ViroidDB: a database of viroids and viroid-like circular RNAs.

We introduce ViroidDB, a value-added database that attempts to collect all known viroid and viroid-like circular RNA sequences into a single resource. Spanning about 10 000 unique sequences, ViroidDB includes viroids, retroviroid-like elements, small circular satellite RNAs, ribozyviruses, and retrozymes. Each sequence's secondary structure, ribozyme content, and cluster membership are predicted via a custom pipeline optimized for handling circular RNAs. The data can be explored via a purpose-built user interface that features visualizations, multiple sequence alignments, and a portal for downloading bulk data. Users can browse the data by sequence type, taxon, or typo-tolerant search of metadata fields. The database is freely accessible at https://viroids.org.

Hovlinc is a recently evolved class of ribozyme found in human lncRNA.

Although naturally occurring catalytic RNA molecules-ribozymes-have attracted a great deal of research interest, very few have been identified in humans. Here, we developed a genome-wide approach to discovering self-cleaving ribozymes and identified a naturally occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to an unidentified class of small, self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path, from its appearance between ~130 and ~65 million years ago (Ma), to acquiring self-cleavage activity very recently, ~13-10 Ma, in the common ancestors of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within a long noncoding RNA belonging to a class of very long intergenic noncoding RNAs. The presence of a catalytic RNA enzyme in lncRNA creates the possibility that these transcripts could function by carrying catalytic RNA domains.

Virus-associated ribozymes and nano carriers against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoo tonic, highly pathogenic virus. The new type of coronavirus with contagious nature spread from Wuhan (China) to the whole world in a very short time and caused the new coronavirus disease (COVID-19). COVID-19 has turned into a global public health crisis due to spreading by close person-to-person contact with high transmission capacity. Thus, research about the treatment of the damages caused by the virus or prevention from infection increases everyday. Besides, there is still no approved and definitive, standardized treatment for COVID-19. However, this disaster experienced by human beings has made us realize the significance of having a system ready for use to prevent humanity from viral attacks without wasting time. As is known, nanocarriers can be targeted to the desired cells in vitro and in vivo. The nano-carrier system targeting a specific protein, containing the enzyme inhibiting the action of the virus can be developed. The system can be used by simple modifications when we encounter another virus epidemic in the future. In this review, we present a potential treatment method consisting of a nanoparticle-ribozyme conjugate, targeting ACE-2 receptors by reviewing the virus-associated ribozymes, their structures, types and working mechanisms.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB).

BACKGROUND: The rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P's sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date. RESULTS: Of the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation. CONCLUSIONS: The coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

Group I introns are widespread in archaea.

Group I catalytic introns have been found in bacterial, viral, organellar, and some eukaryotic genomes, but not in archaea. All known archaeal introns are bulge-helix-bulge (BHB) introns, with the exception of a few group II introns. It has been proposed that BHB introns arose from extinct group I intron ancestors, much like eukaryotic spliceosomal introns are thought to have descended from group II introns. However, group I introns have little sequence conservation, making them difficult to detect with standard sequence similarity searches. Taking advantage of recent improvements in a computational homology search method that accounts for both conserved sequence and RNA secondary structure, we have identified 39 group I introns in a wide range of archaeal phyla, including examples of group I introns and BHB introns in the same host gene.

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes.

Recent structural and mechanistic studies have shed considerable light on the catalytic mechanisms of nucleolytic ribozymes. The discovery of several new ribozymes in this class has now allowed comparisons to be made, and the beginnings of mechanistic groupings to emerge.

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 ŠX-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

The RNA origin of transfer RNA aminoacylation and beyond.

Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA aminoacylation, in the primitive translation system RNA molecules could have catalysed aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.

An expanded collection and refined consensus model of glmS ribozymes.

Self-cleaving glmS ribozymes selectively bind glucosamine-6-phosphate (GlcN6P) and use this metabolite as a cofactor to promote self-cleavage by internal phosphoester transfer. Representatives of the glmS ribozyme class are found in Gram-positive bacteria where they reside in the 5' untranslated regions (UTRs) of glmS messenger RNAs that code for the essential enzyme L-glutamine:D-fructose-6-phosphate aminotransferase. By using comparative sequence analyses, we have expanded the number of glmS ribozyme representatives from 160 to 463. All but two glmS ribozymes are present in glmS mRNAs and most exhibit striking uniformity in sequence and structure, which are features that make representatives attractive targets for antibacterial drug development. However, our discovery of rare variants broadens the consensus sequence and structure model. For example, in the Deinococcus-Thermus phylum, several structural variants exist that carry additional stems within the catalytic core and changes to the architecture of core-supporting substructures. These findings reveal that glmS ribozymes have a broader phylogenetic distribution than previously known and suggest that additional rare structural variants may remain to be discovered.

MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches.

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines.

Group II introns in eubacteria and archaea: ORF-less introns and new varieties.

Group II introns are a major class of ribozymes found in bacteria, mitochondria, and plastids. Many introns contain reverse transcriptase open reading frames (ORFs) that confer mobility to the introns and allow them to persist as selfish DNAs. Here, we report an updated compilation of group II introns in Eubacteria and Archaea comprising 234 introns. One new phylogenetic class is identified, as well as several specialized lineages. In addition, we undertake a detailed search for ORF-less group II introns in bacterial genomes in order to find undiscovered introns that either entirely lack an ORF or encode a novel ORF. Unlike organellar group II introns, we find only a handful of ORF-less introns in bacteria, suggesting that if a substantial number exist, they must be divergent from known introns. Together, these results highlight the retroelement character of bacterial group II introns, and suggest that their long-term survival is dependent upon retromobility.

