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PubMed1999

Characterization of the Azoarcus ribozyme: tight binding to guanosine and substrate by an unusually small group I ribozyme.

We report novel chemical properties of the ribozyme derived from the smallest group I intron (subgroup IC3) that comes from the pre-tRNA(Ile) of the bacterium Azoarcus sp. BH72. Despite the small size of the Azoarcus ribozyme (195 nucleotides (nt)), it binds tightly to the guanosine nucleophile (Kd = 15 +/- 3 microM) and exhibits activity at high temperatures (approximately 60-70 degrees C). These features may be due to the two GA3 tetraloop interactions postulated in the intron and the high GC content of the secondary structure. The second order rate constant for the Azoarcus ribozyme, ((k(cat)/Km)S = 8.4 +/- 2.1 x 10(-5) M(-1) min(-1)) is close to that found for the related ribozyme derived from the pre-tRNA(Ile) of the cyanobacterium Anabaena PCC7120. pH dependence studies and kinetic analyses of deoxy-substituted substrates suggest that the chemical cleavage step is the rate-determining process in the Azoarcus ribozyme. This may be due to the short 3-nt guide sequence-substrate pairing present in the Azoarcus ribozyme. Finally, the Azoarcus ribozyme shares features conserved in other group I ribozymes including the pH profile, the stereospecificity for the Rp-phosphorothioate at the cleavage site and the 1000-fold decrease in cleavage rate with a deoxyribonucleoside leaving group.

Azoarcus↗

PubMed1996

Optimizing the cell efficacy of synthetic ribozymes. Site selection and chemical modifications of ribozymes targeting the proto-oncogene c-myb.

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5'-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2'-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

Animals↗

PubMed1994

Intracellular susceptibility to ribozymes in a tethered substrate-ribozyme provirus model is not predicted by secondary structures of human immunodeficiency virus type 1 RNAs in vitro.

We have assessed the sensitivity of different sites in HIV-1 genomic RNA to ribozymes. Ribozymes targeted to sequences in U5, Pol, Env, RRE, or R were positioned into nef of an infectious HIV-1 provirus. When these proviral DNAs were introduced into HeLa CD4+ cells, recombinant viruses that contain ribozymes tethered to genomic RNA or viral mRNAs were produced. The growth kinetics of ribozyme-containing viruses in CD4+ lymphocytes (MT4 cells) were distinctly delayed when compared to control viruses. On the basis of the ability of a particular ribozyme to inhibit virus replication, we inferred intracellular ribozyme-sensitive sites. We found that although ribozyme sensitivity in vitro could be correlated with predicted secondary structures of target RNAs, such in vivo correlations could not be made when using the HIV provirus model. We conclude that both Zuker algorithm computer modeling of substrate RNA secondary structures and in vitro cleavage efficiencies cannot be reliably used to determine HIV-1 ribozyme sensitive sites in vivo.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease.

BACKGROUND: Because the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-beta, a selective decrease in TGF-beta may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-beta expression. METHODS AND RESULTS: We constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-beta gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-beta gene was confirmed by selective inhibition of TGF-beta mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-beta resulted in significant inhibition of neointimal formation after vascular injury in a rat carotid artery model (P:<0.01), whereas DNA-based control ONs and mismatched ribozyme ONs did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by (1) a reduction in collagen synthesis and mRNA expression of collagen I and III and (2) a significant decrease in DNA synthesis as assessed by proliferating cell nuclear antigen staining. Moreover, we modified ribozyme ONs containing phosphorothioate DNA and RNA targeted to the TGF-beta gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-beta expression. CONCLUSIONS: Overall, this study provides the first evidence that selective blockade of TGF-beta resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that modification of ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.

Animals↗

PubMed2002

Multitarget ribozyme against the S1 genome segment of reovirus possesses novel cleavage activities and is more efficacious than its constituent mono-ribozymes.

