Ribozyme Activity Research Articles

A simple physical mechanism enables homeostasis in primitive cells.

The emergence of homeostatic mechanisms that enabled maintenance of an intracellular steady-state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles; ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation. This simple physical system enables a primitive homeostatic behavior: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.

Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution.

The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23% in single turnover experiments at 25°C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43% were observed in one freeze-thaw cycle and a maximum of 60% was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24% in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

The Earliest Discovery of the Role of Magnesium Ions on Stabilizing the Tertiary Structure of the Transfer RNA and Its Biological Significance <br>—A Short Memoir

In early of 1960s, I was a graduate student studying on tRNA biochemistry. In the course of the research, the magnesium ions stabilized the tertiary structure of tRNA, resulting in its resistance to enzymatic degradation was discovered independently. The experiment of deaminated (denatured) tRNA obtained from native tRNA was designed and conducted and further proved the validity of this finding. It was found that magnesium ions could stabilize the tertiary structure of the natrive tRNA but could not stabilize structure of the deaminated tRNA. In term of the methodology, this stabilization technique has been widely applied in sequencing analysis of RNA and has greatly promoted the progress in the study of primary structure of RNA. More importantly, the stabilization of the tertiary structure of RNA by magnesium ions plays a key role both in the processing of messenger RNAs and the ribozyme activity. After our first article in Chinese was published in 1963, a paper of Nishimura & Novelli came into our note. The received date of their paper was March 22 of 1963, only 4 days earlier than that of our first paper. Thus, we and Nishimura & Novelli made almost at the same time the earliest discovery of the role of magnesium ions on stabilizing the tertiary structure of the transfer RNA and thus resulted in resistance of tRNA degradation by enzymes. However, this discovery was not initially appreciated for a period of time but was finally “visualized” and proved by X-ray crystal structure of yeast phenylalanine tRNA, which has provided more accurate information on the geometry of the magnesium-binding sites in tRNA.

Bridging the gap between in vitro and in vivo RNA folding.

Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functions in vivo. Within the last few years, methods have been developed to probe RNA structures in vivo and genome-wide. These studies reveal that RNA often adopts very different structures in vivo and in vitro, and provide profound insights into RNA biology. Nonetheless, both in vitro and in vivo approaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studies in vitro often lack direct cellular relevance, leaving a gap in our knowledge of RNA folding in vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under such in vivo-like conditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA folding in vitro and in vivo and discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.

Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

RNA recombination in Hepatitis delta virus: Identification of a novel naturally occurring recombinant.

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity. It replicates in the nucleus by host RNA polymerase via a rolling circle mechanism. Similar to many RNA viruses encoding their own RNA-dependent RNA polymerases, homologous recombination of HDV occurs in mixed-genotype infections and in cultured cells cotransfected with two HDV sequences, as demonstrated by molecular analyses. Among 237 published complete genomic sequences, 34 sequences were reported from the small and isolated Miyako Island, Japan, and belonged to the Asia-specific genotypes, HDV-2 and HDV-4 (the majority of them belonged to the known Miyako Island-specific subgroup, HDV-4M). We investigated the presence of naturally occurring HDV recombinant in Miyako Island using phylogenetic and recombination analyses. We identified a two-switch HDV-4/4M intersubtype recombinant with an unbranched rod-like RNA genome. Our data suggest that RNA recombination plays an important role in the rapid evolution of HDV, allowing the production of new HDV strains with correct genomic structures.

Thiosulfate-Hydrogen Peroxide Redox Oscillator as pH Driver for Ribozyme Activity in the RNA World.

The RNA world of more than 3.7 billion years ago may have drawn on thermal and pH oscillations set up by the oxidation of thiosulfate by hydrogen peroxide (the THP oscillator) as a power source to drive replication. Since this primordial RNA also must have developed enzyme functionalities, in this work we examine the responses of two simple ribozymes to a THP periodic drive, using experimental rate and thermochemical data in a dynamical model for the coupled, self-consistent evolution of all reactants and intermediates. The resulting time traces show that ribozyme performance can be enhanced under pH cycling, and that thermal cycling may have been necessary to achieve large performance gains. We discuss three important ways in which the dynamic hydrogen peroxide medium may have acted as an agent for development of the RNA world towards a cellular world: proton gradients, resolution of the ribozyme versus replication paradox, and vesicle formation.

DNAzyme-based therapeutics for cancer treatment.

