Cross-catalytic replication of an RNA ligase ribozyme

A self-replicating RNA ligase ribozyme was converted to a cross-catalytic format whereby two ribozymes direct each other's synthesis from a total of four component substrates. Each ribozyme binds two RNA substrates and catalyzes their ligation to form the opposing ribozyme. The two ribozymes are not perfectly complementary, as is the case for replicating nucleic acid genomes in biology. Rather, the ribozymes contain both template elements, which are complementary, and catalytic elements, which are identical. The specificity of the template interactions allows the cross-catalytic pathway to dominate over all other reaction pathways. As the concentration of the two ribozymes increases, the rate of formation of additional ribozyme molecules increases, consistent with the overall autocatalytic behavior of the reaction system.     

Generalized RNA-directed recombination of RNA

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.     

Novel RNA catalysts for the Michael reaction

BACKGROUND: In vitro selected ribozymes with nucleotide synthase, peptide and carbon-carbon bond forming activity provide insight into possible scenarios on how chemical transformations may have been catalyzed before protein enzymes had evolved. Metabolic pathways based on ribozymes may have existed at an early stage of evolution. RESULTS: We have isolated a novel ribozyme that mediates Michael-adduct formation at a Michael-acceptor substrate, similar to the rate-limiting step of the mechanistic sequence of thymidylate synthase. The kinetic characterization of this catalyst revealed a rate enhancement by a factor of approximately 10(5). The ribozyme shows substrate specificity and can act as an intermolecular catalyst which transfers the Michael-donor substrate onto an external 20-mer RNA oligonucleotide containing the Michael-acceptor system. CONCLUSION: The ribozyme described here is the first example of a catalytic RNA with Michael-adduct forming activity which represents a key mechanistic step in metabolic pathways and other biochemical reactions. Therefore, previously unforeseen RNA-evolution pathways can be considered, for example the formation of dTMP from dUMP. The substrate specificity of this ribozyme may also render it useful in organic syntheses.     

Isolation of fast purine nucleotide synthase ribozymes

Here we report the in vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6-thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine ((6S)Gua). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M(-1) min(-1) and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a K(m) of approximately 80 muM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an 'RNA World' and provides a context from which to study small molecule RNA catalysis.     

Generation and development of RNA ligase ribozymes with modular architecture through "design and selection"

In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed "design and selection," new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection.     

RNA-catalyzed RNA ligation on an external RNA template

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside 5'-triphosphates onto the 3' end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.     

Peptidyl-transferase ribozymes: trans reactions,structural characterization and ribosomal RNA-like features

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.     

Single-cell detection of trans-splicing ribozyme in vivo activity

The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants.     

Rational design of allosteric ribozymes

Background: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes.Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created.Conclusions: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation — a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.     

Ligand requirements for glmS ribozyme self-cleavage

Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage. Herein, we demonstrate that ligand is absolutely requisite for glmS ribozyme self-cleavage activity. Furthermore, catalysis both requires and is dependent upon the acid dissociation constant (pKa) of the amine functionality of GlcN6P and related compounds. The data demonstrate that ligand is integral to catalysis, consistent with a coenzyme role for GlcN6P and illustrating an expanded capacity for biological RNA catalysis.     

A general RNA-capping ribozyme retains stereochemistry during cap exchange

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.     

In Vitro Selection of Specific Ligand-binding Nucleic Acids

In vitro selection is a method that allows the simultaneous screening of very large numbers of nucleic acid molecules for a wide range of properties from binding characteristics to catalytic properties; moreover, the isolation of the very rare functional molecules becomes possible. Binding sites between proteins and nucleic acids, for example, have been evaluated by this methodology in order to gain information about protein/nucleic acid interactions. Structure and function of catalytic RNA (“ribozymes”) has been studied by in vitro selection and has led to new ribozymes with improved catalytic function. Substrate specificity of catalytic RNA has been changed and has led to a ribozyme that cleaves DNA. Other applications include the isolation of nucleic acids that bind specifically to small organic molecules and of RNA molecules that form triple helices with double-stranded DNA. In this article we discuss the background, design, and results of in vitro genetic experiments, which bridge biochemical/molecular biological and organic chemical approaches to molecular recognition.     

