Analysis of metal ion dependence in glmS ribozyme self-cleavage and coenzyme binding

The bacterial glmS ribozyme is mechanistically unique among both riboswitches and RNA catalysts. Its self-cleavage activity is the basis of riboswitch regulation of glucosamine-6-phosphate (GlcN6P) production, and catalysis requires GlcN6P as a coenzyme. Previous work has shown that the coenzyme amine of GlcN6P is essential for glmS ribozyme self-cleavage, as is its protonation state. Metal ions are also essential within the glmS ribozyme core for both structure and function of the ribozyme. Although metal ions do not directly promote catalysis, we show that metal ion identity and the varying physicochemical properties of metal ions have an impact on the rate of glmS ribozyme self-cleavage. Specifically, these studies demonstrate that metal ion identity influences the overall apparent pK(a) of ribozyme self-cleavage, and metal ion binding largely reflects phosphate oxygen affinity. Results suggest that metal ions take alternative roles in supporting the mechanism of catalysis.     

Enhancement of the Catalytic Activity of Hammerhead Ribozymes by Organic Cations

Catalytic turnover is important for the application of ribozymes to biotechnology. However, the turnover is often impaired because of the intrinsic high stability of base pairs with cleaved RNA products. Here, organic cations were used as additives to improve the catalytic performance of hammerhead ribozyme constructs that exhibit different kinetic behaviors. Kinetic analysis of substrate cleavage demonstrated that bulky cations, specifically tetra-substituted ammonium ions containing pentyl groups or a benzyl group, have the ability to greatly increase the turnover rate of the ribozymes. Thermal stability analysis of RNA structures revealed that the bulky cations promote the dissociation of cleaved products and refolding of incorrectly folded structures with small disruption of the catalytic structure. The use of bulky cations is a convenient method for enhancing the catalytic activity of hammerhead ribozymes, and the approach may be useful for advancing ribozyme technologies.     

A Hexameric Ribozyme Nanostructure Formed by Double-Decker Assembly of a Pair of Triangular Ribozyme Trimers

The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units. The resulting double-decker nanostructures containing six ribozyme units were characterized biochemically and their structures were observed by atomic force microscopy. The double-decker hexamers exhibited higher catalytic activity than the parent ribozyme trimers.     

RNA Aminoacylation Mediated by Sequential Action of Two Ribozymes and a Nonactivated Amino Acid

In the transition from the RNA world to the modern DNA/protein world, RNA‐catalyzed aminoacylation might have been a key step towards early translation. A number of ribozymes capable of aminoacylating their own 3′ termini have been developed by in vitro selection. However, all of those catalysts require a previously activated amino acid—typically an aminoacyl‐AMP—as substrate. Here we present two ribozymes connected by intermolecular base pairing and carrying out the two steps of aminoacylation: ribozyme 1 loads nonactivated phenylalanine onto its phosphorylated 5′ terminus, thereby forming a high‐energy mixed anhydride. Thereafter, a complex of ribozymes 1 and 2 is formed by intermolecular base pairing, and the “activated” phenylalanine is transferred from the 5′ terminus of ribozyme 1 to the 3′ terminus of ribozyme 2. This kind of simple RNA aminoacylase complex was engineered from previously selected ribozymes possessing the two required activities. RNA aminoacylation with a nonactivated amino acid as described here is advantageous to RNA world scenarios because initial amino acid activation by an additional reagent (in most cases, ATP) and an additional ribozyme would not be necessary.     

Mapping the Conformational Landscape of the Neutral Network of RNA Sequences That Connect Two Functional Distinctly Different Ribozymes

During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000 , 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network.     

Stereoselective synthesis using immobilized Diels-Alderase ribozymes

Development of artificial ribozymes by in vitro selection has so far, mostly been addressed from the viewpoint of fundamental research. However, such ribozymes also have high potential as selective catalysts in practical syntheses. Immobilization of an active and selective ribozyme is an important step towards this end. A 49-nucleotide RNA molecule that was previously found to stereoselectively catalyze Diels-Alder reactions between various anthracene dienes and maleimide dienophiles was quantitatively immobilized on an agarose matrix by periodate oxidation of the 3'-terminal ribose and coupling to a hydrazide moiety. Typical loadings were 45 pmol microL(-1) gel. The specific activity was comparable to that of soluble ribozyme, and high enantioselectivities were obtained in catalyzed cycloadditions. The catalytic matrix was found to be stable and could be regenerated about 40 times with only minimal reduction of catalytic activity. Like the soluble ribozyme, the immobilized catalyst stereoselectively converts various diene and dienophile substrates. By using either natural D-RNA or enantiomeric L-RNA, both product enantiomers were made synthetically accessible with similar selectivities.     

