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An in vitro selection was carried out to identify hammerhead ribozymes cleaving 3' to GAC triplets more efficiently than the wild type ribozyme. A double-stranded DNA containing the sequence for the hammerhead ribozyme with 10 randomizations in the catalytic core, designed for in cis cleavage, was transcribed and the cleavage product amplified by reverse transcription and PCR. After seven selection cycles, the DNA was cloned and 50 colonies sequenced. One sequence, appearing six times, was active for in cis cleavage of GAC. It was identical to the consensus sequence except for a mutation at position 7. Another cleaved GUC and two more, cleaved GUA. The cleavage rates of these ribozymes for in trans cleavage were slower than the rate of the consensus ribozyme. Interestingly, the consensus sequence was not found in the selection. This strongly suggests that the consensus hammerhead ribozyme has evolved to an optimal sequence.

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%).

In vitro selection was used to isolate Mg 2+ -dependent self-cleaving ribozymes from random sequence. Characterization of representative clones revealed the emergence of at least 12 classes of ribozymes that adopt distinct secondary structure motifs. Only one class corresponds to a previously known structural motif, that of the naturally occurring hammerhead ribozyme. Each ribozyme promotes self-cleavage via an internal phosphoester transfer reaction involving the adjacent 2′-hydroxyl group with a chemical rate enhancement of between 10 3 - and 10 6 -fold greater than the corresponding uncatalyzed rate. These findings indicate that RNA can form a multitude of secondary and tertiary structures that promote cleavage by internal phosphoester transfer. Upon further in vitro selection, a class I ribozyme that adopts an “X motif” structure dominates over all other ribozymes in the population. Thus, self-cleaving RNAs isolated by in vitro selection from random-sequence populations can rival the catalytic efficiency of natural ribozymes.

DiGIR1 is a group I-like ribozyme derived from the mobile twin ribozyme group I intron DiSSU1 in the nuclear ribosomal DNA of the myxomycete Didymium iridis. This ribozyme is responsible for intron RNA processing in vitro and in vivo at two internal sites close to the 5'-end of the intron endo-nuclease open reading frame and is a unique example of a group I ribozyme with an evolved biological function. DiGIR1 is the smallest functional group I ribozyme known from nature and has an unusual core organization including the 6 bp P15 pseudoknot. Here we report results of functional and structural analyses that identify RNA elements critical for hydrolysis outside the DiGIR1 ribozyme core moiety. Results from deletion analysis, disruption/compensation mutagenesis and RNA structure probing analysis all support the existence of two new segments, named P2 and P2.1, involved in the hydrolysis of DiGIR1. Significant decreases in the hydrolysis rate, k (obs), were observed in disruption mutants involving both segments. These effects were restored by compensatory base pairing mutants. The possible role of P2 is to tether the ribozyme core, whereas P2.1 appears to be more directly involved in catalysis.

A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable.

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5' end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.

Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors.

Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.

Ribozymes are catalytic RNAs that offer several advantages as specific therapeutic genes against human immunodeficiency virus type 1 (HIV-1). Significant challenges in antiviral uses of ribozymes include (1) how best to express and to deliver this agent and (2) what is the best locale to target ribozymes against HIV-1 RNA. To explore the former, we have previously characterized several vector systems for efficient expression/delivery of anti-HIV-1 ribozymes (Dropulic et al., 1992; Dropulic and Jeang, 1994a; Smith et al., 1997). Here, to investigate an optimal locale for ribozyme-targeting, we asked whether it might be advantageous to direct ribozymes into HIV-1 virions as opposed to the more conventional approach of targeting ribozymes into infected cells. Two series of experiments were performed. First, we demonstrated that anti-HIV-1 ribozymes could indeed be packaged specifically and efficiently into virions. Second, we compared the virus suppressing activity of a packageable ribozyme with its counterpart, which cannot be packaged into HIV-1 virions. Our results showed that although both ribozymes cleaved HIV-1 genomic RNA in vitro with equivalent efficiencies, the former ribozyme demonstrated significantly higher virus-suppressing activity than the latter. These findings provide proof-of-principle that to combat productive HIV-1 replication, intravirion targeting is more effective than intracellular targeting of ribozymes.