Gene-silencing strategies based on catalytic nucleic acids have been rapidly developed in the past decades. Ribozymes, antisense oligonucleotides and RNA interference have been actively pursued for years due to their potential application in gene inactivation. Pioneered by Joyce et al., a new class of catalytic nucleic acid composed of deoxyribonucleotides has emerged via an in vitro selection system. The therapeutic potential of these RNA-cleaving DNAzymes have been shown both in vitro and in vivo. Although they rival the activity and stability of synthetic ribozymes, they are limited by inefficient delivery to the intracellular targets. Recent successes in clinical testing of the DNAzymes in cancer patients have revitalized the potential clinical utility of DNAzymes.

Effect of Terminal Labeling of Fluorescent Dyes on Ribozyme Activity

Effect of Terminal Labeling of Fluorescent Dyes on Ribozyme Activity

How do metal ions direct ribozyme folding?

Ribozymes, which carry out phosphoryl-transfer reactions, often require Mg(2+) ions for catalytic activity. The correct folding of the active site and ribozyme tertiary structure is also regulated by metal ions in a manner that is not fully understood. Here we employ coarse-grained molecular simulations to show that individual structural elements of the group I ribozyme from the bacterium Azoarcus form spontaneously in the unfolded ribozyme even at very low Mg(2+) concentrations, and are transiently stabilized by the coordination of Mg(2+) ions to specific nucleotides. However, competition for scarce Mg(2+) and topological constraints that arise from chain connectivity prevent the complete folding of the ribozyme. A much higher Mg(2+) concentration is required for complete folding of the ribozyme and stabilization of the active site. When Mg(2+) is replaced by Ca(2+) the ribozyme folds, but the active site remains unstable. Our results suggest that group I ribozymes utilize the same interactions with specific metal ligands for both structural stability and chemical activity.

Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

Effects of background anionic compounds on the activity of the hammerhead ribozyme in Mg(2+)-unsaturated solutions.

Cellular ribozymes exhibit catalytic activity in media containing large numbers of anionic compounds and macromolecules. In this study, the RNA cleavage activity of the hammerhead ribozyme induced by Mg(2+) was investigated using the solutions containing background nucleic acids, small phosphate and carboxylic acid compounds, and neutral polymers. Analysis of the substrate cleavage kinetics showed that the anionic compounds do not affect the ribozyme activity in Mg(2+)-saturated solutions and there is almost no effect of the anion-Mg(2+) complexes formed. On the other hand, the rate of substrate cleavage in Mg(2+)-unsaturated solutions was reduced under conditions of a high background of anionic compounds found in cells. The extent of the reduction was more with a greater net negative charge, caused by decreased amounts of Mg(2+) that could be used for the ribozyme reaction. It was remarkable that background DNA and RNA molecules having phosphodiester bonds reduced the cleavage rate as much as adenosine monophosphates having a charge of -2 when the effects of the same amount of phosphate groups were compared. Greater reductions in rates than those expected from the molecular charge were also observed in the background of fatty acids that form micelles. An addition of poly(ethylene glycol) to the solutions partially restored the ribozyme activity, suggesting a possible role of macromolecular crowding in counteracting the inhibitory effects of background anions on the ribozyme reaction. The results have biological and practical implications with respect to the effects of molecular environment on the efficiency of ion binding to RNA.

Targeted Regression of Hepatocellular Carcinoma by Cancer-Specific RNA Replacement through MicroRNA Regulation.

Hepatocellular carcinoma (HCC) has a high fatality rate and limited therapeutic options with side effects and low efficacy. Here, we proposed a new anti-HCC approach based on cancer-specific post-transcriptional targeting. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically induce therapeutic gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To circumvent side effects due to TERT expression in regenerating liver tissue, liver-specific microRNA-regulated ribozymes were constructed by incorporating complementary binding sites for the hepatocyte-selective microRNA-122a (miR-122a), which is down-regulated in HCC. The ribozyme activity in vivo was assessed in mouse models orthotopically implanted with HCC. Systemic administration of adenovirus encoding the developed ribozymes caused efficient anti-cancer effect and the least hepatotoxicity with regulation of ribozyme expression by miR-122a in both xenografted and syngeneic orthotopic murine model of multifocal HCC. Of note, the ribozyme induced local and systemic antitumor immunity, thereby completely suppressing secondary tumor challenge in the syngeneic mouse. The cancer specific trans-splicing ribozyme system, which mediates tissue-specific microRNA-regulated RNA replacement, provides a clinically relevant, safe, and efficient strategy for HCC treatment.

