Ribozyme Cleavage Research Articles

New Insights into the Dependence of CPEB3 Ribozyme Cleavage on Mn2+ and Mg2.

CPEB3 ribozyme is a self-cleaving RNA that occurs naturally in mammals and requires divalent metal ions for efficient activity. Ribozymes exhibit preferences for specific metal ions, but the exact differences in the catalytic mechanisms of various metal ions on the CPEB3 ribozyme remain unclear. Our findings reveal that Mn2+ functions as a more effective cofactor for CPEB3 ribozyme catalysis compared to Mg2+, as confirmed by its stronger binding affinity to CPEB3 by EPR. Cleavage assays of CPEB3 mutants and molecular docking analyses further showed that excessive Mn2+ ions can bind to a second binding site near the catalytic site, hindering CPEB3 catalytic efficiency and contributing to the Mn2+ bell-shaped curve. These results implicate a pivotal role for the local nucleobase-Mn2+ interactions in facilitating RNA folding and modulating the directed attack of nucleophilic reagents. Our study provides new insights and experimental evidence for exploring the divalent cation dependent cleavage mechanism of the CPEB3 ribozyme.

A novel viral RNA detection method based on the combined use of trans-acting ribozymes and HCR-FRET analyses.

The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis.

Cell-Free Biosensing Genetic Circuit Coupled with Ribozyme Cleavage Reaction for Rapid and Sensitive Detection of Small Molecules.

Synthetic biological systems have been utilized to develop a wide range of genetic circuits and components that enhance the performance of biosensing systems. Among them, cell-free systems are emerging as important platforms for synthetic biology applications. Genetic circuits play an essential role in cell-free systems, mainly consisting of sensing modules, regulation modules, and signal output modules. Currently, fluorescent proteins and aptamers are commonly used as signal outputs. However, these signal output modes cannot simultaneously achieve faster signal output, more accurate and reliable performance, and signal amplification. Ribozyme is a highly structured and catalytic RNA molecule that can specifically recognize and cut specific substrate sequences. Here, by adopting ribozyme as the signal output, we developed a cell-free biosensing genetic circuit coupled with the ribozyme cleavage reaction, enabling rapid and sensitive detection of small molecules. More importantly, we have also successfully constructed a 3D-printed sensor array and thereby achieved high-throughput analysis of an inhibitory drug. Furthermore, our method will help expand the application range of ribozyme in the field of synthetic biology and also optimize the signal output system of cell-free biosensing, thus promoting the development of cell-free synthetic biology in biomedical research, clinical diagnosis, environmental monitoring, and food inspection.

Kinetic competition between theophylline sensitive ribozyme cleavage and termination in living cells by 3D orbital tracking-fluorescent cross-correlation spectroscopy.

Synthetic RNA-based regulatory tools such as riboswitches are promising candidates for the realization of complex engineered genetic circuits. However, previous efforts to implement such tools in synthetic biology have yielded inconsistent results. Often the quantification of performance is measured using protein expression, even though these devices act at the RNA level. To overcome this, we use in vivo RNA labeling of Hemoglobin B in Human Epithelial Kidney T-Rex Cells to determine two objectives. First, we will determine whether a theophylline-dependent hammerhead ribozyme riboswitch alters gene expression by inducing early termination of transcription or via post-transcriptional mRNA cleavage. The second objective will use a self-cleaving hammerhead ribozyme to separate transcription elongation from termination. This will allow for the determination of transcription termination kinetics. Quantification for both objectives will be completed using 3D Orbital Tracking-Fluorescent Cross-Correlation Spectroscopy (3DOT-FCCS). To determine the levels of post-transcription mRNA cleavage, we will use Raster Image Correlation Spectroscopy (RICS) to measure mRNA stability via mRNA lifetime. Preliminary data suggests that the theophylline aptamer stabilizes the ribozyme, correlating to an increased RNA dwell time and termination. However, when the aptamer is absent, there is a decrease. This indicates early termination and premature transcript release from the DNA template are occurring with the ribozyme. In the future, we will examine the transcribing order of Intron 1 and Intron 2, splicing behaviors, post-termination splicing using RICS, and RNA lifetime.

Carba-Sugar Analogs of Glucosamine-6-Phosphate: New Activators for the glmS Riboswitch.