The RNA world hypothesis implies that coded protein synthesis evolved from a set of ribozyme catalyzed acyl-transfer reactions, including those of aminoacyl-tRNA synthetase ribozymes. We report here that a bifunctional ribozyme generated by directed in vitro evolution can specifically recognize an activated glutaminyl ester and aminoacylate a targeted tRNA, via a covalent aminoacyl-ribozyme intermediate. The ribozyme consists of two distinct catalytic domains; one domain recognizes the glutamine substrate and self-aminoacylates its own 5'-hydroxyl group, and the other recognizes the tRNA and transfers the aminoacyl group to the 3'-end. The interaction of these domains results in a unique pseudoknotted structure, and the ribozyme requires a change in conformation to perform the sequential aminoacylation reactions. Our result supports the idea that aminoacyl-tRNA synthetase ribozymes could have played a key role in the evolution of the genetic code and RNA-directed translation.

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5' and 3' hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5' arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix + loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5' and the 3' arms of a miniribozyme containing an optimized sequence for helix + loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of gene-length RNA substrates may be best achieved by miniribozymes.

Using electrophoresis, sequencing and enzymatic digestion, we show that the group I intron from the cyanobacterium Anabaena sp. PCC 7120 catalyzes phosphodiester bond formation using a triphosphate on the 5'-terminal nucleotide, much like protein polymerases and engineered ribozymes. In the process, this ribozyme forms a unique circular RNA that incorporates the exogenous guanosine cofactor added during self-splicing. This finding may have relevance to a prebiotic RNA world and to modern biology.

A ribozyme based gene control element enabled the spatio-temporal regulation of gene function in mammalian cell culture with light.

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10 16 different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3′,5′-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3′ hydroxyl and the other a 5′ triphosphate. Ligation occurs in the context of a Watson–Crick duplex, with a catalytic rate of 0.26 min −1 under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.

Recent reports have demonstrated that the group I ribozyme from Tetrahymena thermophila can perform trans-splicing reactions to repair mutant RNAs. For therapeutic use, such ribozymes must function efficiently when transcribed from genes delivered to human cells, yet it is unclear how group I splicing reactions are influenced by intracellular expression of the ribozyme. Here we evaluate the self-splicing efficiency of group I introns from transcripts expressed by RNA polymerase II in human cells to directly measure ribozyme catalysis in a therapeutically relevant setting. Intron-containing expression cassettes were transfected into a human cell line, and RNA transcripts were analyzed for intron removal. The percentage of transcripts that underwent self-splicing ranged from 0 to 50%, depending on the construct being tested. Thus, self-splicing activity is supported in the mammalian cellular environment. However, we find that the extent of self-splicing is greatly influenced by sequences flanking the intron and presumably reflects differences in the intron’s ability to fold into an active conformation inside the cell. In support of this hypothesis, we show that the ability of the intron to fold and self-splice from cellular transcripts in vitro correlates well with the catalytic efficiency observed from the same transcripts expressed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozymes. The self-splicing system that we describe should facilitate these efforts as well as aid in efforts at enhancing in vivo ribozyme activity for various applications of RNA repair.

Ribozymes use a number of the same catalytic strategies as protein enzymes. However, general base catalysis by a ribozyme has not been demonstrated. In the hepatitis delta virus antigenomic ribozyme, imidazole buffer rescued activity of a mutant with a cytosine-76 (C76) to uracil substitution. In addition, a C76 to adenine substitution reduced the apparent pKa(whereKais the acid constant) of the self-cleavage reaction by an amount consistent with differences in the pKavalues of these two side chains. These results suggest that, in the wild-type ribozyme, C76 acts as a general base. This finding has implications for potential catalytic functions of conserved cytosines and adenines in other ribozymes and in ribonuclear proteins with enzymatic activity.

