A cis and trans adenine-dependent hairpin ribozyme against Tpl-2 target

Ribozymes are catalytic RNAs that possess the property of cutting an RNA target via site-specific cleavage after sequence-specific recognition. Ribozymes can moreover cleave multiple substrate molecules. An increasing number of studies show that ribozymes are particularly well adapted tools against cancer, silencing or down-regulating gene expression at the RNA level. We have constructed an adenine-dependent hairpin ribozyme that cleaves the sequence at nucleotides A(225)(downward arrow)G(226) relative to the start codon of translation of the Tpl-2 kinase mRNA; this serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in breast cancer. An adenine-dependent hairpin ribozyme 1 (ADHR1) was previously isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure. Switch on/switch off ribozymes are particularly useful since high amounts of stable ribozyme can be produced in the absence of adenine and the ribozyme specifically cleaves its target in the presence of adenine. The ADHR1 target sequence was replaced by a sequence derived from the Tpl-2 kinase mRNA. The resulting Tpl-2 ribozyme is active in cis cleavage: kinetic studies have been performed as a function of Mg2+ concentration, adenine concentration, as well as at different pH and with various cofactors. Finally, the Tpl-2 ribozyme was shown to cleave its target in trans successfully. These findings demonstrate that a potential therapeutic ribozyme can be produced by simple sequence modification.     

The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.     

RNA double cleavage by a hairpin-derived twin ribozyme

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.     

The many faces of the hairpin ribozyme: structural and functional variants of a small catalytic RNA

The hairpin ribozyme is a small catalytic RNA that has been reengineered resulting in a number of variants with extended or even new functions. Thus, manipulation of the hairpin ribozyme structure has allowed for activity control by external effectors, namely oligonucleotides, flavine mononucleotide, and adenine. Hairpin ribozyme-derived twin ribozymes that mediate RNA fragment exchange reactions as well as self-processing hairpin ribozymes were designed. Furthermore, several hairpin ribozyme variants have been engineered for knock down of specific RNA substrates by adapting the substrate-binding domain to the specific target sequence. This review will focus on hairpin ribozymes possessing structural extensions/variations and thus functionally differing from the parent hairpin ribozyme.     

Ribozymes in the age of molecular therapeutics

Ribozymes are RNA molecules capable of sequence-specific cleavage of other RNA molecules. Since the discovery of the first group I intron ribozyme in 1982, new classes of ribozymes, each with their own unique reaction, target site specifications, and potential applications, have been identified. These include hammerhead, hairpin, hepatitis delta, varkud satellite, groups I and II intron, and RNase P ribozymes, as well as the ribosome and spliceosome. Meanwhile, ribozyme engineering has enabled the in vitro selection of synthetic ribozymes with unique properties. This, along with advances in ribozyme delivery methods and expression systems, has led to an explosion in the potential therapeutic applications of ribozymes, whether for anti-cancer or anti-viral therapy, or for gene repair.     

Structure and function of the hairpin ribozyme

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.     

Self-association of adenine-dependent hairpin ribozymes

Hairpin ribozymes are flexible molecules that catalyse reversible self-cleavage after the docking of two independently folded internal loops, A and B. The activities, self-association and structures in solution of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) were studied by native gel electrophoresis, analytical centrifugation, and small angle neutron scattering. Bi-molecular RNA interactions such as linear–linear, loop–loop, loop–linear or kissing interactions have been found to be important in the control of various biological functions, and hairpin loops present rich potential for establishing both intra- and intermolecular interactions through standard Watson-Crick base pairing or non-canonical interactions. Similar results were obtained for ADHR1 and ADHR2. At room temperature, they indicated end-to-end self-association of the ribozymes in rod-like structures with a cross-section corresponding to two double strands side-by-side. Dimers, which predominate at low concentration (∼0.1 mg/ml), associate into longer rods, with increasing concentration (∼1 mg/ml). Above 65°C, the dimers and rods dissociated into compact monomers, with a radius of gyration similar to that of tRNA (about 70 bases). The dimers were non-active for catalysis, which suggests that dimer formation, probably by preventing the correct docking of loops A and B, could act as an inhibition mechanism for the regulation of hairpin ribozyme catalysis.     

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.     

Tertiary structure stabilization promotes hairpin ribozyme ligation.

The hairpin ribozyme catalyzes a reversible RNA cleavage reaction that participates in processing intermediates of viral satellite RNA replication in plants. A minimal hairpin ribozyme consists of two helix-loop-helix segments. These segments associate noncoaxially in the active folded structure in a way that brings catalytically important loop nucleotides into close proximity. The hairpin ribozyme in the satellite RNA of Tobacco Ringspot Virus assembles in the context of a four-way helical junction. Recent physical characterization of hairpin ribozyme structures using fluorescence resonance energy transfer demonstrated enhanced stability of the folded structure in the context of a four-way helical junction compared to minimal hairpin ribozyme variants. Analysis of the functional consequences of this modification of the helical junction has revealed two changes in the hairpin ribozyme kinetic mechanism. First, ribozymes with a four-way helical junction bind 3' cleavage products with much higher affinity than minimal hairpin ribozymes, evidence that tertiary interactions within the folded structure contribute to product binding energy. Second, the balance between ligation and cleavage shifts in favor of ligation. The enhanced ligation activity of hairpin ribozymes that contain a four-way helical junction supports the notion that tertiary structure stability is a major determinant of the hairpin ribozyme proficiency as a ligase and illustrates the link between RNA structure and biological function.     