A kinetic study to characterize the effect of peripheral elements on the catalytic activity of hairpin ribozymes

A kinetic study to characterize the effect of peripheral elements on the catalytic activity of hairpin ribozymes

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 Mg(2+) 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 Mg(2+) -induced structural transitions. The results suggest that the solution environment has various effects on the Mg(2+) interactions involved in the catalysis and global folding of the ribozyme.

In silico ribozyme evolution in a metabolically coupled RNA population

BackgroundThe RNA World hypothesis offers a plausible bridge from no-life to life on prebiotic Earth, by assuming that RNA, the only known molecule type capable of playing genetic and catalytic roles at the same time, could have been the first evolvable entity on the evolutionary path to the first living cell. We have developed the Metabolically Coupled Replicator System (MCRS), a spatially explicit simulation modelling approach to prebiotic RNA-World evolution on mineral surfaces, in which we incorporate the most important experimental facts and theoretical considerations to comply with recent knowledge on RNA and prebiotic evolution. In this paper the MCRS model framework has been extended in order to investigate the dynamical and evolutionary consequences of adding an important physico-chemical detail, namely explicit replicator structure – nucleotide sequence and 2D folding calculated from thermodynamical criteria – and their possible mutational changes, to the assumptions of a previously less detailed toy model.ResultsFor each mutable nucleotide sequence the corresponding 2D folded structure with minimum free energy is calculated, which in turn is used to determine the fitness components (degradation rate, replicability and metabolic enzyme activity) of the replicator. We show that the community of such replicators providing the monomer supply for their own replication by evolving metabolic enzyme activities features an improved propensity for stable coexistence and structural adaptation. These evolutionary advantages are due to the emergent uniformity of metabolic replicator fitnesses imposed on the community by local group selection and attained through replicator trait convergence, i.e., the tendency of replicator lengths, ribozyme activities and population sizes to become similar between the coevolving replicator species that are otherwise both structurally and functionally different.ConclusionsIn the most general terms it is the surprisingly high extra viability of the metabolic replicator system that the present model adds to the MCRS concept of the origin of life. Surface-bound, metabolically coupled RNA replicators tend to evolve different, enzymatically active sites within thermodynamically stable secondary structures, and the system as a whole evolves towards the robust coexistence of a complete set of such ribozymes driving the metabolism producing monomers for their own replication.ReviewersThis article was reviewed by Gáspár Jékely, Anthony Poole and Armen Mulkidjanian

Control of alphavirus-based gene expression using engineered riboswitches

Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust gene expression and immune responses. To further explore their use in vaccination, replicons were engineered to allow conditional control over their gene expression. Riboswitches, comprising a ribozyme actuator and RNA aptamer sensor, were engineered into the replicon 3′ UTR. Binding of ligand to aptamer modulates ribozyme activity and, therefore, gene expression. Expression from DNA-launched and VRP-packaged replicons containing riboswitches was successfully regulated, achieving a 47-fold change in expression and modulation of the resulting type I interferon response. Moreover, we developed a novel control architecture where riboswitches were integrated into the 3′ and 5′ UTR of the subgenomic RNA region of the TC-83 virus, leading to an 1160-fold regulation of viral replication. Our studies demonstrate that the use of riboswitches for control of RNA replicon expression and viral replication holds promise for development of novel and safer vaccination strategies.

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.

Modulation of group I ribozyme activity by cationic porphyrins.

The effects of cationic porphyrins on the catalytic activities of four group I ribozymes were investigated. A cationic porphyrin possessing four pyridinium moieties (pPyP) inhibited two group IC3 ribozymes (Syn Rz and Azo Rz) and a group IC1 ribozyme (Tet Rz). In the case of a group IA2 ribozyme (Td Rz), however, pPyP served not only as an inhibitor but also as an activator, and the effects of pPyP were dependent on its concentration. To analyze the structural and electronic factors determining the effects of pPyP on group I ribozymes, three cationic porphyrins (pPyNCP, pPyF4P, and TMPyP) were also examined. As interactions between small organic molecules and nucleic acids are attractive and important issues in biochemistry and biotechnology, this study contributes to the development of porphyrin-based molecules that can modulate functions of structured RNA molecules.

Dead Element Replicating: Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect (Insecta: Phasmida)

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.