Tecto‐GIRz: Engineered Group I Ribozyme the Catalytic Ability of Which Can Be Controlled by Self‐Dimerization

RNA is a promising biomaterial for self‐assembly of nano‐sized structures with a wide range of applications in nanotechnology and synthetic biology. Several RNA‐based nanostructures have been reported, but most are unrelated to intracellular RNA, which possesses modular structures that are sufficiently large and complex to serve as catalysts to promote sophisticated chemical reactions. In this study, we designed dimeric RNA structures based on the Tetrahymena group I ribozyme. The resulting dimeric RNAs (tecto group I ribozyme; tecto‐GIRz) exhibit catalytic ability that depended on controlled dimerization, by which a pair of ribozymes can be activated to perform cleavage and splicing reactions of two distinct substrates. Modular redesign of complex RNA structures affords large ribozymes for use as modules in RNA nanotechnology and RNA synthetic biology.     

Genome Evolution from Random Ligation of RNAs of Autocatalytic Sets

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis, such as evaluating the ability of ribozyme RNA polymerases to generate random ligation products and testing the catalytic activity of linked ribozyme domains.     

Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA

Fluorescence techniques for the investigation of biomolecules and their folding pathways require an efficient labeling strategy. A common method to internally label large RNAs involves the introduction of long loops for hybridization of fluorophore‐carrying DNA strands. Such loops often disturb the structure, and thus the functionality, of the RNA. Here we show, in a proof of concept study with a >600 nucleotide group II intron ribozyme, that the usage of the nucleic acid analogue peptide nucleic acid (PNA) is more efficient in several aspects, minimizing the required structural modifications of the RNA. We demonstrate by various methods, including smFRET, that much smaller concentrations and shorter PNAs can be applied, compared to DNA, for rapid and specific internal RNA labeling. The folding pathway and catalytic activity of this large ribozyme is only minimally affected by the PNA, but the background signal is significantly reduced.     

Catalytic strategies of self-cleaving ribozymes

[Structure: see text]. Five naturally occurring nucleolytic ribozymes have been identified: the hammerhead, hairpin, glmS, hepatitis delta virus (HDV), and Varkud satellite (VS) ribozymes. All of these RNA enzymes catalyze self-scission of the RNA backbone using a chemical mechanism equivalent to that of RNase A. RNase A uses four basic strategies to promote this reaction: geometric constraints, activation of the nucleophile, transition-state stabilization, and leaving group protonation. In this Account, we discuss the current thinking on how nucleolytic ribozymes harness RNase A's four sources of catalytic power. The geometry of the phosphodiester cleavage reaction constrains the nucleotides flanking the scissile phosphate so that they are unstacked from a canonical A-form helix and thus require alternative stabilization. Crystal structures and mutational analysis reveal that cross-strand base pairing, along with unconventional stacking and tertiary hydrogen-bonding interactions, work to stabilize the splayed conformation in nucleolytic ribozymes. Deprotonation of the 2'-OH nucleophile greatly increases its nucleophilicity in the strand scission reaction. Crystal structures of the hammerhead, hairpin, and glmS ribozymes reveal the N1 of a G residue within hydrogen-bonding distance of the 2'-OH. In each case, this residue has also been shown to be important for catalysis. In the HDV ribozyme, a hydrated magnesium has been implicated as the general base. Catalysis by the VS ribozyme requires both an A and a G, but the precise role of either has not been elucidated. Enzymes can lower the energy of a chemical reaction by binding more tightly to the transition state than to the ground states. Comparison of the hairpin ground- and transition-state mimic structures reveal greater hydrogen bonding to the transition-state mimic structure, suggesting transition-state stabilization as a possible catalytic strategy. However, the hydrogen-bonding pattern in the glmS ribozyme transition-state mimic structure and the ground-state structures are equivalent. Protonation of the 5'-O leaving group by a variety of functional groups can promote the cleavage reaction. In the HDV ribozyme, the general acid is a conserved C residue. In the hairpin ribozyme, a G residue has been implicated in protonation of the leaving group. An A in the hammerhead ribozyme probably plays a similar role. In the glmS ribozyme, an exogenous cofactor may provide the general acid. This diversity is in contrast to the relatively small number of functional groups that serve as a general base, where at least three of the nucleolytic ribozymes may use the N1 of a G.     

Flexizymes: their evolutionary history and the origin of catalytic function

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.     

Imaging target mRNA and siRNA-mediated gene silencing in vivo with ribozyme-based reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.     

