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Besides its primary informational role, the sequence of the mRNA (mRNA) including its 5′- and 3′- untranslated regions (UTRs), contains important features that are relevant for post-transcriptional and translational regulation of gene expression. In this work a number of bacterial twister motifs are characterized both in vitro and in vivo. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. Our data show that the ribozyme-mediated cleavage of the bacterial 3′-UTR has major effects on gene expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they show dependence on motif-specific structural features and on mRNA stabilization properties of the secondary structures that remain on the 3′-UTR after ribozyme cleavage. Using these principles, novel artificial twister-based riboswitches are developed that exert their activity via ligand-dependent cleavage of the 3′-UTR and the removal of the protective intrinsic terminator. Our results provide insights into possible biological functions of these recently discovered and widespread catalytic RNA motifs and offer new tools for applications in biotechnology, synthetic biology and metabolic engineering. published

RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.

The regulation of RNA interference (RNAi) could be a powerful method for the study of temporal and dose dependent effects of gene expression. In this study, we designed the hepatitis delta virus (HDV) ribozyme with an embedded theophylline aptamer as the sensor domain and the pri-miRNA of endogenous gene Bcl-2 as the effector domain to engineer an RNAi-regulatory device in MCF-7 cells. The system allowed us to control gene expression by adding theophylline into the culture media in a dose dependent fashion. This is the pioneering application of ribozyme switches to activate RNAi for modulating endogenous genes in mammalian cells. The platform sets the stage for investigations of other endogenous genes that regulate various biological functions such as differentiation, cell division or cell death, and provides a promising interface with other universal RNAi-based decision-making circuits that operate in mammalian cells. It can be used to study more genes associated with cancer and screen for potential drug targets for gene therapy.

Recent discoveries of new classes of self‐cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self‐cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations.

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.

The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report the crystal structure of a pistol ribozyme termed env25, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2'-O of G for attack on the adjacent to-be-cleaved P-O5' bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2'-OH positions) are aligned to act as a general base and a general acid, respectively, to accelerate cleavage chemistry, with their roles confirmed by cleavage assays on variants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defined how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on variants revealing key residues that participate in acid-base-catalyzed cleavage chemistry.

Engineered ribozymes are powerful tools for manipulating gene expression in living cells. However, identification of active ribozymes in mammalian cells has relied on empirical assays of a limited number of arbitrarily chosen ribozymes. Here, we synthesized 376 natural and 2625 synthetic variants of pistol ribozymes, and screened for active variants directly in mammalian cells, greatly expanding the ribozyme toolbox for biological applications.

Enzymes made of RNA catalyze reactions that are essential for protein synthesis and RNA processing. However, such natural ribozymes are exceedingly rare, as evidenced by the fact that the discovery rate for new classes has dropped to one per decade from about one per year during the 1980s. Indeed, only 11 distinct ribozyme classes have been experimentally validated to date. Recently, we recognized that self-cleaving ribozymes frequently associate with certain types of genes from bacteria. Herein we exploited this association to identify divergent architectures for two previously known ribozyme classes and to discover additional noncoding RNA motifs that are self-cleaving RNA candidates. We identified three new self-cleaving classes, which we named twister sister, pistol and hatchet, from this collection, suggesting that even more ribozymes remain hidden in modern cells.

An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

We have previously engineered new RNase P-based ribozyme variants with improved in vitro catalytic activity. In this study, we employed a novel engineered variant to target a shared mRNA region of human cytomegalovirus (HCMV) immediate early proteins 1 (IE1) and 2 (IE2), which are essential for the expression of viral early and late genes as well as viral growth. Ribozyme F-R228-IE represents a novel variant that possesses three unique base substitution point mutations at the catalytic domain of RNase P catalytic RNA. Compared to F-M1-IE that is the ribozyme derived from the wild type RNase P catalytic RNA sequence, the functional variant F-R228-IE cleaved the target mRNA sequence in vitro at least 100 times more efficiently. In cultured cells, expression of F-R228-IE resulted in IE1/IE2 expression reduction by 98-99% and in HCMV production reduction by 50,000 folds. In contrast, expression of F-M1-IE resulted in IE1/IE2 expression reduction by less than 80% and in viral production reduction by 200 folds. Studies of the ribozyme-mediated antiviral effects in cultured cells suggest that overall viral early and late gene expression and viral growth were inhibited due to the ribozyme-mediated reduction of HCMV IE1 and IE2 expression. Our results provide direct evidence that engineered RNase P ribozymes, such as F-R228-IE, can serve as a novel class of inhibitors for the treatment and prevention of HCMV infection. Moreover, these results suggest that F-R228-IE, with novel and unique mutations at the catalytic domain to enhance ribozyme activity, can be a candidate for the construction of effective agents for anti-HCMV therapy.

