Influence of threose nucleoside units on the catalytic activity of a hammerhead ribozyme

TNA (alpha-L-threose nucleic acids) is potentially a natural nucleic acid, that might have acted as an evolutionary alternative of RNA. We determined the catalytic activity of hammerhead ribozymes containing a threofuranosyl-modified nucleoside at position U4 and U7, and compared these results with those obtained from HNA (hexitol nucleic acids) insertion into the same ribozyme. Our experiments showed that, although the threofuranosyl-modified ribozymes still cleave the substrate strand, cleavage activity is highly decreased. It, therefore, seems that TNA can play a functional role in the RNA world, but only to a limited extent.     

Ribozymes: recent advances in the development of RNA tools

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.     

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

BACKGROUND: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. RESULTS: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. CONCLUSIONS: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.     

Zeptomole detection of a viral nucleic acid using a target-activated ribozyme

We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.     

Ribozyme speed limits

The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2'-oxygen atom carries out an SN2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.     

Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.     

RNA-catalyzed carbon-carbon bond formation.

RNA molecules with catalytic properties have been isolated by in vitro selection from combinatorial libraries. A broad range of chemical reactions can be catalyzed, and nucleic acids can accelerate bond formation between small organic substrates. The catalytic performance of nucleic acids can be enhanced by incorporation of additional functional groups. This minireview focuses on carbon-carbon bond formation accelerated by in vitro selected ribozymes.     

Applications of nucleic acid technology in the CNS

This review examines applications of nucleic acid technology in the form of catalytic nucleic acids (ribozymes and DNAzymes) and RNA interference (RNAi) in the CNS. The basic mechanism of catalytic nucleic acids and RNAi is reviewed, and potentials and problems highlighted. Recent advances in chemical modifications and delivery techniques are summarized. Applications in the CNS, including their use in primary neuronal cells, organotypic slice culture and the brain in vivo are further discussed.     

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.     

Chemical models for ribozyme action

Mechanistic studies of the action of catalytic ribonucleic acids, ribozymes, are highly challenging, because even a slight structural change can dramatically affect the chain folding. This, in turn, alters the binding properties of the catalytic core, making identification of the real origin of the observed influence on rate difficult. Unambiguous structure-reactivity correlations based on studies with structurally simplified chemical models may help to distinguish between alternative mechanistic interpretations. The results of such model studies are reviewed. The topics include intramolecular cleavage of RNA phosphodiester bonds by solvent-derived species, general acids/bases and metal ions, effect of molecular environment on their hydrolytic stability and trinucleoside monophosphates as models for large ribozymes.     

In vitro selection of adenine-dependent hairpin ribozymes

Adenine-dependent hairpin ribozymes were isolated by in vitro selection from a degenerated hairpin ribozyme population. Two new adenine-dependent ribozymes catalyze their own reversible cleavage in the presence of free adenine. Both aptamers have Mg(2+) requirements for adenine-assisted cleavage similar to the wild-type hairpin ribozyme. Cleavage kinetics studies in the presence of various other small molecules were compared. The data suggest that adenine does not induce RNA self-cleavage in the same manner for both aptamers. In addition, investigations of pH effects on catalytic rates show that both adenine-dependent aptamers are more active in basic conditions, suggesting that they use new acid/base catalytic strategies in which adenine could be involved directly. The discovery of hairpin ribozymes dependent on adenine for their reversible self-cleavage presents considerable biochemical and evolutionary interests because we show that RNA is able to use exogenous reactive molecules to enhance its own catalytic activity. Such a mechanism may have been a means by which the ribozymes of the RNA world enlarged their chemical repertoire.     

Generation and selection of ribozyme variants with potential application in protein engineering and synthetic biology

Over the past two decades, RNA catalysis has become a major topic of research. On the one hand, naturally occurring ribozymes have been extensively investigated concerning their structure and functional mechanisms. On the other hand, the knowledge gained from these studies has been used to engineer ribozyme variants with novel properties. In addition to RNA engineering by means of rational design, powerful techniques for selection of ribozymes from large pools of random sequences were developed and have been widely used for the generation of functional nucleic acids. RNA as catalyst has been accompanied by DNA, and nowadays a large number of ribozymes and deoxyribozymes are available. The field of ribozyme generation and selection has been extensively reviewed. With respect to the field of biotechnology, RNA and DNA catalysts working on peptides or proteins, or which are designed to control protein synthesis, are of utmost importance and interest. Therefore, in this review, we will focus on engineered nucleic acid catalysts for peptide synthesis and modification as well as for intracellular control of gene expression.     

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.     

Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme

Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid-base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme-substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions

The glmS ribozyme is a riboswitch class that occurs in certain Gram-positive bacteria, where it resides within mRNAs encoding glucosamine 6-phosphate synthase. Members of this self-cleaving ribozyme class rapidly catalyze RNA transesterification upon binding GlcN6P, and genetic evidence suggests that this cleavage event is important for down-regulating GlmS protein expression. In this report, we present a refined secondary structure model of the glmS ribozyme and determine the importance of a conserved pseudoknot structure for optimal ribozyme function. Analyses of deletion constructs demonstrate that the pseudoknot, together with other structural elements, permits the ribozyme to achieve maximum rate constants for RNA cleavage at physiologically relevant Mg2+ concentrations. In addition, we show that substantial rate enhancements are supported by an exchange-inert cobalt (III) complex and by molar concentrations of monovalent ions. Our findings indicate that the glmS ribozyme forms a complex structure to employ catalytic strategies that do not require the direct participation of divalent metal ions.     

Synthetic metallonucleases for RNA cleavage

Synthetic metallonucleases are versatile metal ion catalysts that use multiple catalytic strategies for the cleavage of RNA. Recent work in the design of more active metallonucleases combines a single metal ion with functional groups that interact with RNA, including amino acid fragments or additional metal ions. Rate enhancements by multifunctional catalysts for cleavage of simple model substrates with good leaving groups are as high as 10(6) but somewhat lower (10(5)) for real RNA. However, cleavage of RNA substrates is complicated by different binding modes and steric interactions that can interfere with catalysis. Antisense oligonucleotides, peptides and small molecules that act as RNA recognition agents increase the strength of substrate binding, but not necessarily the catalytic rate constant. In general, catalytic strategies used by synthetic metallonucleases are probably not optimized. A better grasp of the mechanism of RNA cleavage by metal ions and more effort on positioning the metal ion complex with respect to the cleavage site may lead to improved catalysts.