A region of c-myc mRNA was identified which permitted very efficient antisense effects to be achieved in living cells using chimeric methylphosphonate--phosphodiester antisense effectors. Novel inosine--containing ribozymes (which cleave after NCH triplets) were directed to an ACA triplet within this region and delivered into living cells. No ribozyme intracellular activity could be identified. Very low ribozyme function was also observed in in vitro assays using a 1700nt substrate RNA.

1. How do ribozymes work? 2412. The hammerhead RNA as a prototype ribozyme 2422.1 RNA enzymes 2422.2 Satellite self-cleaving RNAs 2422.3 Hammerhead RNAs and hammerhead ribozymes 2443. The chemical mechanism of hammerhead RNA self-cleavage 2463.1 Phosphodiester isomerization via an SN2(P) reaction 2473.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 2513.3 The hammerhead ribozyme does not actually require metal ions for catalysis 2543.4 Hammerhead RNA enzyme kinetics 2574. Sequence requirements for hammerhead RNA self-cleavage 2604.1 The conserved core, mutagenesis and functional group modifications 2604.2 Ground-state vs. transition-state effects 2615. The three-dimensional structure of the hammerhead ribozyme 2625.1 Enzyme–inhibitor complexes 2625.2 Enzyme–substrate complex in the initial state 2645.3 Hammerhead ribozyme self-cleavage in the crystal 2645.4 The requirement for a conformational change 2655.5 Capture of conformational intermediates using crystallographic freeze-trapping 2665.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 2675.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 2685.8 Is the conformational change pH dependent? 2695.9 Isolating the structure of the cleavage product 2715.10 Evidence for and against additional large-scale conformation changes 2745.11 NMR spectroscopic studies of the hammerhead ribozyme 2786. Concluding remarks 2807. Acknowledgements 2818. References 2811. How do ribozymes work? 241The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?

The structures of the model oligoribonucleotides that mimic the consecutive stages in the transcription of genomic HDV ribozyme have been analyzed by the Pb 2+ -induced cleavage method, partial digestion with specific nucleases and chemical probing. In the transcription intermediates, the P1 and P4 helical segments are found to be present in the final folded forms in which they exist in the full-length transcript. However, the region corresponding to the central hairpin forms another thermodynamically stable hairpin structure. Its correct folding requires the presence of a ribozyme 3′-terminal sequence and the formation of helix P2. This confirms the ribozyme structure of the pseudoknot type and points to the crucial role of helix P2 in its overall folding. Moreover, we show that the J4/2 region can be specifically cleaved in the presence of selected divalent metal ions in the full-length transcript, but not in a shorter one lacking six 3′-terminal nucleotides, which cannot form the pseudoknotted structure. Thus, a particular RNA conformation around that cleavage site is required for specific hydrolysis, and the J4/2 region seems to be involved in the formation of a general metal ion binding site. Recently, it has been proposed that, in the antigenomic ribozyme, a four nucleotide sequence within the J1/2 region may contribute to the folding pathway, being part of a mechanism responsible for controlling ribozyme cleavage activity. Our study shows that in the genomic ribozyme the central hairpin region may contribute to a similar mechanism, providing a barrier to the formation of an active structure in the ribozyme folding pathway.

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a “snorbozyme,” is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo . This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo . The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

The catalytic center of the RNA from the negative strand of the satellite RNA of chicory yellow mottle virus type 1 (sCYMV1) is in the hairpin ribozyme family, has catalytic activity, and cleaves substrates before a preferred GUA sequence. This is different from that of the satellite RNA from the negative strand of tobacco ringspot virus (sTRSV) which prefers a GUC sequence at the site of cleavage. The sCYMV1 hairpin ribozyme has now been developed for cleaving heterologous RNA substrates. When helix 1 was extended from the native 5 bp to 6 bp with a newly added A:U base pair, catalytic activity increased three-fold. The preferred sequence for the substrate loop was the native A*GUA sequence where * is the site of cleavage. When each nucleotide in this sequence was changed to each of the other three nucleotides, catalytic activity decreased 66-100%. RNA targets, containing this A*GUA sequence, were located in both human papillomavirus and HIV-1. Ribozymes were developed which efficiently cleaved these targets in vitro. These results identify a new class of hairpin ribozymes capable of cleaving substrates before a preferred GUA sequence rather than the GUC preferred by the sTRSV hairpin ribozyme. This expands the repertoire of target sites available for gene therapy using the hairpin ribozyme.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3' cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G.U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.