Two hammerhead motif containing ribozymes (Rzs) were constructed through recombinant techniques that were directed to cleave at the conserved sites of the reovirus S1 gene segment which encodes the cell attachment protein sigma1. The two mono-ribozymes 553 and 984 cleaved the target RNA in a sequence specific manner, and Rz-553 being the more efficient. When the mono-Rzs were combined in direct tandem to make it a multitarget-Rz, very efficient cleavage of the S1 RNA was achieved that retained the specificity of the two mono-ribozymes. This cleavage was, as expected, Mg(++)-dependent but protein-independent. Almost complete cleavage of the S1 RNA was observed with multitarget ribozyme alone. Although S1-Rz-984 cleaved the short S1 synthetic RNA, it failed to cleave the full length S1 RNA (1.4 kb). On the contrary, Rz-553 cleaved the short synthetic RNA as well as the full length S1 RNA with equal efficiency. Full length S1 RNA was, however, cleaved efficiently by the multitarget-ribozyme-S1-Rz-984-553 that cleaved at both the target sites. Thus, hybridization of one ribozyme (Rz-553) to a full length S1 RNA potentially opened up the 984-Rz target site that was otherwise inaccessible to the mono-Rz-984. Multitarget ribozyme expressing mammalian cells showed reduced amounts of S1 RNA that correlated well with the levels of reovirus sigma1 protein. Potential uses of such multitarget-Rzs are discussed.

Base Sequence↗

PubMed1991

Ribozyme-mediated cleavage of an HIV-1 gag RNA: the effects of nontargeted sequences and secondary structure on ribozyme cleavage activity.

Catalytic antisense RNAs, or ribozymes, have great potential as inhibitors of gene expression and as antiviral therapeutic agents. The major advantage of ribozymes versus standard antisense RNAs is their catalytic capability, enabling these RNAs to cleave multiple substrates. We have been investigating the antiviral activity of ribozymes targeted to the HIV-1 genome. The successful use of these antisense agents in an intracellular milieu requires stabilization of the ribozymes by flanking, non-base-pairing sequences, or some modification of the sugar-phosphate backbone. We describe a systematic investigation of the effects of flanking, non-base-pairing sequences on the catalytic activity of an anti-HIV-1 gag ribozyme embedded in radically different transcripts. Amazingly, these complex ribozyme-containing transcripts maintain substantial catalytic activity. Finally, we describe a bacterial gene fusion system that has potential for the large scale production of catalytically active ribozymes.

Base Sequence↗

PubMed2003

Ribozyme-based gene-inactivation systems require a fine comprehension of their substrate specificities; the case of delta ribozyme.

The ability of ribozymes (i.e. RNA enzymes) to specifically recognize and subsequently catalyze the cleavage of an RNA substrate makes them attractive for the development of therapeutic tools for the inactivation of both viral RNAs and mRNAs associated with various diseases. Several applicable ribozyme models have been tested both in vitro and in a cellular environment, and have shown significant promise. However, several hurdles remain to be surpassed before we generate a useful gene-inactivation system based on a ribozyme. Among the most important requirements for further progress are a better understanding of the features that contribute to defining the substrate specificity for cleavage by a ribozyme, and the identification of the potential cleavage sites in a given target RNA. The goal of this review is to illustrate the importance of both of these factors at the RNA level in the development of any type of ribozyme based gene-therapy. This is achieved by reviewing the recent progress in both the structure-function relationships and the development of a gene-inactivation system of a model ribozyme, specifically delta ribozyme.

Base Sequence↗

PubMed1992

Activities of HIV-RNA targeted ribozymes transcribed from a 'shot-gun' type ribozyme-trimming plasmid.

We have constructed a "shot-gun" type ribozyme-trimming system. By concatenating several units, each consisting of a trans-acting ribozyme (targeted to HIV-RNA) and cis-acting ribozymes (trimming 5'- and 3'-ends of the trans-acting ribozyme), several kinds of trans-acting ribozymes can be liberated upon transcription and self-cleavage. Since each liberated HIV-RNA-targeted ribozymes can work independently, they can simultaneously cleave HIV-RNA at several different sites. Ribozymes were targeted at relatively conserved GUC-containing sites at LTR, gag and tat regions.

Base Sequence↗

PubMed1996

Toxicity of cationic lipid-ribozyme complexes in human prostate tumor cells can mimic ribozyme activity.

Prostate tumor cell lines have been shown to both produce interleukin-6 (IL-6) and express the IL-6 receptor, suggesting a potential autocrine growth regulatory role for IL-6. We explored the role of IL-6 in the proliferation of the human prostatic carcinoma cell line, DU145, using ribozymes to inhibit IL-6 expression. Hammerhead-type ribozymes targeted against IL-6 mRNA sequences were prepared, and in vitro analyses were used to demonstrate that these molecules catalyzed the cleavage of IL-6 mRNA poly- nucleotide fragments. To test in situ activity, these ribozymes were transfected into DU145 cells using cationic transfection lipids, cytofectins. Treatment of cultured cells with ribozyme/cationic lipid complexes resulted in a reduction of IL-6 protein levels in the supernatant and reduced numbers of DU145 cells 48 h after treatment. However, similar results were also seen following treatment with control RNA/lipid complexes. This reduction in IL-6 levels and cell numbers was a function of the RNA/lipid complexes and was not seen with either lipid or RNA alone. Therefore, the reductions in IL-6 levels and cell numbers observed were not due to ribozyme-mediated cleavage of IL-6 mRNA, but rather reflected a dose-dependent, nonspecific toxic effect of the treatment with ribozyme/cytofectin complexes. This effect can resemble functional ribozyme activity, complicating analysis of the activity of synthetic ribozymes after transfection into cultured cells.

