Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5' end of the substrate to the 3' end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.     

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems.     

Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).     

Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme

The free energy of substrate binding to the hammerhead ribozyme was compared for 10 different hammerheads that differed in the length and sequence of their substrate recognition helices. These hammerheads were selected because neither ribozyme nor substrate oligonucleotide formed detectable alternate secondary structures. The observed free energies of binding varied from -8 to -24 kcal/mol and agreed very well with binding energies calculated from the nearest-neighbor free energies if a constant energetic penalty of DeltaG degreescore = +3.3 +/- 1 kcal/mol is used for the catalytic core. A set of substrates that contained a competing hairpin secondary structure showed weaker binding to the ribozyme by an amount consistent with the predicted free energy for hairpin formation. These thermodynamic conclusions permit the prediction of substrate binding affinities for ribozyme-substrate pairs of any helix length and sequence, and thus, should be very valuable for the rational design of ribozymes directed toward gene inactivation.     

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In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group I ribozyme to obtain individuals with a 10(5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approximately 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiments confirmed the selective advantage of two covarying mutations within the catalytic core of the ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average of 28 mutations relative to the wild-type that are responsible for the altered phenotype.     

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core.     

In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity

NanGIR1 is a member of a new class of group I ribozymes whose putative biological function is site-specific hydrolysis at an internal processing site (IPS). We have previously shown that NanGIR1 requires 1 M KCl for maximal activity, which is nevertheless slow (0.03 min(-1)). We used in vitro selection and an RNA pool with approximately nine mutations per molecule to select for faster hydrolysis at the IPS in 100 mM KCl. After eight rounds of selection, GIR1 variants were isolated that catalyzed hydrolysis at 300-fold greater rates than NanGIR1 RNA. Although not required by the selection, many of the resultant RNAs had increased thermal stability relative to the parent RNA, and had a more compact structure as evidenced by their faster migration in native gels. Although a wide spectrum of mutations was found in generation 8 clones, only two mutations, U149C and U153C, were common to greater than 95% of the molecules. These and one other mutation, G32A, are sufficient to increase activity 50-fold. All three mutations lie within or proximal to the P15 pseudoknot, a structural signature of GIR1 RNAs that was previously shown to be important for catalytic activity. Overall, our findings show that variants of the Naegleria GIR1 ribozyme with dramatically improved activity lie very close to the natural GIR1 in sequence space. Furthermore, the selection for higher activity appeared to select for increased structural stability.     

Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme

Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HCl buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex--analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HCl buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural variation induced by different nucleotides at the cleavage site of the hammerhead ribozyme

The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compared with the X-ray structure of the hammerhead ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.     

Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking

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.     

Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors

The sarcin/ricin domain in 23 S/28 S rRNA is crucial for ribosome function, since it constitutes at least part of the binding site for the elongation factors and hence is essential for binding aminoacyl-tRNA and for translocation. The domain is also the site of action of ricin and sarcin and analysis of the effect of mutations in the RNA on recognition by the cytotoxins has helped to define the structure and to understand the function of the region. We have constructed deletions, separately, of pairs of non-conserved, juxtaposed but non-hydrogen-bonded nucleotides that correspond to C4317 and C4331, and to U4316 and C4332, in an oligoribonucleotide that mimics the sarcin/ricin domain in rat 28 S rRNA. The deletions had no effect on the depurination of A4324 by ricin nor on the cleavage of the phosphodiester bond on the 3' side of G4325 by sarcin. However, simultaneous deletion of the four nucleotides decreased cleavage by sarcin but did not affect depurination by ricin. Removal of the non-canonical A4318.A4330 pair abolished recognition by both toxins. Deletion from oligoribonucleotides, that reproduce the sarcin/ricin domain of Escherichia coli 23 S rRNA, of U2653 and C2667 (equivalent to U4316, C4317 and C4331, C4332 in 28 S rRNA), or substitution of guanosine for U2653 (designed to form a Watson-Crick G2653.C2667 pair), reduced cleavage by sarcin whereas depurination by ricin was slightly increased. An increase in the stability of the mutant oligoribonucleotides may be the basis of the impairment in sarcin action. The tm for the wild-type RNA is 60 degreesC; for the double-deletion mutant U2653Delta/C2667Delta it is 65 degreesC; and for the U2653G transversion it is 69 degreesC. Expression of a mutant 23 S rRNA gene lacking U2653 and C2667 is lethal and a U2653G transversion mutation impairs growth. The mutant ribosomes are less active in protein synthesis than the wild-type and ribosomes with the U2653G mutation are resistant to sarcin. The binding of EF-G to oligoribonucleotides with a U2653/C2667 double deletion is reduced and an effect on the affinity of the factor for the sarcin/ricin domain may account in part for the decrease in ribosome efficiency. The results stress the potential importance in rRNA structure and function of non-conserved nucleotides, and suggest that the sarcin/ricin domain in ribosomes requires a region of structural flexibility for optimal efficiency.     