Recent bioinformatics studies have demonstrated a wide-spread occurrence of the hammerhead ribozyme (HHR) and similar small endonucleolytic RNA motifs in all domains of life. It is becoming increasingly evident that such ribozyme motifs participate in important genetic processes in diverse organisms. Although the HHR motif has been studied for more than three decades, only little is known about the consequences of ribozyme activity on gene expression. In the present study we analysed eight different naturally occurring HHR sequences in diverse genetic and organismal contexts. We investigated the influence of active ribozymes incorporated into mRNAs in mammalian, yeast and bacterial expression systems. The experiments show an unexpectedly high degree of organism-specific variability of ribozyme-mediated effects on gene expression. The presented findings demonstrate that ribozyme cleavage profoundly affect gene expression. However, the extent of this effect varies and depends strongly on the respective genetic context. The fast-cleaving type 3 HHRs [CChMVd(-) and sLTSV(-)] generally tended to cause the strongest effects on intracellular gene expression. The presented results are important in order to address potential functions of naturally occurring ribozymes in RNA processing and post-transcriptional regulation of gene expression. Additionally, our results are of interest for biotechnology and synthetic biology approaches that aim at the utilisation of self-cleaving ribozymes as widely applicable tools for controlling genetic processes. published

Catalysis in the biological context was largely thought to be a protein-based phenomenon until the discovery of RNA catalysts called ribozymes. These discoveries demonstrated that many RNA molecules exhibit remarkable structural and functional versatility. By virtue of these features, naturally occurring ribozymes have been found to be involved in catalyzing reactions for fundamentally important cellular processes such as translation and RNA processing. Another class of RNAs called riboswitches directly binds ligands to control downstream gene expression. Most riboswitches regulate downstream gene expression by controlling premature transcription termination or by affecting the efficiency of translation initiation. However, one riboswitch class couples ligand-sensing to ribozyme activity. Specifically, the glmS riboswitch is a nucleolytic ribozyme, whose self-cleavage activity is triggered by the binding of GlcN6P. The products of this self-cleavage reaction are then targeted by cellular RNases for rapid degradation, thereby reducing glmS expression under conditions of sufficient GlcN6P. Since the discovery of the glmS ribozyme, other metabolite-binding ribozymes have been identified. Together, these discoveries have expanded the general understanding of noncoding RNAs and provided insights that will assist future development of synthetic riboswitch-ribozymes. A very broad overview of natural and synthetic ribozymes is presented herein with an emphasis on the structure and function of the glmS ribozyme as a paradigm for metabolite-binding ribozymes that control gene expression. This article is part of a Special Issue entitled: Riboswitches.

RNA repair is an emerging strategy for gene therapy. Conventional gene therapy typically relies on the addition of the corrected DNA sequence of a defective gene to restore gene function. As an additional option, RNA repair allows alteration of the sequence of endogenous messenger RNAs (mRNAs). mRNA sequence alteration is either facilitated by intracellular spliceosome machinery or by the intrinsic catalytic activity of trans-acting ribozymes. Previously we developed twin ribozymes, derived from the hairpin ribozyme, by tandem duplication and demonstrated their potential for patchwise RNA repair. Herein we describe the development of such a twin ribozyme for potential repair of a deletion mutation in the oncogenic CTNNB1-ΔS45 mRNA. We demonstrate that hairpin ribozyme units within the twin ribozyme can be adapted to efficiently cleave/ligate non-consensus substrates by introduction of compensatory mutations in the ribozyme. Thus, we show the twin ribozyme mediated repair of truncated CTNNB1 transcripts (up to 1000 nt length). Repair of the entire CTNNB1-ΔS45 mRNA, although apparently possible in general, is hampered in vitro by the secondary structure of the transcript.