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

The active centers of the hairpin and VS ribozymes are both generated by the interaction of two internal loops, and both ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid-base catalysis involving the adenine (A38) and guanine (G8). It appears that the two ribozymes are mechanistically equivalent.     

Hydrostatic and osmotic pressure study of the hairpin ribozyme

The recent discovery of numerous catalytically active RNAs in various living species as well as the in vitro selection of a large series of RNA aptamers able to bind specifically various molecules such as metabolites and co-factors, emphasize the adaptability of RNAs through the plasticity of their secondary structure. Furthermore, all these observations give support to the "RNA world" hypothesis as a step in the primitive development of life on Earth. On this background, we used high pressure to study the mechanism of action of a model hairpin ribozyme which exhibits self-cleavage and ligation. The activation volume (DeltaV( not equal)) of the cleavage reaction (34+/-4 ml/mol) indicates that an important compaction of the RNA molecule occurs during the reaction and must be accompanied by a significant movement of water molecules . Indeed, such a release of 78+/-4 water molecules per RNA molecule could be measured by complementary osmotic shock experiments. These results are consistent with the information provided by the structural studies which indicate that two loops of the RNA molecule should come into contact for the reaction to occur .The high pressure study of a modified form of the ribozyme whose activity is strictly dependent on the presence of adenine as a co-factor should bring some information about the structural significance of this important DeltaV( not equal) of activation.     

Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse-joined hairpin ribozyme units

In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into useful tools. Work in our laboratory has focused on the development of twin ribozymes for site-specific RNA sequence alteration. To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme). Here, we present reverse-joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation. The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min(-1) and a final yield of 70%. We combined a reverse-joined hairpin ribozyme with a conventional hairpin ribozyme to create a twin ribozyme that mediates the insertion of four additional nucleotides into a predetermined position of a substrate RNA, and thus mimics, at the RNA level, the repair of a short deletion mutation; 17% of the initial substrate was converted to the insertion product.     

The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure

The discovery of ribozymes strengthened the RNA world hypothesis, which assumes that these precursors of modern life both stored information and acted as catalysts. For the first time among extensive studies on ribozymes, we have investigated the influence of hydrostatic pressure on the hairpin ribozyme catalytic activity. High pressures are of interest when studying life under extreme conditions and may help to understand the behavior of macromolecules at the origins of life. Kinetic studies of the hairpin ribozyme self-cleavage were performed under high hydrostatic pressure. The activation volume of the reaction (34 ± 5 ml/mol) calculated from these experiments is of the same order of magnitude as those of common protein enzymes, and reflects an important compaction of the RNA molecule during catalysis, associated to a water release. Kinetic studies were also carried out under osmotic pressure and confirmed this interpretation and the involvement of water movements (78 ± 4 water molecules per RNA molecule). Taken together, these results are consistent with structural studies indicating that loops A and B of the ribozyme come into close contact during the formation of the transition state. While validating baro-biochemistry as an efficient tool for investigating dynamics at work during RNA catalysis, these results provide a complementary view of ribozyme catalytic mechanisms.     

Sequence elements outside the catalytic core of natural hairpin ribozymes modulate the reactions differentially

Abstract Hairpin ribozymes occur naturally only in the satellite RNAs of tobacco ringspot virus (TRsV), chicory yellow mottle virus (CYMoV) and arabis mosaic virus (ArMV). The catalytic centre of the predominantly studied sTRsV hairpin ribozyme, and of sArMV is organised around a four-way helical junction. We show here that sCYMoV features a five-way helical junction instead. Mutational analysis indicates that the fifth stem does not influence kinetic parameters of the sCYMoV hairpin ribozyme in vitro reactions, and therefore seems an appendix to that junction in the other ribozymes. We report further that all three ribozymes feature a three-way helical junction outside the catalytic core in stem A, with Watson-Crick complementarity to loop nucleotides in stem B. Kinetic analyses of cleavage and ligation reactions of several variants of the sTRsV and sCYMoV hairpin ribozymes in vitro show that the presence of this junction interferes with their reactions, particularly the ligation. We provide evidence that this is not due to a presumed interaction of the afore-mentioned elements in stems A and B. The evolutionary survival of this cis-inhibiting element seems rather to be caused by the coincidence of its position with that of the hammerhead ribozyme in the other RNA polarity.     

