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Nucleic acid-based gene interference and editing strategies, such as antisense oligonucleotides, ribozymes, RNA interference (RNAi), and CRISPR/Cas9 coupled with guide RNAs, are exciting research tools and show great promise for clinical applications in treating various illnesses. RNase P ribozymes have been engineered for therapeutic applications against human viruses such as human cytomegalovirus (HCMV). M1 ribozyme, the catalytic RNA subunit of RNase P from Escherichia coli, can be converted into a sequence-specific endonuclease, M1GS ribozyme, which is capable of hydrolyzing an mRNA target base-pairing with the guide sequence. M1GS RNAs have been shown to hydrolyze essential HCMV mRNAs and block viral progeny production in virus-infected cell cultures. Furthermore, RNase P ribozyme variants with enhanced hydrolyzing activity can be generated by employing in vitro selection procedures and exhibit better ability in suppressing HCMV gene expression and replication in cultured cells. Additional studies have also examined the antiviral activity of RNase P ribozymes in mice in vivo. Using cytomegalovirus infection as an example, this review summarizes the principles underlying RNase P ribozyme-mediated gene inactivation, presents recent progress in engineering RNase P ribozymes for applications in vitro and in mice, and discusses the prospects of using M1GS technology for therapeutic applications against HCMV as well as other pathogenic viruses.

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Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.

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Sidney Altman's discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P-related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere.

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Ribonuclease P (RNase P), which may consist of both protein subunits and a catalytic RNA part, is responsible for 5' maturation of tRNA by cleaving the 5'-leader sequence. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 RNA) and a protein factor (C5 protein). In human cells, RNase P holoenzyme consists of an RNA subunit (H1 RNA) and multiple protein subunits that include human RPP29 protein. M1GS, a sequence specific targeting ribozyme derived from M1 RNA, can be constructed to target a specific mRNA to degrade it in vitro. Recent studies have shown that M1GS ribozymes are efficient in blocking the expression of viral mRNAs in cultured cells and in animals. These results suggest that RNase P ribozymes have the potential to be useful in basic research and in clinical applications. It has been shown that RNase P binding proteins, such as C5 protein and RPP29, can enhance the activities of M1GS RNA in processing a natural tRNA substrate and a target mRNA. Understanding how RPP29 binds to M1GS RNA and enhances the enzyme's catalytic activity will provide great insight into developing more robust gene-targeting ribozymes for in vivo application. In this chapter, we describe the methods of using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to determine the regions of a M1GS ribozyme that are in proximity to RPP29 protein.

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Kaposi's sarcoma, an AIDS-defining illness, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus. In this study, we engineered ribozymes derived from ribonuclease P (RNase P) catalytic RNA with targeting against the mRNA encoding KSHV immediate early replication and transcription activator (RTA), which is vital for KSHV gene expression. The functional ribozyme F-RTA efficiently sliced the RTA mRNA sequence in vitro. In cells, KSHV production was suppressed with ribozyme F-RTA expression by 250-fold, and RTA expression was suppressed by 92-94%. In contrast, expression of control ribozymes hardly affected RTA expression or viral production. Further studies revealed both overall KSHV early and late gene expression and viral growth decreased because of F-RTA-facilitated suppression of RTA expression. Our results indicate the first instance of RNase P ribozymes having potential for use in anti-KSHV therapy.