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.

Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5′ splice site located 8 nt upstream of the usual 5′ GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1–EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5′ splice site is shown to be affected by structures in addition to IBS1–EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3′ exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.

Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2,700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.

Hammerhead ribozyme is a versatile tool for down-regulation of gene expression in vivo. Owing to its small size and high activity, it is used as a model for RNA structure–function relationship studies. In the present paper we describe a new extended hammerhead ribozyme HH-2 with a tertiary stabilizing motif constructed on the basis of the tetraloop receptor sequence. This ribozyme is very active in living cells, but shows low activity in vitro. To understand it, we analysed tertiary structure models of substrate–ribozyme complexes. We calculated six unique catalytic core geometry parameters as distances and angles between particular atoms that we call the ribozyme fingerprint. A flanking sequence and tertiary motif change the geometry of the general base, general acid, nucleophile and leaving group. We found almost complete correlation between these parameters and the decrease of target gene expression in the cells. The tertiary structure model calculations allow us to predict ribozyme intracellular activity. Our approach could be widely adapted to characterize catalytic properties of other RNAs.

Riboswitches are functional non-coding RNA regulatory components that play an important role in the regulation of gene expression in diverse organisms. In particular, using riboswitches to modulate RNA interference (RNAi) enables temporal and spatial control of gene expression in mammalian cells. Herein, a ribozyme gene switch to activate RNAi was fabricated for the artificial regulation of versatile gene silencing through the interaction of an RNA aptamer with small molecules. The device comprised an allosteric HDV ribozyme with an embedded theophylline aptamer and a primary miRNA (pri-miRNA) to silence the MAP4K4 gene in hepatic (HepG2) cells, aiming to achieve dose-dependent control of the activation of RNAi, and then the regulation of the MAP4K4 gene by theophylline. Finally, we demonstrated the feasibility and applicability of utilizing HDV ribozyme switches to activate RNAi for regulating an endogenous gene in mammalian cells.

The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer–template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells. Lysine-rich peptides from the ribosomal core and derived homolysine decapeptides of either L-, D- or mixed chirality have now been shown to enhance RNA polymerase ribozyme activity at low magnesium concentrations, accelerate ribozyme evolution and enable templated RNA synthesis within membranous protocells.

When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to generate sufficient amounts of lariat intron for the latter to be purified and used in kinetic assays in which folding and reaction are uncoupled.

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure–function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ∼20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.

Group I intron ribozymes can repair mutated mRNAs by replacing the 3′-terminal portion of the mRNA with their own 3′-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 5′-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 × 106 ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

The rescue of recombinant rabies virus (RV) from cloned cDNA is an inefficient process because it relies on the de novo formation within cells of functional ribonucleoprotein (RNP) complexes from plasmid-expressed viral-like antigenome RNAs and three helper proteins. In the standard RV reverse genetics systems, bacteriophage T7 RNA polymerase drives the transcription of virus antigenome-like RNAs containing three nonviral G residues at the 5'-end and a correct 3'-end generated by the autocatalytic activity of an 85 nucleotides long hepatitis delta virus antigenomic "core" ribozyme (HDVagrz). Here, we show that employing optimized ribozyme sequences significantly improves RV rescue. Substitution of the "core" HDVagrz by a ribozyme with an enhanced cleavage activity resulted in an approximately 10-fold higher number of rescue events and faster initiation of an infectious cycle. The alternative use of a hammerhead ribozyme for the generation of an exact 5'-end similarly enhanced rescue efficiency. Notably, RV cDNA clones containing the combination of optimized 3'- and 5'-ribozymes were rescued at an at least 100-fold increase. In addition to virus rescue, reporter gene expression from transfected minigenome cDNAs was significantly enhanced by the novel ribozymes. The improved RV reverse genetics system greatly facilitates recovery of strongly attenuated viruses and vectors for biomedical applications.