Functional analysis of hairpin ribozyme active site architecture

The hairpin ribozyme is a small catalytic motif found in plant satellite RNAs where it catalyzes a reversible self-cleavage reaction during processing of replication intermediates. Crystallographic studies of hairpin ribozymes have provided high resolution views of the RNA functional groups that comprise the active site and stimulated biochemical studies that probed the contributions of nucleobase functional groups to catalytic chemistry. The dramatic loss of activity that results from perturbation of active site architecture points to the importance of positioning and orientation in catalytic rate acceleration. The current study focuses on the network of noncovalent interactions that align nucleophilic and leaving group oxygens in the orientation required for the S(N)2-type reaction mechanism and orient the active site nucleobases near the reactive phosphate to facilitate catalytic chemistry. Nucleotide modifications that alter or eliminate individual hydrogen bonding partners had different effects on the activation barrier to catalysis, the stability of ribozyme complexes in the ground state, and the internal equilibrium between cleavage and ligation of bound products. Furthermore, substitution of hydrogen bond donors and acceptors with seemingly equivalent pairs sometimes had very different functional consequences. These biochemical analyses augment high resolution structural information to provide insights into the functional significance of active site architecture.     

Catalysis by the nucleolytic ribozymes

The nucleolytic ribozymes use general acid-base catalysis to contribute significantly to their rate enhancement. The VS (Varkud satellite) ribozyme uses a guanine and an adenine nucleobase as general base and acid respectively in the cleavage reaction. The hairpin ribozyme is probably closely similar, while the remaining nucleolytic ribozymes provide some interesting contrasts.     

Examination of the structural and functional versatility of glmS ribozymes by using in vitro selection

Self-cleaving ribozymes associated with the glmS genes of many Gram-positive bacteria are activated by binding to glucosamine-6-phosphate (GlcN6P). Representatives of the glmS ribozyme class function as metabolite-sensing riboswitches whose self-cleavage activities down-regulate the expression of GlmS enzymes that synthesizes GlcN6P. As with other riboswitches, natural glmS ribozyme isolates are highly specific for their target metabolite. Other small molecules closely related to GlcN6P, such as glucose-6-phosphate, cannot activate self-cleavage. We applied in vitro selection methods in an attempt to identify variants of a Bacillus cereus glmS ribozyme that expand the range of compounds that induce self-cleavage. In addition, we sought to increase the number of variant ribozymes of this class to further examine the proposed secondary structure model. Although numerous variant ribozymes were obtained that efficiently self-cleave, none exhibited changes in target specificity. These findings are consistent with the hypothesis that GlcN6P is used by the ribozyme as a coenzyme for RNA cleavage, rather than an allosteric effector.     

Cross-ligation and exchange reactions catalyzed by hairpin ribozymes.

The negative strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) contains a hairpin catalytic domain that shows self-cleavage and self-ligation activities in the presence of magnesium ions. We describe here that the minimal catalytic domain can catalyze a cross-ligation reaction between two kinds of substrates in trans. The cross-ligated product increased when the reaction temperature was decreased during the reaction from 37 degrees C to 4 degrees C. A two-stranded hairpin ribozyme, divided into two fragments between G45 and U46 in a hairpin loop, showed higher ligation activity than the nondivided ribozyme. The two stranded ribozyme also catalyzed an exchange reaction of the 3'-portion of the cleavage site.     

Mechanisms of RNA catalysis

Ribozymes are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. The nucleolytic ribozymes comprise a class of five structurally-distinct species that bring about site-specific cleavage by nucleophilic attack of the 2'-O on the adjacent 3'-P to form a cyclic 2',3'-phosphate. In general, they will also catalyse the reverse reaction. As a class, all these ribozymes appear to use general acid-base catalysis to accelerate these reactions by about a million-fold. In the Varkud satellite ribozyme, we have shown that the cleavage reaction is catalysed by guanine and adenine nucleobases acting as general base and acid, respectively. The hairpin ribozyme most probably uses a closely similar mechanism. Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes.     

Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme

Several small ribozymes carry out self-cleavage at a specific phosphodiester bond to yield 2',3'-cyclic phosphate and 5'-hydroxyl termini. Prior mechanistic and structural studies on the HDV ribozymes led to the proposal that the pK(a) of C75 is shifted toward neutrality, making it an effective general acid. Recent mechanistic studies on the hairpin ribozyme have led to models in which protonation of G8 is required for phosphodiester cleavage, either for general acid catalysis or for electrostatic stabilization. Inspection of recent crystal structures of the hairpin ribozyme, including a complex with a vanadate transition state mimic, suggests an alternative model involving general acid-base catalysis with G8 serving as the general base and A38 as the general acid. This model is consistent with the literature on the hairpin ribozyme, including pH-rate profiles of wild-type and mutant ribozymes and solvent isotope effects. General mechanistic considerations for RNA catalysis suggest that the penalty for having general acids and bases with pK(a)s removed from neutrality is not as severe as expected. These considerations suggest that general acid-base catalysis may be a common mechanistic strategy of RNA enzymes.