Riboswitches are 5'-untranslated mRNA regions mostly found in bacteria. They are promising drug targets to overcome emerging bacterial resistance against commonly used antibiotics. The glmS riboswitch is unique among the family of riboswitches as it is a ribozyme that undergoes self-cleavage upon binding to glucosamine-6-phosphate (GlcN6P). Previously, we showed that carba glucosamine-6-phosphate (carba-GlcN6P) induces self-cleavage of the riboswitch with a potency similar to that of GlcN6P. Here, we report a synthetic approach to a new class of carba-GlcN6P derivatives with an alkoxy substituent in the carba position. Key features of the synthesis are a ring closing metathesis followed by a hydroboration. The strategy gives access to libraries of carba-GlcN6P derivatives. Ribozyme cleavage assays unraveled new activators for the glmS riboswitch from Listeria monocytogenes and Clostridium difficile.

Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes.

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labeled with a fluorophore at its 5'-end, while the other strand is labeled with a quencher at its 3'-end. These two DNA strands are perfectly complementary, but with a 3'-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

CyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2ʹ,3ʹ cyclic phosphates and 5ʹ hydroxyl ends

ABSTRACT Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5′-fragment with a 2′,3′-cyclic phosphate and a 3′-fragment with a 5′-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5′-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3′-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms.

Biochemical analysis of cleavage and ligation activities of the pistol ribozyme from Paenibacillus polymyxa

ABSTRACT Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium Paenibacillus polymyxa. We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg2+ concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the P. polymyxa pistol ribozyme – an aspect that has not been previously analysed for this ribozyme class. We determined that the P. polymyxa pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from P. polymyxa. We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.

Engineering Allosteric Ribozymes to Detect Thiamine Pyrophosphate in Whole Blood.

Thiamine deficiency contributes to several human diseases including Alzheimer's. As its biologically active form, thiamine pyrophosphate (TPP) has been considered as a potential biomarker for Alzheimer's disease (AD) based on several clinical reports that apparently lower blood TPP levels were found in patients with mild cognitive impairment to AD. However, highly sensitive and high-throughput detection of TPP in biological fluids remains an analytical challenge. Here, we report engineering RNA-based sensors to quantitatively measure TPP concentrations in whole blood samples with a detection limit down to a few nM. By fusing a TPP-specific aptamer with the hammerhead ribozyme for in vitro selection, we isolated an allosteric ribozyme with an EC50 value (68 nM) similar to the aptamer's KD value (50 nM) for TPP, which for the first time demonstrates the possibility to maintain the effector binding affinity of the aptamer in such engineered allosteric RNA constructs. Meanwhile, we developed a new blood sample preparation protocol to be compatible with RNA. By coupling the TPP-induced ribozyme cleavage event with isothermal amplification, we achieved fluorescence monitoring of whole blood TPP levels through the "mix-and-read" operation with high-throughput potential. We expect that the engineered TPP-sensing RNAs will facilitate clinical research on AD as well as other thiamine-related diseases.

Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage

Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA-binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2'-hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase-mediated general acid-base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small-molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme.

A Modified In Vitro Transcription Approach to Improve RNA Synthesis and Ribozyme Cleavage Efficiency.

RNA elements such as catalytic RNA, riboswitch, microRNA, and long non-coding RNA perform a major role in cellular processes. A complete understanding of cellular processes is impossible without knowing the structure-function relationship of participating RNA molecules that ultimately requires large quantities of pure RNAs. Thus, structural/functional analyses of emerging RNAs necessitate revised protocols for improved RNA quantity and quality. Here we present a modified in vitro transcription protocol to enhance ribozyme cleaving efficiency and RNA yield by working on two variables, i.e., incubation temperature and limiting GTPs. Following an improved RNA synthesis, the target RNA is purified from transcription mixture components through denaturing size-exclusion chromatography. The protocol confirms that cyclic elevated incubation temperatures during transcription and increased concentrations of GTPs improve the production rate of RNA. Our modified in vitro transcription method improves the ribozyme cleaving efficiency and targets RNA yield by four- to fivefold that can benefit almost any RNA-related study from protein-RNA interaction analysis to crystallography.

Engineering a Glucosamine-6-phosphate Responsive glmS Ribozyme Switch Enables Dynamic Control of Metabolic Flux in Bacillus subtilis for Overproduction of N-Acetylglucosamine.