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.

We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.

Oligonucleotides were inserted into the Lp3 region of a hepatitis delta virus (HDV) cis-cleaving ribozyme and the effect of Lp3 enlargement on ribozyme folding was examined by assaying the activity of each mutant. The location of insertion and the sequence of the inserted oligonucleotide had distinct effects on ribozyme activity, and the HDV ribozyme was capable of adopting the active structure for cis-cleavage when a pentanucleotide was inserted into the 3′ portion of Lp3. Furthermore, whether the insertion mutant cis-cleaved or not, the structure of Lp3 was altered by the inserted oligonucleotide. These findings disclose that the 5′ rather than the 3′ portion of Lp3 is critical for ribozyme folding and catalysis.

The bacterial RNase P ribozyme is a site-specific endonuclease that catalyzes the removal of pre-tRNA leader sequences to form the 5' end of mature tRNA. While several specific interactions between enzyme and substrate that direct this process have been determined, nucleotides on the ribozyme that interact directly with functional groups at the cleavage site are not well-defined. To identify individual nucleotides in the ribozyme that are in close proximity to the pre-tRNA cleavage site, we introduced the short-range photoaffinity cross-linking reagent 6-thioguanosine (s6G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence [tRNA(G-1)] and examined cross-linking in representatives of the two structural classes of bacterial RNase P RNA (from Escherichia coli and Bacillus subtilis). These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s6G upstream of the cleavage (-1) site is cleaved accurately. Interestingly, s6G at position +1 of tRNA cross-links with high efficiency to homologous positions in J5/15 in both E. coli and B. subtilis RNase P RNAs, while s6G at position -1 of tRNA(G-1) cross-links to homologous nucleotides in J18/2. Both cross-links are detected over a range of ribozyme and substrate concentrations, and importantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cleave the covalently attached substrate. These data indicate that the conserved guanosine at the 5' end of tRNA is adjacent to A248 (E. coli) of J5/15, while the base upstream of the substrate phosphate is adjacent to G332 (E. coli) of J18/2 and, along with available biochemical data, suggest that these nucleotides play a direct role in binding the substrate at the cleavage site.

The ability of delta ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single- and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (kcat/KM) was higher at 37 degreesC than at 56 degreesC. The cleavage reaction seems to be limited by the product release step at 37 degreesC and by the chemical cleavage step at 56 degreesC. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (kcat and KM) and dissociation constant (Kd) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2'-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the delta ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Polymerase III (pol III)-dependent genes, like the adenoviral VA1 gene, are of particular interest for expressing small therapeutic RNAs into cells. A new VA1 RNA carrier molecule was generated through the deletion of the VA1 RNA central domain to give rise to the VAdeltaIV RNA vector that was devoid of undesirable physiologic activity (i.e., inhibition of the interferon-induced protein kinase, PKR). This vector was used to express in human cells hammerhead ribozymes targeted against the human immunodeficiency virus (HIV). Eight anti-HIV ribozymes were inserted at the 3'-end of this vector immediately before the four T-residues that serve as a transcription termination signal. Although the constructs were active in vitro, they failed to inhibit HIV replication in transient assays. Analysis of the intracellular ribozyme expression in cells revealed several anomalies. First, using mutant derivatives, we showed that the presence of two or three consecutive T-residues in the ribozyme portion was sufficient to promote the release of anomalous short transcripts. Second, when the ribozyme did not contain T-rich sequence, full-length transcripts were produced, but these transcripts were very unstable and were retained in the cell nucleus. In contrast, insertion of the ribozyme in place of the central domain of VA1 RNA led to production of full-length transcripts that were stable and located in the cytoplasm but that were not found to be active in vitro. Taken together, these results have important consequences for the future design of active intracellular ribozymes based on the use of pol III-transcribed genes.

Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.

The CCR5 chemokine receptor plays a crucial role in the initiation of in vivo HIV infection, acting as a critical coreceptor molecule for primary strains. Individuals with mutations in the CCR5 gene that reduce its level of expression are resistant to HIV-1 infection. Since these mutations are not associated with any known clinical condition, CCR5 may be an ideal target for anti-HIV therapy. We have designed an artificial hammerhead ribozyme, denoted RzR5-76, targeted to exon 2 of the human CCR5 mRNA. When RzR5-76 activity is induced in HEK 293 cells transfected with a CCR5 expression plasmid, the surface levels of this chemokine receptor are reduced up to 60%. The results indicate that this inhibitory effect is mainly due to the catalytic activity of the ribozyme and not to its antisense properties. These preliminary data suggest that intracellular ribozymes could be used in vivo to block HIV-1 entry into human cells.

The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3 A resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5'-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.

Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. However, particular attention must be paid to the following points: the identification of regions on the mRNA accessible to the ribozyme; the delivery of ribozymes to cells by either exogenous or endogenous delivery; colocalization of the ribozyme with the target RNA in the cell; and differentiation between closely related sequences. This field is advancing rapidly, and results obtained with transgenic animals demonstrate the power of this strategy for the inhibition of gene expression.

Background: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an ‘RNA world' was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids. Results: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular (‘ trans ') peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety. Conclusions: An in vitro -selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the ‘helical wheel' portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.

Ribozyme Pharmaceuticals, Inc. is based on a distinctive technology which uses antisense molecules that are also enzymes. An obvious use of these molecules is in the cleavage of mRNA for genes expressed in disease states or those encoded by infecting viruses. The Company has an impressive portfolio of patented ribozyme sequences relevant to a wide range of disorders, although questions remain over the degree of homology needed to target the enzyme and their stability and activity.

The uniform 2'-pyrimidine modifications have been found to inhibit ribozyme cleavage activity. However, in the present study we show that a good cleavage activity can be achieved for the 2'-amino modified ribozymes when their sequences were designed to contain only a few pyrimidines in helix I. In particular, ribozymes with no pyrimidines in helix I cleaved their target RNAs with almost the same efficacy as their unmodified versions. Interestingly, selective 2'-amino modification at positions 2.1 and 2.2 reduced the ribozyme cleavage activity by 8-fold, suggesting that the 2'-amino groups at these two positions may interfere with the formation of the ribozyme active conformation. In addition, uniformly modified ribozymes showed a remarkable stability in serum. Taken together these results should facilitate the design of stable ribozymes with sustained cleavage activity.

Combinatorial libraries of hairpin ribozymes representing all possible cleavage specificities (>10(5)) were used to evaluate all ribozyme cleavage sites within a large (4.2-kilobase) and highly structured viral mRNA, the 26 S subgenomic RNA of Sindbis virus. The combinatorial approach simultaneously accounts for target site structure and dynamics, together with ribozyme folding, and the sequences that result in a ribozyme-substrate complex with maximal activity. Primer extension was used to map and rank the relative activities of the ribozyme pool against individual sites and revealed two striking findings. First, only a small fraction of potential recognition sites are effectively cleaved (activity-selected sites). Second, nearly all of the most effectively cleaved sites deviated substantially from the established consensus selection rules for the hairpin ribozyme and were not predicted by examining the sequence, or through the use of computer-assisted predictions of RNA secondary structure. In vitro selection methods were used to isolate ribozymes with increased activity against substrates that deviate from the GUC consensus sequence. trans-Acting ribozymes targeting nine of the activity-selected sites were synthesized, together with ribozymes targeting four sites with a perfect match to the cleavage site consensus (sequence-selected sites). Activity-selected ribozymes have much higher cleavage activity against the long, structured RNA molecules than do sequence-selected ribozymes, although the latter are effective in cleaving oligoribonucleotides, as predicted. These results imply that, for Sindbis virus 26 S RNA, designing ribozymes based on matches to the consensus sequence may be an ineffective strategy.