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Retrozymes are nonautonomous retrotransposons with hammerhead ribozymes in their long terminal repeats (LTRs). Retrozyme transcripts can be self-cleaved by the LTR ribozyme, circularized, and can undergo RNA-to-RNA replication. Here, we demonstrate that the Nicotiana benthamiana genome contains hundreds of retrozyme loci, of which nine represent full-length retrozymes. The LTR contains a promoter directing retrozyme transcription. Although retrozyme RNA is easily detected in plants, the LTR region is heavily methylated, pointing to its transcriptional silencing, which can be mediated by 24 nucleotide-long retrozyme-specific RNAs identified in N. benthamiana. A transcriptome analysis revealed that half of the retrozyme-specific RNAs in plant leaves have no exact matches to genomic retrozyme loci, containing up to 13% mismatches with the closest genomic sequences, and could arise as a result of many rounds of RNA-to-RNA replication leading to error accumulation. Using a cloned retrozyme copy, we show that retrozyme RNA is capable of replication and systemic transport in plants. The presented data suggest that retrozyme loci in the N. benthamiana genome are transcriptionally inactive, and that circular retrozyme RNA can persist in cells due to its RNA-to-RNA replication and be transported systemically, emphasizing functional and, possibly, evolutionary links of retrozymes to viroids-noncoding circular RNAs that infect plants.

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Naturally occurring ribozymes with defined three-dimensional (3D) structures serve as promising platforms for the design and construction of artificial RNA nanostructures. We constructed a hexameric ribozyme nanostructure by face-to-face dimerization of a pair of triangular ribozyme trimers, unit RNAs of which were derived from the Tetrahymena group I ribozyme. In this study, we have expanded the dimerization strategy to a square-shaped ribozyme tetramer by introducing four pillar units. The resulting box-shaped nanostructures, which contained eight ribozyme units, can be assembled from either four or two components of their unit RNAs.

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Ribozymes are folded catalytic RNA molecules that perform important biological functions. Since the discovery of the first RNA with catalytic activity in 1982, a large number of ribozymes have been reported. While most catalytic RNA molecules act alone, some RNA-based catalysts, such as RNase P, the ribosome, and the spliceosome, need protein components to perform their functions in the cell. In the last decades, the structure and mechanism of several ribozymes have been studied in detail. Aside from the ribosome, which catalyzes peptide bond formation during protein synthesis, the majority of known ribozymes carry out mostly phosphoryl transfer reactions, notably trans-esterification or hydrolysis reactions. In this review, we describe the main features of the mechanisms of various types of ribozymes that can function with or without the help of proteins to perform their biological functions.

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Human hepatitis delta virus (HDV) is a unique infectious agent whose genome is composed of a small circular RNA. Recent data, however, have reported the existence of highly divergent HDV-like circRNAs in the transcriptomes of diverse vertebrate and invertebrate species. The HDV-like genomes described in amniotes such as birds and reptiles encode self-cleaving RNA motifs or ribozymes similar to the ones present in the human HDV, whereas no catalytic RNA domains have been reported for the HDV-like genomes detected in metagenomic data from some amphibians, fish, and invertebrates. Herein, we describe the self-cleaving motifs of the HDV-like genomes reported in newts and fish, which belong to the characteristic class of HDV ribozymes. Surprisingly, HDV-like genomes from a toad and a termite show conserved type III hammerhead ribozymes, which belong to an unrelated class of catalytic RNAs characteristic of plant genomes and plant subviral circRNAs, such as some viral satellites and viroids. Sequence analyses revealed the presence of similar HDV-like hammerhead ribozymes encoded in two termite genomes, but also in the genomes of several dipteran species. In vitro transcriptions confirmed the cleaving activity for these motifs, with moderate rates of self-cleavage. These data indicate that all described HDV-like agents contain self-cleaving motifs from either the HDV or the hammerhead class. Autocatalytic ribozymes in HDV-like genomes could be regarded as interchangeable domains and may have arisen from cellular transcriptomes, although we still cannot rule out some other evolutionary explanations.

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Retrozymes are a novel family of non-autonomous retrotransposable elements that contain hammerhead ribozyme motifs. These retroelements are found widespread in eukaryotic genomes, with active copies present in many species, which rely on other autonomous transposons for mobilization. Contrary to other retrotransposons, transcription of retrozymes in vivo leads to the formation and accumulation of circular RNAs, which can be readily detected by RNA blotting. In this chapter, we describe the procedures needed to carry out the cloning of genomic retrozymes, and to detect by northern blot their circular RNA retrotransposition intermediates.