Charged nucleobases and their potential for RNA catalysis

Catalysis in living cells is carried out by both proteins and RNA. Protein enzymes have been known for over 200 years, but RNA enzymes, or "ribozymes", were discovered only 30 years ago. Developing insight into RNA enzyme mechanisms is invaluable for better understanding both extant biological catalysis as well as the primitive catalysis envisioned in an early RNA-catalyzed life. Natural ribozymes include large RNAs such as the group I and II introns; small RNAs such as the hepatitis delta virus and the hairpin, hammerhead, VS, and glmS ribozymes; and the RNA portion of the ribosome and spliceosome. RNA enzymes use many of the same catalytic strategies as protein enzymes, but do so with much simpler side chains. Among these strategies are metal ion, general acid-base, and electrostatic catalysis. In this Account, we examine evidence for participation of charged nucleobases in RNA catalysis. Our overall approach is to integrate direct measurements on catalytic RNAs with thermodynamic studies on oligonucleotide model systems. The charged amino acids make critical contributions to the mechanisms of nearly all protein enzymes. Ionized nucleobases should be critical for RNA catalysis as well. Indeed, charged nucleobases have been implicated in RNA catalysis as general acid-bases and oxyanion holes. We provide an overview of ribozyme studies involving nucleobase catalysis and the complications involved in developing these mechanisms. We also consider driving forces for perturbation of the pK(a) values of the bases. Mechanisms for pK(a) values shifting toward neutrality involve electrostatic stabilization and the addition of hydrogen bonding. Both mechanisms couple protonation with RNA folding, which we treat with a thermodynamic formalism and conceptual models. Furthermore, ribozyme reaction mechanisms can be multichannel, which demonstrates the versatility of ribozymes but makes analysis of experimental data challenging. We examine advances in measuring and analyzing perturbed pK(a) values in RNA. Raman crystallography and fluorescence spectroscopy have been especially important for pK(a) measurement. These methods reveal pK(a) values for the nucleobases A or C equal to or greater than neutrality, conferring potential histidine- and lysine/arginine-like behavior on them. Structural support for ionization of the nucleobases also exists: an analysis of RNA structures in the databases conducted herein suggests that charging of the bases is neither especially uncommon nor difficult to achieve under cellular conditions. Our major conclusions are that cationic and anionic charge states of the nucleobases occur in RNA enzymes and that these states make important catalytic contributions to ribozyme activity. We conclude by considering outstanding questions and possible experimental and theoretical approaches for further advances.     

A Hairpin Ribozyme Derived Spliceozyme

The vast majority of RNA splicing in today‘s organisms is achieved by the highly regulated and precise removal of introns from pre-mRNAs via the spliceosome. Here we present a model of how RNA splicing may have occurred in earlier life forms. We have designed a hairpin ribozyme derived spliceozyme that mediates two RNA cleavages and one ligation event at specific positions and thus cuts a segment (intron) out of a parent RNA and ligates the remaining fragments (exons). The cut-out intron then performs a downstream function, acting as a positive regulator of the activity of a bipartite DNAzyme. This simple scenario shows how small RNAs can perform complex RNA processing dynamics, involving the generation of new phenotypes by restructuring segments of given RNA species, as well as delivering small RNAs that may play a functional role in downstream processes.     

Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II

Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg(2+) concentrations were in vitro selected from a library with 18 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had the same sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mM Mg(2+) concentration in the context of several substrates with differences in the lengths of stem I and III, including the well-characterised HH16 substrate and a derivative thereof with a GUA triplet at the cleavage site, as well as a substrate used previously in a related study. For the HH16 substrate, a change of base pair 10.2-11.2 to C-G in stem II further improved activity by about 2.5-fold to 0.8 min(-1) (at 1 mM Mg(2+) concentration, 25 degrees C, pH 7.5). Interestingly, this very active variant was not identified by the selection procedure. Changing loop II from UUUU to GCAA or extension of stem II to three or four base pairs reduced the cleavage rate by 2.0-2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so far at low Mg(2+) concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degrees C and pH 7.5 in monophasic reactions, with endpoints between 75 and 90 %. Such constructs promise to be advantageous for the inhibition of gene expression in vivo.     

Effects of Cosolvents on the Folding and Catalytic Activities of the Hammerhead Ribozyme

The RNA cleavage activity of the hammerhead ribozyme has been compared in various mixed aqueous solutions containing cosolvents. Kinetic analysis revealed that the tested cosolvents enhanced the ribozyme activity, particularly at low MgCl₂ concentrations. These enhancements, in some cases of more than tenfold, resulted from a reduction in the Mg²⁺ concentration required for substrate cleavage. An inverse correlation was found between the MgCl₂ concentration essential for efficient catalysis and the dielectric constant values. In contrast, FRET measurements showed no substantial influence of cosolvents on the Mg²⁺‐induced structural transitions. The results suggest that the solution environment has various effects on the Mg²⁺ interactions involved in the catalysis and global folding of the ribozyme.     

Development and Applications of Coarse-Grained Models for RNA

In contrast to proteins, much less attention has been focused on the development of computational models for describing RNA molecules, which are being recognized as playing key roles in many cellular functions. Current atomically detailed force fields are not accurate enough to capture the properties of even simple nucleic acid constructs. In this article, we review our efforts to develop coarse-grained (CG) models that capture the underlying physics for the particular length scale of interest. Two models are discussed. One of them is the three interaction site (TIS) model, in which each nucleotide is represented by three beads corresponding to sugar, phosphate, and base. The other is the self-organized polymer (SOP) model, in which each nucleotide is represented as a single interaction center. Applications of the TIS model to study the complexity of hairpin formation and the effects of crowding in a shifting equilibrium between two conformations in human telomerase pseudoknot are described. The work on crowding illustrates a direct link to the activity of telomerase. We use the SOP model to describe the response of the Tetrahymena ribozyme to force. The simulated unfolding pathways agree well with single molecule pulling experiments. We also review predictions for the unfolding pathways for the Azoarcus ribozyme. The success of the CG applications to describe dynamics in RNA gives hope that more complex processes involving RNA-protein interactions can be tackled using variants of the proposed models.