A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage

Background: Ribozymes are biological catalysts that promote the hydrolysis and transesterification of phosphate diesters of RNA. They typically require divalent magnesium ions for activation, although it has proven difficult to differentiate structural from catalytic roles for the magnesium ions and to identify the molecular mechanism of catalysis. Direct inner-sphere coordination is usually invoked in the catalytic step, although there is no evidence to support the generality of such a pathway for all ribozymes.Results: We studied the catalytic pathway for the hairpin class of ribozyme. The substitutionally inert transition metal complex cobalt hexaammine [Co(NH₃)₆³⁺) was shown to be as active as Mg²⁺(aq) in promoting hairpin ribozyme activity, demonstrating that inner-sphere pathways are not used by this class of ribozyme. These results were confirmed by studies with R_P- and S_P-phosphorothioate substrate analogs which show a similar reactivity to that of the native substrate towards the magnesium-activated ribozyme. Monovalent cations enhance the activity of Co(NH₃)₆³⁺-promoted reactions, but inhibit Mg²⁺-activated catalysis, demonstrating a requirement for hydrated cations at several key sites in the ribozyme.Conclusions: These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pK_a values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.     

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

Background: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5′-5′-pyrophosphate ‘capped’ RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5′-monophosphate (AMP) may be a vestige of ‘RNA world’ catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated.Results: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10¹⁵ RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5′-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of ∼ 5 × 10⁵ over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3′-5′-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.Conclusions: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.     

Continuous in vitro evolution of a ribozyme that catalyzes three successive nucleotidyl addition reactions

Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5'-->3' or a 3'-->5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.     

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.     

A molecular description of the evolution of resistance

BACKGROUND: In vitro evolution has been used to obtain nucleic acid molecules with interesting functional properties. The evolution process usually is carried out in a stepwise manner, involving successive rounds of selection, amplification and mutation. Recently, a continuous in vitro evolution system was devised for RNAs that catalyze the ligation of oligonucleotide substrates, allowing the evolution of catalytic function to be studied in real time. RESULTS: Continuous in vitro evolution of an RNA ligase ribozyme was carried out in the presence of a DNA enzyme that was capable of cleaving, and thereby inactivating, the ribozyme. The DNA concentration was increased steadily over 33.5 hours of evolution, reaching a final concentration that would have been sufficient to inactivate the starting population in one second. The evolved population of ribozymes developed resistance to the DNA enzyme, reducing their vulnerability to cleavage by 2000-fold but retaining their own catalytic function. Based on sequencing and kinetic analysis of the ribozymes, two mechanisms are proposed for this resistance. One involves three nucleotide substitutions, together with two compensatory mutations, that alter the site at which the DNA enzyme binds the ribozyme. The other involves enhancement of the ribozyme's ability to bind its own substrate in a way that protects it from cleavage by the DNA enzyme. CONCLUSIONS: The ability to direct the evolution of an enzyme's biochemical properties in response to the behavior of another macromolecule provides insight into the evolution of resistance and may be useful in developing enzymes with novel or enhanced function.     

Relationship between catalytic activity and secondary structure of a hammerhead ribozyme: A study using thermodynamic parameters for RNA structure prediction

Abstract

The stabilization energy for the secondary structures of wild-type hammerhead and mutant ribozymes has been calculated at different salt conditions and temperatures by using the thermodynamic parameters for RNA structure prediction. The most stable structure at each condition has been searched and the obtained secondary structure is compared with the structure suggested phylogenetically or experimentally. The results indicate that the hammerhead-type secondary structure of the ribozyme and its reactivity correlate with each other. The multibranched loop containing the self-cleavage site of the ribozyme particularly should be a key structure in the hammerhead ribozyme reaction. The predicted secondary structures also suggest that the reactivity of the hammerhead ribozyme should be very much lower at 10°C than that at 37°C.