In vitro selection for altered divalent metal specificity in the RNase P RNA

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.     

Substrate specificity of delta ribozyme cleavage

The specificity of delta ribozyme cleavage was investigated using a trans-acting antigenomic delta ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min-1, an apparent Km of 17.9 nM and an apparent second-order rate constant of 1.89 x 10(7) min-1 M-1. The substrate specificity of the delta ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of delta ribozyme cleavage.     

Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme

The L-21 ScaI ribozyme catalyzes sequence-specific cleavage of an oligonucleotide substrate. Cleavage is preceded by base pairing of the substrate to the internal guide sequence (IGS) at the 5' end of the ribozyme to form a short RNA duplex (P1). Tertiary interactions between P1 and the catalytic core dock P1 into the active site of the ribozyme. These include interactions between the catalytic core and 2'-hydroxyls of the substrate at nucleotide positions -3u and perhaps -2c. In this study, 2'-hydroxyls of the IGS strand that contribute to P1 recognition by the ribozyme are identified. IGS 2'-hydroxyls (nucleotide positions 22-27) were individually modified to either 2'-deoxy or 2'-methoxynucleotides within full-length semisynthetic L-21 ScaI ribozymes generated using T4 DNA ligase. Thermodynamic and kinetic characterization of the resulting IGS variant ribozymes justify the following conclusions: (i) 2'-Hydroxyls at nucleotide positions G22 and G25 play a critical energetic role in docking P1 into the catalytic core, contributing 2.6 and 2.1 kcal.mol-1, respectively. (ii) The loss of binding energy is manifest primarily as an increase in the rate of dissociation. Because turnover for the wild-type ribozyme is limited by product dissociation, G22 and G25 deoxy variants display up to a 20-fold increase in the multiple-turnover rate at saturating substrate. (iii) IGS tertiary interactions are energetically coupled with the tertiary interactions made to the substrate, consistent with P1 becoming undocked from its binding site in J8/7 upon substitution of either the G22 or G25 2'-hydroxyl. (iv) The G22 deoxy variant loses energetic coupling between guanosine and substrate binding, suggesting that in this variant the P1 helix is also undocked from its binding site in J4/5, the proposed site of guanosine and substrate interaction. Therefore, in combinations with previous studies four P1 2'-hydroxyls are implicated as important for docking. The contributions of the 2'-hydroxyl tertiary interactions are not equivalent and follow the hierarchical order G22 > G25 > -3u > -2c. Because the G22 2'-hydroxyl appears to mediate P1 docking into both J8/7 and J4/5, it may serve as the molecular linchpin for the recognition of P1 by the catalytic core.     

Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb

Terbium(III) [Tb(III)] was shown to inhibit the hammerhead ribozyme by competing with a single magnesium(II) ion. X-ray crystallography revealed that the Tb(III) ion binds to a site adjacent to an essential guanosine in the catalytic core of the ribozyme, approximately 10 angstroms from the cleavage site. Synthetic modifications near this binding site yielded an RNA substrate that was resistant to Tb(III) binding and capable of being cleaved, even in the presence of up to 20 micromolar Tb(III). It is suggested that the magnesium(II) ion thought to bind at this site may act as a switch, affecting the conformational changes required to achieve the transition state.     

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.     

Delta ribozyme has the ability to cleave in transan mRNA

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.     

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.     

Hammerhead ribozymes targeted to the FBN1 mRNA can discriminate a single base mismatch between ribozyme and target

Hammerhead ribozymes are catalytic RNA molecules that can act in trans, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome. The majority of mutations are single-base changes, many of which exert their effect via a dominant-negative mechanism. Previously we have shown that an antisense hammerhead ribozyme, targeted to the FBN1 mRNA can reduce deposition of fibrillin to the extracellular matrix of cultured fibroblasts, suggesting it may be possible to utilize ribozymes to down regulate the production of mutant protein and thus restore normal fibrillin function. This might be achieved by the mutation creating a ribozyme cleavage site that is not present in the normal allele, however this is likely to limit the number of mutations that could be targeted. Alternatively, it might be possible to target the mutant allele via the ribozyme binding arms. To determine the potential of ribozymes to preferentially target mutant FBN1 alleles via the latter approach, the effect of mismatches in helix I of a hammerhead ribozyme, on the cleavage of fibrillin (FBN1) mRNA was investigated. A single base mismatch significantly reduced ribozyme cleavage efficiency both in vitro and in vivo. The discrimination between fully-matched and mismatched ribozyme varied with the length of helix I, with the discrimination being more pronounced in ribozymes with a shorter helix. These data suggest that it should be possible to design hammerhead ribozymes that can discriminate between closely related (mutant and normal) target RNAs varying in as little as a single nucleotide, even if the mutation does not create a ribozyme cleavage site.