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.

The Hepatitis Delta Virus (HDV) ribozyme, which is well adapted to the environment of the human cell, is an excellent candidate for the future development of gene-inactivation systems. On top of this, a new generation of HDV ribozymes now exists that benefits from the addition of a specific on/off adaptor (specifically the SOFA-HDV ribozymes) which greatly increases both the ribozyme’s specificity and its cleavage activity. Unlike RNAi and hammerhead ribozymes, the designing of SOFA-HDV ribozymes to cleave, in trans, given RNA species has never been the object of a systematic optimization study, even with their recent use for the gene knockdown of various targets. This report aims at both improving and clarifying the design process of SOFA-HDV ribozymes. Both the ribozyme and the targeted RNA substrate were analyzed in order to provide new criteria that are useful in the selection of the most potent SOFA-HDV ribozymes. The crucial features present in both the ribozyme’s biosensor and blocker, as well as at the target site, were identified and characterized. Simple rules were derived and tested using hepatitis C virus NS5B RNA as a model target. Overall, this method should promote the use of the SOFA-HDV ribozymes in a plethora of applications in both functional genomics and gene therapy.

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y−2N−1*G+1U+2Y+3B+4, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions −2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U−2G−1*G+1U+2A+3A+4 sites. Substrates with G−1*G+1U+2A+3 sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In particular, we show that A+3 and A+4 are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G6U, owing to restoration of a Watson–Crick base pair in helix 1. In this ribozyme–substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A7 with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A7, C7 and G7. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3’-untranslated region (3’-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans-splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3’-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a transsplicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3–P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

Hammerhead (HH) ribozymes can be used for highly specific inhibition of gene expression through the degradation of target mRNA. In vitro experiments with minimal HH domains demonstrated that the efficiency of catalysis is highly dependent on concentration of magnesium ions. Optimal ion requirements for HH-catalysed RNA cleavage are far from these found in the cell. Recently, it has been proposed that the efficiency of HH ribozymes can be increased at low magnesium concentration through stabilization of a catalytically active conformation by tertiary interactions between helices I and II. We designed a ribozyme stabilized by GAAA tetraloop and its receptor motifs and demonstrated that it can efficiently catalyse target RNA hydrolysis at submillimolar Mg(2+) concentrations in vitro as well as in cultured cells. Both unmodified and locked nucleic acid-modified extended ribozymes proved superior to the minimal core ribozyme and DNAzyme against the same target sequence.

Hairpin ribozymes possess the properties of RNA sequence-specific recognition and site-specific cleavage. These properties make them a powerful extension of the antisense approach for the inhibition of gene expression. From a randomized RNA pool of hairpin ribozymes, using the systematic evolution of ligands by exponential enrichment, we have obtained an adenine-dependent hairpin ribozyme, Tpl2/Cot (tumour progression locus 2) ribozyme, which cleaves the Tpl2/Cot kinase mRNA sequence at nucleotides A225G226 relative to the start codon of translation. This serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in cancer. The selected ‘Tpl2/Cot-YL ribozyme’ efficiently cleaves its target sequence in cis and in trans; furthermore, the ribozyme efficiently cleaves a longer target sequence of 54 nucleotides in trans, as well as the full-length mRNA.

The trans insertion-splicing reaction, catalyzed by a group I intron-derived from Pneumocystis carinii, was recently developed for the site-specific insertion of a segment of RNA into a separate RNA substrate. The molecular determinants of this reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. To demonstrate proof-of-concept, we now report that the P. carinii ribozyme can except modified oligonucleotides as substrates for catalyzing the trans insertion-splicing reaction. Oligonucleotides that contain one or more sugar modifications (deoxy or methoxy substitution), a backbone modification (phosphorothioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in this reaction. Apparently, trans insertion-splicing is a unique and viable reaction for the site-specific incorporation of modified oligonucleotides into RNAs. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a modified oligonucleotide into RNA.

