhibited self-cleavage activity in cellulo. The titer of ribozyme was significantly increased in the cells expressing the multimeric M8Rz547 compared to the monomeric Rz547. Ribozymes were expressed in the nucleus and transported to the cytoplasm. The monomeric Rz547 produced a modest reduction of the target RNA and protein, compared to the dramatic elimination of target transcript and protein in the cells expressing the multimeric M8Rz547 ribozyme. The osteoblasts co-expressing M8Rz547 and the COL1A1 minigene target showed a reversion to a normal collagen phenotype, demonstrated by increased collagen fibril diameter and increased collagen deposition To examine the activity of the multimeric ribozyme in vivo, a transgenic mouse expressing the M8Rz547 has been generated. Self cleavage of the multimeric ribozyme was seen in tail RNA from these mice, demonstrating that the multimer is active in vivo. These experiments demonstrate the increased activity that multimerization confers to ribozymes targeting specific transcript sequences, and demonstrates the utility that multimeric ribozymes may have as a gene therapy tool.

In most in vitro run-off transcription reactions with T7 RNA polymerase, transcripts with heterogeneous ends are commonly obtained. Towards the goal of finding a simple and effective procedure for correct processing of their 3' ends we propose the use of trans-acting antigenomic delta ribozyme. We demonstrate that the extension of nascent transcripts with only seven nucleotides complementary to the ribozyme's recognition site, and subsequently, the removal of those nucleotides with the ribozyme acting in trans, is an efficient procedure for generating transcripts with homogenous 3' ends. This approach was tested on two model RNA molecules: an in vitro transcript of yeast tRNA(Phe) and a delta ribozyme, which processed itself during transcription. The proposed procedure is a simple alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends. As there is little possibility that the required additional stretch, only seven nucleotides long, enters into stable interactions with other parts of the transcripts, it can be cleaved off with high efficacy.

AIM: To design a hammerhead ribozyme targeting human telomerase reverse transcriptase (hTERT) and clone it’s gene for future use in the study of tumor gene therapy. METHODS: Using the software RNAstructure, the secondary structure of hTERT mRNA was predicted and the cleavage site of ribozyme was selected. A hammerhead ribozyme targeting this site was designed and bimolecular fold between the ribozyme and hTERT was predicted. The DNA encoding the ribozyme was synthesized and cloned into pGEMEX-1 and the sequence of the ribozyme gene was confirmed by DNA sequencing. RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosen as the cleavage site of the ribozyme. The designed ribozyme was comprised of 22 nt catalytic core and 17 nt flanking sequence. Computer-aided prediction suggested that the ribozyme and hTERT mRNA could cofold into a proper conformation. Endonuclease restriction and DNA sequencing confirmed the correct insertion of the ribozyme gene into the vector pGEMEX-1. CONCLUSION: This fundamental work of successful designing and cloning of an anti-hTERT hammerhead ribozyme has paved the way for further study of inhibiting tumor cell growth by cleaving hTERT mRNA with ribozyme.

To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro.TGFbeta1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. (32)p-labeled TGFbeta1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFbeta1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. (32)p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 degrees; at 37 degrees, it was active, K(m)=34.48 nmol/L, K(cat)=0.14 min(-1); while the point mutant ribozyme U1Rz803(m) had no cleavage activity, so these indicated the design of U1Rz803 was correct.U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

A method is described that allows a general drawback of in vitro transcription assays to be overcome: RNA polymerases tend to add extra nucleotides to the RNA 3′ end that are not encoded in the linearized DNA template. Furthermore, these polymerases show a considerable rate of premature termination close to the RNA’s 3′ end. These features lead to a decreased yield of full-length transcripts and often make it difficult to determine and isolate the correctly transcribed full-length RNA. The hammerhead ribozyme is frequently used in cis to cleave off these extra nucleotides. However, the upstream sequence requirements of this ribozyme restrict its general usability. In contrast, the hepatitis delta virus ribozyme has no such requirements and can therefore be applied to any RNA sequence in cis. Due to the catalytic activity of the ribozyme, the desired transcript is released as an RNA molecule with a homogeneous 3′ end. The resulting 2′,3′-cyclo-phosphate group of the released RNA can be easily and efficiently removed by T4 polynucleotide kinase treatment. The presented method can be applied for virtually any sequence to be transcribed and is therefore superior to other ribozyme strategies, suggesting possible applications in every field where transcripts with homogeneous 3′ ends are required.

The discovery of ribozymes in 1982 boosted a hypothesis in which RNA evolved and carried out biological reactions before proteins developed. Now, researchers have found a ribozyme that can replicate RNA – more evidence for this ‘RNA world’.For such a world to have existed, RNA must have catalyzed its own formation, but no known natural ribozyme can do this. So David Bartel and his colleagues at the Whitehead Research Institute set out to make artificial ribozymes that were more versatile and efficient than the natural ones. Such ribozymes would suggest that natural ones could have existed during the RNA era, but have become extinct.The group has isolated a ribozyme from a pool of >1 × 1015 randomly generated RNA sequences. This ribozyme does not require a particular template, but builds a complimentary strand of RNA using any general RNA template. The ribozyme also matches bases accurately to the RNA template more than 95% of the time, better than any previously isolated ribozyme. [Johnston, W.K. et al. (2001) Science 292, 1319–1325] PL

In a novel approach to gene therapy, researchers at Duke University Medical Center have shown that ribozymes can "edit" genetic information encoded in messenger RNA molecules inside mammalian cells. If further development is successful, the technique could, in effect, bypass a defective gene and instead correct the RNA blueprint the gene produces to direct protein synthesis. Ribozymes are RNA segments that catalyze reactions within cells. Because they selectively bind and react with other RNA segments, they have drawn attention as potential tools that could protect against viral infection by cleaving and destroying an invading virus's RNA. Clinical trials are now testing the potential of several modified ribozymes to protect against HIV infection. But the Duke team's approach is quite different. Assistant professor of surgery and genetics Bruce A. Sullenger, research assistant Joshua T. Jones, and postdoctoral fellow Seong-Wook Lee modified a ribozyme from a different class than those that cleave viral RN...