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Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.

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RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA's resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme (L-Rz, L-HH) that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson-Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics.

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Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates. The morphologies of the assembled ribozyme structures were observed directly by atomic force microscopy (AFM).

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The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing (nonenzymatically) to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication.

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The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient biological assemblies that were already present in the last universal common ancestor of all life. The large subunit rRNA in the ribosome and the RNA subunit of RNase P are the ribozyme components required for catalysis. Here, we explore the idea that these two large ribozymes may have begun their evolutionary odyssey as an assemblage of RNA "fragments" smaller than the contemporary full-length versions and that they transitioned through distinct stages along a pathway that may also be relevant for the evolution of other non-coding RNAs.

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Ribozymes are functional RNA molecules that can catalyze biochemical reactions. Since the discovery of the first catalytic RNA, various functional ribozymes (e.g., self-cleaving ribozymes, splicing ribozymes, RNase P, etc.) have been uncovered, and their structures and mechanisms have been identified. Ribozymes have the advantage of possessing features of "RNA" molecules; hence, they are highly applicable for manipulating various biological systems. To fully employ ribozymes in a broad range of biological applications in synthetic biology, a variety of ribozymes have been developed and engineered. Here, we summarize the main features of ribozymes and the methods used for engineering their functions. We also describe the past and recent efforts towards exploiting ribozymes for effective and novel applications in synthetic biology. Based on studies on their significance in biological applications till date, ribozymes are expected to advance technologies in artificial biological systems.

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BACKGROUND: The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial RNA processing is currently not known. Here, we report experimental support for unconventional processing events of ND5-717 containing RNA. RESULTS: We obtained the complete mitochondrial genome sequences and corresponding mitochondrial transcriptomes of the two distantly related corallimorpharian species Ricordea yuma and Amplexidiscus fenestrafer. All mitochondrial genes were found to be expressed at the RNA-level. Both introns were perfectly removed by autocatalytic splicing, but COI-884 excision appeared more efficient than ND5-717. ND5-717 was organized into giant group I intron elements of 18.1 kb and 19.3 kb in A. fenestrafer and R. yuma, respectively. The intron harbored almost the entire mitochondrial genome embedded within the P8 peripheral segment. CONCLUSION: ND5-717 was removed by group I intron splicing from a small primary transcript that contained a permutated intron-exon arrangement. The splicing pathway involved a circular exon-containing RNA intermediate, which is a hallmark of RNA back-splicing. ND5-717 represents the first reported natural group I intron that becomes excised by back-splicing from a permuted precursor RNA. Back-splicing may explain why Corallimorpharia mitochondrial genomes tolerate giant group I introns.

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Bimolecular ribozymes derived by physical dissection of unimolecular ribozymes consisting of two structural modules are promising platforms for the design and construction of assembled RNA nanostructures. Unit RNAs to be assembled intermolecularly into one-dimensional (1D) oligomers are designed by reconnecting the two structural modules in a manner different from the parent ribozymes. This strategy was applied to the Tetrahymena group I ribozyme. We constructed 1D ribozyme oligomers the assembly of which was observed by atomic force microscopy (AFM) and also controlled rationally to design a heterooctamer by differentiating the interface between the two modules.

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Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causal Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite's growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene of interest to understand its importance and function. However, these approaches can be limited by the availability of selectable markers and the time required to generate transgenic parasites. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a ribozyme-guide-ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free parasites with homology arms as short as 80-100 bp. Additionally, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and desired locus replacement editing events, whereas the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically dead SpCas9 (dSpCas9) to the region upstream of a gene of interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates efficient genetic characterizations of rodent-infectious Plasmodium species.

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The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNALeu differed by about 104-fold. We further established the critical role for the A76 3'-OH group of the tRNALeu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNALeu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.

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