Base Sequence↗

PubMed1994

Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex.

We have used chemical modification analysis to probe the solution structure of the hairpin ribozyme. The modifying reagents dimethylsulfate, 1-cyclohexyl-N'-[2-(N-methylmorpholino) ethyl-carbodiimide-p-toluenesulfonate, kethoxal, diethylpyrocarbonate and (2,12-dimethyl-3,7,11,17- tetraazabicyclo [11.3.1]heptadeca-1(17),2,11,13,15-pentaenato) nickel(II) perchlorate were used to probe functional groups that participate in Watson-Crick and non-canonical base-pairs. Our results confirm the existence of four short helices (3 to 6 bp) within the ribozyme-substrate complex, and demonstrate that one intramolecular helix (helix 4) is comprised of three base-pairs rather than the previously suggested five. In the absence of magnesium, the ribozyme is observed to fold into its secondary structure. Upon addition of magnesium, a striking difference in chemical modification is observed, particularly at sites within the ribozyme's large internal loop (loop B) that are essential for catalytic function (bases 21 to 26). Moreover, magnesium-dependent folding clearly destabilizes an A-U base-pair in a region where a proposed bend is required to juxtapose the catalytically essential loops A and B. Upon addition of substrate, no changes are observed in the structure of helix 3, loop B or helix 4. However, strong protection of bases in the substrate-binding domain is observed, including those located across internal loop A. The modification data are consistent with the formation of a previously proposed tertiary structure motif within loop B that includes non-canonical G-A, A-U and A-A base-pairs, and that is identical with those identified by NMR analysis of loop E of 5 S rRNA and the sarcin/ricin loop of 28 S rRNA. Our results indicate that the hairpin ribozyme adopts a stable magnesium-dependent tertiary structure to which the substrate binds without inducing major conformational changes, and that substrate recognition is likely to involve non-canonical base-pairs between the ribozyme and substrate sequences adjacent to the cleavage site.

Base Sequence↗

PubMed2000

Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators.

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus, has a bisegmented double-stranded RNA genome consisting of a 3.2-kb A segment and a 2.9-kb B segment. To determine the function of IPNV's viral proteins and to study the effects of viral RNA cleavage by hammerhead ribozymes, we cloned and sequenced the IPNV E1S strain of the A segment. After sequencing, we continued to study the virus pathogens inhibited by ribozyme cleavage and analyzed the cleavage of the virus RNA in vitro. The templates (VP2, 1220 bp) for in vitro transcription of S569 and S969 (substrates 569 and 969 bp in length) were synthesized by polymerase chain reaction. The DNA templates of hammerhead ribozymes targeted different sites in the partial sense RNA of IPNV. These templates were chemically synthesized RNAs prepared by runoff transcription of amplification products or synthetic DNA templates containing a T7 RNA polymerase promoter, and were used to characterize several properties of the cleavage reaction at 25 degrees C in 12 mM Mg(2+). Under this condition (25 degrees C, 12 mM Mg(2+)), the hammerhead ribozymes formed an estimated fraction of product during the reaction of only 30% in cleaving long RNA substrates in vitro. Short DNA facilitators (12 or 24-mers) that bind adjacent to either the 3' or 5' end of the ribozyme enhanced the rate of cleavage of the long RNA substrates containing 569 and 969 nucleotides, respectively, in trans. The hammerhead ribozymes with 3'-end facilitators reacted more efficiently (i.e., 65%).

Journal Article↗

PubMed1994

An intron in the nuclear ribosomal DNA of Didymium iridis codes for a group I ribozyme and a novel ribozyme that cooperate in self-splicing.