Bacillus subtilis is a typical industrial microorganism and is widely used in industrial biotechnology, particularly for nutraceutical production. There are many studies on the static metabolic engineering of B.subtilis, whereas there are few reports on dynamic metabolic engineering due to the lack of appropriate elements. Here, we established a dynamic reprogramming strategy for reconstructing metabolic networks in B.subtilis, using a typical nutraceutical, N-acetylglucosamine (GlcNAc), as a model product and the glmS (encoding glucosamine-6-phosphate synthase) ribozyme as an engineering element. First, a trp terminator was introduced to effectively release the glmS ribozyme feedback inhibition. Further, we engineered the native glucosamine-6-phosphate (GlcN6P) responsive glmS ribozyme switch to dynamically control the metabolic flux in B.subtilis for overproduction of GlcNAc. With GlcN6P as a ligand, the native sensor glmS ribozyme is integrated at the 5'- of phosphoglucosamine mutase and 6-phosphofructokinase genes to decrease the flux dynamically toward the peptidoglycan synthesis and glycolysis pathway, respectively. The glmS ribozyme mutant M5 ( glmS ribozyme cleavage site AG → GG) with decreased ribozyme activity is integrated at the 5'- of glucose-6-phosphate isomerase gene to increase the flux dynamically toward the GlcNAc synthesis pathway. This strategy increased the GlcNAc titer from 9.24 to 18.45 g/L, and the specific GlcNAc productivity from 0.53 to 1.21 g GlcNAc/g cell. Since GlcN6P is involved in the biosynthesis of various products, here the developed strategy for multiple target dynamic engineering of metabolic pathways can be generally used in B.subtilis and other industrial microbes for chemical production.

Highly motif- and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression

ABSTRACTRecent 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.

In Planta Processing of the SpCas9-gRNA Complex.

In CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)-mediated genome editing in plants, Streptococcus pyogenes Cas9 (SpCas9) protein and the required guide RNA (gRNA) are, in most cases, expressed from a stably integrated transgene. Generally, SpCas9 protein is expressed from an RNA polymerase (pol) II promoter, while gRNA is expressed from a pol III promoter. However, pol III promoters have not been much characterized other than in model plants, making it difficult to select appropriate promoters for specific applications, while pol II transcripts have to be processed to generate functional gRNAs. Recently, successful processing of a pol II transcript into functional gRNAs using ribozyme or Csy4-RNA cleavage systems has been demonstrated. Here, we show that functional gRNAs can be efficiently processed using SpCas9 protein and plant endogenous RNA cleavage systems without the need for a specific RNA processing system. In our system, SpCas9 RNA and gRNA are both transcribed as a single RNA using a single pol II promoter; translated SpCas9 protein can be bound to this RNA and, finally, extra RNA sequences are trimmed by plant RNA processing systems to form a functional SpCas9–gRNA complex. The efficiency of targeted mutagenesis using our novel SpCas9–gRNA fused system was comparable with that of the SpCas9–gRNA system with ribozyme sequence, achieving rates of up to 100% in rice. Our results could be useful in developing stable SpCas9–gRNA expression systems and in RNA virus vector-mediated genome editing systems in plants.

The 3′-untranslated region of mRNAs as a site for ribozyme cleavage-dependent processing and control in bacteria

ABSTRACTBesides 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.

Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.

The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

BackgroundRecovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus.MethodsIn this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5′ termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus.ResultsOptimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency.ConclusionOur study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

The structure of a nucleolytic ribozyme that employs a catalytic metal ion.

The TS ribozyme (originally called twister-sister) is a nucleolytic catalytic RNA. We present a crystal structure of the ribozyme in a pre-reactive conformation. Two co-axial helical stacks are organized by a three-way junction and two tertiary contacts. Five divalent metal ions are directly coordinated to RNA ligands, making important contributions to the RNA architecture. The scissile phosphate lies in a quasi-helical loop region that is organized by a network of hydrogen bonding. A divalent metal ion is directly bound to the nucleobase 5' to the scissile phosphate, with an inner-sphere water molecule positioned to interact with the O2' nucleophile. The rate of ribozyme cleavage correlated in a log-linear manner with divalent metal ion pKa, consistent with proton transfer in the transition state, and we propose the bound metal ion is a likely general base for the cleavage reaction. Our data indicate that the TS ribozyme functions predominantly as a metalloenzyme.