P5abc domain of Tetrahymena LSU intron functions as an activator that is not essential for but enhances the activity of the ribozyme either when present in cis or when added in trans. This domain contains three regions (A-rich bulge, L5b, and L5c) that have been demonstrated to interact with the rest of the intron. Although these regions are presumably important for efficient activation, the role of each element is not understood in the mechanism of activation. We employed circularly permuted introns and examined the roles of each element. The results show that each of the three elements can activate the intron independently. We also found that a correlation between the activation by P5abc and the physical affinity of P5abc to the intron exists.

We have been developing triple ribozyme (TRz) constructs which consist of two cis-acting ribozymes flanking an internal trans-acting ribozyme, which is targeted to a cellular RNA. Actions of the two cis-acting ribozymes efficiently liberate the internal ribozyme with minimal non-specific flanking sequences. The liberated internal targeted ribozyme shows substantially greater catalytic activity than TRz preparations, constructs which cannot undergo self-liberation or than single ribozymes with flanking vector sequences. Here we construct a TRz which was targeted to retinoblastoma gene (Rb) mRNA, which cleaved Rb target RNA in vitro as expected. A number of tetracycline-regulatable clones stably transfected with the Rb-targeted TRz were developed and analyzed. The internal targeted ribozymes were efficiently liberated in vivo and the stably transfected clones showed varied reductions in Rb mRNA, which were contingent upon ribozyme expression and catalytic activity. The two clones showing major reductions in Rb mRNA (and pRb) levels (>70% reduction) showed abnormal morphology, loss of contact inhibition and the ability to grow in soft agar, as well as altered compartmentation of repetitive B2 transcripts, a phenomenon previously associated with immortalization and/or transformation. TRz constructs coupled with tissue-specific promoters should allow development of in vivo models in which Rb function is markedly reduced in a tissue-specific manner.

The antigenomic RNA of hepatitis delta virus (HDV) can form a short duplex, P2a, in which a four-nucleotide sequence within the self-cleaving domain pairs with a sequence just outside the previously defined 3′-boundary of the ribozyme. Both sequences that would participate in forming P2a were previously determined to be non-essential for self-cleavage activity. Ribozymes able to form P2a were less active than those lacking the 3′ P2a sequence when preincubated under the standard low-Na + conditions. Chemical probing of the RNA correlated base-pairing in P2a with this inhibition. Furthermore, mutagenesis and 3′ truncation experiments mapped the inhibitory sequence to P2a. However, raising the NaCl concentration in the preincubation prior to adding Mg 2+ reversed the inhibitory effect. Moreover, with NaCl preincubation, the P2a-containing ribozyme was more active than an otherwise identical ribozyme lacking the 3′ P2a sequence. Non-denaturing gels provided evidence for alternative conformations of the P2a-containing precursor with only the faster-migrating species correlating with the active form. A difference in the temperature-dependence for the rate of cleavage of the P2a-containing ribozyme with and without NaCl, together with a difference in the melting behavior of the RNA in NaCl with and without P2a, suggested that P2a favors the native structure in NaCl. Many derivatives of the HDV ribozymes form inactive conformers; however, this study reveals details of a specific structure that stabilizes both inactive and active conformations of the HDV ribozyme.

▪ Abstract The hammerhead ribozyme is a small catalytic RNA that cleaves a target phosphodiester bond in a reaction dependent on divalent metal ions. Crystal structures of the hammerhead reveal the tertiary fold of an enzymatic “ground state” of the molecule; however, they do not clarify the catalytic mechanism of the ribozyme, presumably because a significant conformational rearrangement is required to reach an enzymatic transition state. The structural domains seen in the hammerhead can be related to sequence or structural motifs in transfer and ribosomal RNAs, suggesting that they represent tertiary building blocks that will be found in large, complex RNAs.

gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione synthesis and is thought to play a significant role in intracellular detoxification systems. To specifically suppress gamma-GCS gene expression, we constructed two different hammerhead ribozymes against gamma-GCS mRNA transcripts. Two cleavage sites were targeted as follows: site 1 for anti-gamma-GCS ribozyme (H), a GUU triplet located from +348 to +350 of the gamma-GCS heavy chain, and site 2 for anti-gamma-GCS ribozyme (L), a GUU triplet located from +235 to +237 of the gamma-GCS light chain. The anti-gamma-GCS ribozymes effectively cleaved gamma-GCS mRNA in a cell-free system. When transfected into a Min-6 mouse islet cell line, these anti-gamma-GCS ribozymes not only suppressed gamma-GCS gene expression, but also reduced intracellular glutathione concentration. These results suggest that the ribozyme-mediated down-regulation of gamma-GCS gene expression may be useful for analyzing the glutathione-associated cellular defense systems of pancreatic islet cells.

In order to determine the minimum structure of the human hepatitis delta virus (HDV) ribozyme, several variants were constructed by replacing stem IV with UU, CG, A, C, G or U (MTS-N). Cleavage assays of these cis- and trans-acting shortened HDV ribozymes (cis- and trans-MTS-N) showed that almost all had cleavage activity except MTS-CG and MTS-G. Trans-MTS-CG/G seem to have different stable secondary structures from others by RNA folding program. Among them, both cis- and trans-MTS-U showed the most efficient activity. As a result of point mutations of trans-MTS-U in single-stranded regions, there are more strict base requirements at that position than that of cis-acting HDV ribozyme (HDV88). Therefore, stem IV plays a role to support an active conformation, and it is not directly involved in the catalytic core of the HDV ribozyme. Both cis- and trans-acting MTS-U are, to our knowledge, the smallest HDV ribozyme thus reported.

Trans-acting hammerhead ribozymes are usually efficient in cleaving short RNA model substrates under both single-turnover and multiple-turnover conditions. In contrast, when long RNAs are the substrates, the cleavage efficiency of these ribozymes decreases, including a loss of multiple-turnover activity in many cases. Since target substrates for potential therapeutical purposes are mostly long RNAs, a multiple-turnover cleavage of long RNAs would essentially increase the efficiency of hammerhead ribozymes. Therefore, we explored if oligonucleotide facilitators, capable of enhancing multiple-turnover activity with short substrates, can also affect or cause multiple turnover with long substrates. We examined the effects of 12-base and 24-base oligonucleotide facilitators on the multiple-turnover activity with substrates of different length containing 39-, 452- and 942-base sequences of the human tissue factor (HTF) mRNA. In the absence of facilitator, the ribozyme cleaved only the 39-base substrate with multiple-turnover activity, but not the long 452-base and 942-base substrates. However, facilitator addition enabled the ribozyme to cleave even the 452-base and the 942-base substrates with multiple-turnover activity. All facilitators tested showed a remarkable activating effect with the long substrates. The data demonstrate that a hammerhead ribozyme which, by itself, can only act as a single-turnover catalyst with long substrates, can be switched by facilitators into a multiple-turnover catalyst. Thus, the inactivation of long target RNAs in multiple-turnover reactions may be achieved by addition of oligonucleotide facilitators.

The selective inactivation of genes, in a tissue-specific or temporally controlled manner, is now an important requirement for the analysis of nervous system development and function. Hammerhead ribozymes--catalytic RNA enzymes that specifically bind to and then cleave target RNAs--may provide a way to meet this requirement, particularly for organisms in which gene inactivation by homologous recombination is not feasible. However, ribozyme application has to some extent been hampered by limited knowledge as to the base-pairing accessibility of RNA target sites in vivo. In an attempt to circumvent this limitation, we have used a computer program based on free energy minimization to predict secondary structures for 128 RNA sequences for which corresponding ribozymes or antisense oligonucleotides have been synthesized, tested, and reported in the literature. A comparative analysis of the predicted structures of these targets with the reported efficacy of their corresponding antisense reagents has allowed us to formulate a set of rules for the rational choice of hammerhead ribozyme flanking arms and cleavage sites.