We have discovered a unique group I intron-like insertion (DiSSU) in the nuclear small subunit ribosomal RNA gene of the myxomycete Didymium iridis. By sequence, DiSSU consists of a group I ribozyme at the 5' end, an open reading frame (ORF) in the middle, and a novel element at the 3' end. Intron RNA self-splices in vitro to yield ten major processed RNAs, including a full-length circle. The group I ribozyme can efficiently cleave at an internal processing site, which separates the group I ribozyme from the ORF. Surprisingly, deletion that remove the entire group I ribozyme do not impair cleavage at the 3' splice site, implying that the 3' element itself is a catalytic RNA. Deletions that remove portions of the 3' element prevent utilization of the 5' splice site, suggesting that this element cooperates with the upstream group I ribozyme in splicing. DiSSU appears to be the first example for the cooperative interaction of distinct ribozymes in RNA splicing.

Amino Acid Sequence↗

PubMed2000

Significant activity of a modified ribozyme with N7-deazaguanine at g10.1: the double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes.

BACKGROUND: Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance ( approximately 20 A) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site. RESULTS: In earlier discussions, it was difficult to completely exclude the possibility that La3+ ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell-shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd2+ ions, the added La3+ ions did not replace the pre-existing P9 metal ion, and that the replacement of N7 by C7 at G10.1 reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg2+ ion to N7 at G10.1 is catalytically important but not indispensable. Most importantly, 7-deaza-R34 also yielded a bell-shaped curve upon addition of La3+ ions to the reaction mixture. CONCLUSIONS: Since the data based on our experiments with 7-deaza-R34 are completely free from potential artefacts, due to the binding of a La3+ ion to N7 at G10.1, our results, that 7-deaza-R34 yielded a bell-shaped curve following the addition of La3+ ions to the Mg2+-background reaction mixture, strongly supports the proposal that a double-metal-ion mechanism is operative in the cleavage reaction which is catalysed by hammerhead ribozymes.

Catalysis↗

PubMed1991

Non-ribozyme sequences enhance self-cleavage of ribozymes derived from Hepatitis delta virus.

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.

Base Sequence↗

PubMed1999

Induction of ribozyme activity by anti-ribozyme oligonucleotides.

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.

Base Sequence↗

PubMed2003

[Preparation and characterization of ribozyme for tissue inhibitor of metalloproteinases-1 mRNA ribozymes in vitro].

To study the activity of the U6 driven ribozymes for tissue inhibitor of metalloproteinases-1(TIMP-1) mRNA and explore their use to cure hypertrophic scar, the ribozyme gene was designed according to the hammerhead structure described by Symons and then was cloned into pBSKneorU6, a vector containing mutant human U6 gene. TIMP-1 cDNA fragments were cloned into T-vector pGEM-T, to form the plasmid pTIMP-1. [(32)P]-labeled TIMP-1 transcripts as target RNAs and [(32)P]-labeled ribozyme, which was transcribed from the template that had been amplified from the corresponding cloning plasmids in vitro, were incubated together under various conditions for cleavage reactions. The reaction mixtures were electrophoresed on PAGE and autoradiographed. The results showed that the ribozyme(U6-Rz358) cleaved the mRNA successfully at 37 degrees; and the cleavage activity was best at 50 degrees ( K(m)=39.6 nmol/L, k(cat)=0.21 min(-1)), and the cleavage efficiencies were up to 76.34% at 50 degrees and 55.21% at 37 degrees. The designed ribozymes possessed perfect specific cleavage activity to TIMP-1. These findings suggested the potential application of this ribozyme as a new therapeutic agent against hypertrophic scar.

Base Sequence↗

PubMed2000

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.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1988

Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena acts as a sequence-specific endoribonuclease. Specificity of cleavage is determined by Watson-Crick base pairing between the active site of the RNA enzyme (ribozyme) and its RNA substrate [Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature (London) 324, 429-433]. Surprisingly, single-base changes in the substrate RNA 3 nucleotides preceding the cleavage site, giving a mismatched substrate-ribozyme complex, enhance the rate of cleavage. Mismatched substrates show up to a 100-fold increase in kcat and, in some cases, in kcat/Km. A mismatch introduced by changing a nucleotide in the active site of the ribozyme has a similar effect. Addition of 2.5 M urea or 3.8 M formamide or decreasing the divalent metal ion concentration from 10 to 2 mM reverses the substrate specificity, allowing the ribozyme to discriminate against the mismatched substrate. The effect of urea is to decrease kcat and kcat/Km for cleavage of the mismatched substrate; Km is not significantly affected at 0-2.5 M urea. Thus, progressive destabilization of ribozyme-substrate pairing by mismatches or by addition of a denaturant such as urea first increases the rate of cleavage to an optimum value and then decreases the rate.

Animals↗