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

Hammerhead ribozymes have been shown to silence human immunodeficiency virus-1 (HIV-1) gene expression by site-specific cleavage of viral mRNA. The two major factors that determine whether ribozymes will be effective for post-transcriptional gene silencing are colocalization of the ribozyme and the target RNAs, and the choice of an appropriate target site on the mRNA. An effective screening strategy for potential targets on the viral genome is the use of ribozyme libraries in cell culture. Capitalizing on previous findings that HIV-1 and ribozymes can be colocalized in the nucleolus, we created a novel hammerhead ribozyme library by inserting hammerhead ribozymes with fully randomized stems 1 and 2 into the body of the U16 small nucleolar RNA (snoRNA). Following three rounds of cotransfection with an HIV-1 proviral DNA harboring the herpes simplex virus thymidine kinase (HSV-TK) gene, we selected for gancyclovir-resistant cells and identified a ribozyme sequence that could potentially target both the U5 and gag genes of HIV-1 regions on the HIV-1 genome through partial homologies with these targets. When the ribozymes were converted to full complementarity with the targets, they provided potent inhibition of HIV-1 replication in cell culture. These results provide a novel approach for identifying ribozyme targets in HIV-1.

Ribozyme and small interfering RNA (siRNA) now are widely used to suppress target genes bearing homologous sequences. In this study, commonly used cell lines (e.g., HEK, HeLa, H1299) were stably transfected with gene encoding T7 RNA polymerase. The cytoplasm-restricted transcription activity of T7 RNA polymerase confers a continuous and robust transcription from T7 promoter-containing oligonucleotide (ODN) template for siRNA or ribozyme and leads to 70 to 80% inhibition of the tested target genes. ODN template offers the advantages of being more stable and economical than synthetic or in vitro-transcribed siRNA or ribozyme. Compared with the use of siRNA/ribozyme-expressing plasmids, our system does not require procedures with preparations of recombinant plasmids and enrichment of transfected cells and can be applied to synthesize protein in which different levels of translation could be modulated via variations in the presence of polyA tail or internal ribosome entry site (IRES) in the T7-transcribed RNAs. The results of our current study provide a rapid and efficient system for the assay of in vivo synthesis and expression of RNAs and proteins.

The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells.

The potential utility of catalytic RNAs and DNAs (ribozymes and deoxyribozymes, respectively) as reagents in molecular biology as well as therapeutic agents for a variety of human diseases, has long been recognized. Although naturally occurring RNA-cleaving ribozymes are typically not subject to feedback control, rational methodologies for the creation of allosteric ribozymes, by functional combination of ribozyme and ligand-responsive aptamer RNA elements, have existed for some years. Here, we report the in vitro selection of RNA aptamers specific for binding one but not the other of two light-induced isomers of a dihydropyrene photo-switch compound, and the utilization of such an aptamer for the construction of the UG-dihydropyrene ribozyme, an allosteric hammerhead ribozyme whose catalysis is controllable by irradiation with visible versus ultraviolet light. In the presence of micromolar concentrations of the photo-switch compound, the ribozyme behaves as a two-state switch, exhibiting a >900-fold difference in catalytic rates between the two irradiation regimes. We anticipate that the UG-dihydropyrene, and other ribozymes like it, may find significant application in the developmental biology of model organisms such as Drosophila melanogaster and Caenorhabditis elegans, as well as in the biomedical sciences.

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes.

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.