A glimpse into the active site of a group II intron and maybe the spliceosome, too.

The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.

LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes.

Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg(2+) concentrations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios.

Bacterial group II introns: not just splicing.

Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.

Construction of an artificial ribozyme which ligates an RNA fragment activated by nicotinamide mononucleotide.

A new strategy termed "Design & Selection" has been developed for construction of artificial ribozymes. In this strategy, two distinct approaches (de novo rational design and in vitro selection) were successfully combined. De novo rational design was employed for construction of a structural scaffold of a new ribozyme whereas in vitro selection was adopted for isolation of a catalytic unit, sequence and structure of which were unpredictable. Using this strategy, a ligase ribozyme (DSL ribozyme) has been isolated. To validate versatility of Design & Selection strategy, we attempted to isolate a new catalytic unit utilizing a nicotinamide mononucleotide as a leaving group in RNA-RNA ligation reaction. Into the de novo designed scaffold RNA, 45 random nucleotides were inserted. The resulting RNA library consisting of the scaffold and randomized regions was subjected to in vitro selection. Using the library with 10(14) sequence diversity, a new class of ribozyme bearing a novel catalytic unit sequence was successfully isolated.

Gene targeting in the Gram-Positive bacterium Lactococcus lactis, using various delta ribozymes.

The trans-acting antigenomic delta ribozyme, isolated from the human hepatitis delta virus, was shown to be highly stable and active in vitro, as well as in mammalian cell lines. However, the stability and gene-targeting competence of this small ribozyme have not been studied previously in bacterial cells. In this paper we describe the use of two variants of the trans-acting antigenomic delta ribozyme targeting the abundant EF-Tu mRNA in the industrially important gram-positive bacterium Lactococcus lactis. These two delta ribozyme variants were expressed at significant levels and were shown to be highly stable in vivo. The half-life of the EF-Tu mRNA was slightly but consistently reduced in the presence of the classical delta ribozymes (7 to 13%). In contrast, delta ribozymes harboring a specific on/off riboswitch (SOFA-delta ribozymes) targeting the same sites on the EF-Tu mRNA considerably reduced the half-life of this mRNA (22 to 47%). The rates of catalysis of the SOFA-delta ribozymes in L. lactis were similar to the rates determined in vitro, showing that this new generation of delta ribozymes was highly efficient in these bacterial cells. Clearly, SOFA-delta ribozymes appear to be an ideal means for development of gene inactivation systems in bacteria.

Competitive regulation of modular allosteric aptazymes by a small molecule and oligonucleotide effector.

The hairpin ribozyme can catalyze the cleavage of RNA substrates by employing its conformational flexibility. To form a catalytic complex, the two domains A and B of the hairpin-ribozyme complex must interact with one another in a folding step called docking. We have constructed hairpin ribozyme variants harboring an aptamer sequence that can be allosterically induced by flavin mononucleotide (FMN). Domains A and B are separated by distinct bridge sequences that communicate the formation of the FMN-aptamer complex to domains A and B, facilitating their docking. In the presence of a short oligonucleotide that is complementary to the aptamer, catalytic activity of the ribozyme is completely abolished, due to the formation of an extended conformer that cannot perform catalysis. However, in the presence of the small molecule effector FMN, the inhibitory effect of the oligonucleotide is competitively neutralized and the ribozyme is activated 150-fold. We thus have established a new principle for the regulation of ribozyme catalysis in which two regulatory factors (an oligonucleotide and a small molecule) that switch the ribozyme's activity in opposite directions compete for the same binding site in the aptamer domain.

Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities.

BACKGROUND: Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization. RESULTS: Stem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl2 concentrations as low as 0.2 mM at 25 degrees C and 0.5 mM at 37 degrees C. Under multiple-turnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl2 concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active. CONCLUSION: Hammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the stabilization and substrate recognition functions of stem 1 increases cleavage activity at physiological concentrations of divalent magnesium while retaining recognition of exogenous targets. Trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of all natural hammerhead topologies can therefore be used in intracellular applications.

The promise and peril of continuous in vitro evolution.

Experimental evolution methods can be used to address and illuminate issues central to the understanding of evolutionary theory. One of the most powerful of these methods involves the in vitro evolution of nucleic acid enzymes, taking advantage of the direct relationship between the genotype of a nucleic acid sequence and the phenotype of its associated catalytic function. This review and commentary focuses on the past, present, and future potential of systems for the continuous in vitro evolution of nucleic acid enzymes as tools for modeling evolutionary processes in biology. It offers a candid appraisal of both the strengths and the limitations of these systems.

Structure and assembly of group I introns.

Self-splicing group I introns have served as a model for RNA catalysis and folding for over two decades. New three-dimensional structures now bring the details into view. Revelations include an unanticipated turn in the RNA backbone around the guanosine-binding pocket. Two metal ions in the active site coordinate the substrate and phosphates from all three helical domains.