The nonmevalonate pathway of isoprenoid biosynthesis in the apicoplast of the human malaria parasite Plasmodium falciparum is distinct from the mevalonate-dependent pathway of humans and thus a good drug target. We describe here the hammerhead ribozyme based cleavage of the ispH (lytB) gene transcript involved in the last step of this nonmevalonate pathway. Using RNA folding program, three hammerhead ribozymes named as RZ(876), RZ(1260), and RZ(1331) were predicted against ispH (lytB) mRNA. Messenger walk screening (RNaseH) assay confirmed the target accessibility for these ribozymes. All three ribozymes cleaved the target RNA in vitro but RZ(876) exhibited the highest catalytic potential (62.92%). Therefore, RZ(876) was chemically synthesized with appropriate chemical modifications to protect it from nuclease attack while using it for in vitro parasite growth inhibition assay. This ribozyme RZ(876) was able to inhibit 87.36% parasite growth at 30 microM concentration compared to the untreated culture. However, an absolute inhibition of 29.41% was achieved compared to the control ribozyme (RZ(ctrl)). Nonetheless, the growth inhibition effect was found to be sequence-specific as indicated by the decreased level of ispH (lytB) transcript after ribozyme treatment. In conclusion, we have identified the ispH (lytB) as a potential target whose transcript can be cleaved by a ribozyme RZ(876).

The development of gene-inactivation systems is an active and important field for both functional genomics and gene therapy. Towards this end, ribozymes (i.e. RNA enzymes), that specifically recognize and subsequently catalyze the cleavage of other target RNA molecules, are attractive molecular tools. Ribozymes represent an interesting alternative to the RNA interference (RNAi) approach for gene inactivation, especially given the fact that RNAi seems to trigger an immunological response and has demonstrated off-target effects. However, the design and optimization of a ribozyme-based gene-inactivation system is not a straightforward procedure. Several aspects need to be considered in the experimental design in order to provide a suitable suppression system. In this review we present the advances in this domain made available from work using the hepatitis delta virus (HDV) ribozyme as a cis-acting RNA motif in molecular biology, as well as a trans-acting molecular scissor for the development of a gene-inactivation system. This HDV ribozyme technology possesses several unique features that are all related to the fact that it is the only catalytic cleaving RNA motif that has been discovered in humans.

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme escorted by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5'/3' helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganizes into a circularly permuted form, and the 5'/3' ends are relocated and buried in the original 71'/75' positions. Effective silencing of the anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozyme's strength in medical application.

Endonucleolytic hammerhead ribozymes have advantages of inhibiting gene expression by acting specifically, independently of cellular pathways, and within all cell compartments. However, there are concerns about inefficient silencing because of reduced intracellular cleavage of target RNA by ribozymes. To enable production of defined single-unit ribozymes and thereby increase effectiveness, we developed self-cleaving multimeric cassettes that generate several trans-acting ribozyme units from a single transcript. cis and trans ribozyme cleavage, as assessed in vitro against three different sites within the X sequence of hepatitis B virus (HBV), occurred efficiently and precisely according to predictions deduced from the ribozyme designs. Significant knockdown of markers of viral replication in transfected cultured liver-derived cells was achieved by multiribozyme Pol II expression cassettes. To assess silencing efficacy of RNA prepared in vitro, transcription and cis cleavage reactions were carried out to prepare defined single-unit ribozymes. Transfection of ribozyme RNA was capable of inhibiting HBV surface antigen secretion from liver-derived cells without associated elevation of interferon-alpha or interferon-beta secretion into the culture upernatants. The approach described here is potentially useful for several applications, such as generation of RNA interference (RNAi) effectors, which require rapid and inexpensive generation of defined RNA sequences.

The formation of lariat intermediates during the first step of splicing of group II introns and spliceosomal introns is a well-studied fundamental reaction in molecular biology. Apart from this prominent example, there are surprisingly few occurrences of branched nucleotides or even 2′,5′-phosphodiester bonds in biology. We recently described a new ribozyme, the GIR1 branching ribozyme, which catalyzes the formation of a tiny lariat that caps an mRNA. This new example together with work on artificial branching ribozymes and deoxyribozymes shows that branching is facile and points to the possibility that branching reactions could be more prevalent than previously recognized.

Self-cleaving ribozymes were discovered thirty years ago, but their biological distribution and catalytic mechanisms are only beginning to be defined. Each ribozyme family is defined by a distinct structure with unique active sites accelerating the same transesterification reaction across the families. Biochemical studies show that general acid-base catalysis is the most common mechanism of self-cleavage, but metal ions and metabolites can be employed as cofactors. Ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered.