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Η RNase P είναι το ένζυμο που ωριμάζει το 5΄ άκρο των πρόδρομων μορίων tRNA, ενώ έχει βρεθεί και στις τρεις φυλογενετικές περιοχές, καθώς και σε υποκυτταρικά οργανίδια. Είναι ριβονουκλεοπρωτεϊνικής φύσεως στις περισσότερες περιπτώσεις, ενώ έχουν βρεθεί και ένζυμα RNase P αποκλειστικά πρωτεϊνικής φύσεως. Η υπομονάδα RNA των βακτηριακών ολοενζύμων είναι καταλυτικά ενεργή απουσία πρωτεϊνικών παραγόντων in vitro, καθιστώντας την ένα πραγματικό ριβοένζυμο. Η ικανότητα της RNase P να αναγνωρίζει συγκεκριμένες δομές στα μόρια των υποστρωμάτων της και όχι αλληλουχίες, δημιούργησε τη δυνατότητα χρήσης αυτού του ενζύμου ως ενός μοριακού εργαλείου για τη στόχευση πολλών ιικών και παθολογικών μορίων RNA in vitro και in vivo, καταστέλλοντας την έκφραση των μορίων αυτών, μέσω της τεχνολογίας των μορίων EGS (external guide sequence) και των ριβοενζύμων M1GS.Η RNase P, σύμφωνα με πολλές μελέτες, έχει δειχθεί ότι αποτελεί στόχο πολλών φαρμακευτικών παραγόντων, συμπεριλαμβανομένων πολλών γνωστών αντιβιοτικών, οι οποίοι κατά κύριο λόγο αναστέλλουν τη δραστικότητα του ενζύμου. Πρόσφατα δείχτηκε, μέσω αναλυτικής κινητικής μελέτης, ότι ένα μακρολίδιο, η σπιραμυκίνη, ενεργοποιεί σημαντικά τη δραστικότητα της βακτηριακής RNase P και του M1 RNA κατά ένα δοσοεξαρτώμενο τρόπο, λειτουργώντας έτσι ως μη-ειδικός ενεργοποιητής μικτού τύπου. Μέχρι σήμερα, στη διεθνή βιβλιογραφία, δεν έχει αναφερθεί άλλη ουσία η οποία προκαλεί θετική επίδραση στη δραστικότητα της RNase P. Στην παρούσα μελέτη, αρχικά μελετήθηκε η ενεργοποίηση της δραστικότητας της βακτηριακής RNase P και αποκαλύφθηκε ότι η σπιραμυκίνη δεν αλληλεπιδρά με ιοντικούς δεσμούς με το μόριο Μ1 RNA, αλλά προκαλεί αλλαγή διαμόρφωσης στο δομικό στοιχείο P10/11 του ριβοενζύμου. Το δομικό αυτό στοιχείο εμπλέκεται στην αναγνώριση του υποστρώματος, αποτέλεσμα το οποίο έρχεται σε συμφωνία με τις τιμές KD που προσδιορίστηκαν για το σύμπλοκο ριβοενζύμου–υποστρώματος, απουσία και παρουσία σπιραμυκίνης.Με δεδομένο ότι η σπιραμυκίνη δεν επηρεάζει την πρωτεϊνοσύνθεση ή τη δραστικότητα της RNase P των ευκαρυωτικών κυττάρων, κατασκευάστηκε ένα ριβοένζυμο M1GS, ώστε να ελεγχθεί η επίδραση του αντιβιοτικού στη δραστικότητα αυτού του ριβοενζύμου in vivo, σε καλλιεργούμενα ανθρώπινα κύτταρα ΗΕΚ293. Ως στόχος του συγκεκριμένου M1GS, επιλέχτηκε ο μεταγραφικός παράγοντας Ets2 λόγω της μεγάλης κλινικής σημασίας του, εφόσον έχει συσχετιστεί με αρκετούς τύπους καρκίνου και παθολογικές καταστάσεις, καθώς και με διαδικασίες διαφοροποίησης. Ο σπουδαίος ρόλος του Ets2, σε συνδυασμό με τα ελλιπή δεδομένα σχετικά με την έκφρασή του, είχαν αποτρέψει μέχρι σήμερα την αποτελεσματική στόχευσή του με τη χρήση των υπαρχουσών μεθοδολογιών που βασίζονται στο RNA, όπως το RNAi.Μετά από ανάλυση της δευτεροταγούς δομής του Ets2 mRNA, σχεδιάστηκαν δύο οδηγοί αλληλουχίες. Οι αλληλουχίες αυτές, αρχικά, δοκιμάστηκαν ως εξωτερικές οδηγοί αλληλουχίες (EGS) σε συνδυασμό με το βακτηριακό ολοένζυμο της RNase P. Η EGS303 (το νούμερο υποδεικνύει το νουκλεοτιδικό κατάλοιπο του στόχου που δρα η RNase P), εμφάνισε τη μεγαλύτερη ικανότητα να επάγει τη δράση της RNase P in vitro. Η οδηγός αυτή αλληλουχία, στη συνέχεια κλωνοποιήθηκε στο 3΄ άκρο του M1 RNA, παράγοντας το ριβοένζυμο M1GS303, το οποίο είναι δραστικό έναντι του μορίου–στόχου του in vitro. Η δραστικότητα του συγκεκριμένου ριβοενζύμου ενεργοποιείται εντυπωσιακά κατά 160% παρουσία σπιραμυκίνης. Προκειμένου να ελεγχθεί η δραστικότητα αυτού του ριβοενζύμου in vivo, το μόριο–στόχος και το ριβοένζυμο εκφράστηκαν ελεγχόμενα σε κύτταρα E. coli, προκαλώντας μείωση της έκφρασης του μορίου–στόχου από το M1GS303 κατά 95% μετά από 12 ώρες έκφρασης των μορίων. Μείωση στα ίδια επίπεδα ανιχνεύτηκε μόλις μετά από 4 ώρες έκφρασης εφόσον στα κύτταρα είχε προστεθεί σπιραμυκίνη, γεγονός που υποστηρίζει την εντυπωσιακά θετική επίδραση της σπιραμυκίνης επί της δραστικότητας του ριβοενζύμου.Η ίδια σειρά πειραμάτων επαναλήφθηκε σε ευκαρυωτικά κύτταρα, με έκφραση του ριβοενζύμου σε HEK293 κύτταρα. Η δραστικότητα του ριβοενζύμου προσδιορίστηκε ποιοτικά και ποσοτικά, από την έκφραση της χιμαιρικής φθορίζουσας πρωτεΐνης Ets2–EGFP (μόριο–στόχος), σε διαφορετικούς χρόνους έκφρασης. Παρατηρήθηκε ότι το M1GS δρα αποτελεσματικά έναντι του μορίου–στόχου του και σε ευκαρυωτικά κύτταρα in vivo, προκαλώντας μείωση στην έκφραση του Ets2, η οποία αυξάνεται επιπλέον παρουσία σπιραμυκίνης. Τα παραπάνω αποτελέσματα δείχνουν τη σημαντική ενεργοποίηση της δραστικότητας του M1GS σε ανθρώπινα κύτταρα και καθιστούν τη σπιραμυκίνη ένα σημαντικό ενεργοποιητή στη χρήση των ριβοενζύμων M1GS ως εργαλεία γονιδιακής αποσιώπησης. Ο συνδυασμός βελτιωμένων ριβοενζύμων M1GS με την παρουσία σπιραμυκίνης αυξάνει ακόμα περισσότερο την πρακτική χρήση της συγκεκριμένης τεχνολογίας τόσο in vitro όσο και in vivo, επιτυγχάνοντας ακόμα πιο αποτελεσματική αποσιώπηση της γονιδιακής έκφρασης.

A number of small, self-cleaving ribozyme classes have been identified including the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), glmS, twister, hatchet, pistol, and twister sister ribozymes. Within the active sites of these ribozymes, myriad functional groups contribute to catalysis. There has been extensive structure-function analysis of individual ribozymes, but the extent to which catalytic devices are shared across different ribozyme classes is unclear. As such, emergent catalytic principles for ribozymes may await discovery. Identification of conserved catalytic devices can deepen our understanding of RNA catalysis specifically and of enzymic catalysis generally. To probe similarities and differences amongst ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities amongst ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2′OH nucleophile (γ), neutralization of the non-bridging oxygens of the scissile phosphate (β), neutralization of the O5′ leaving group (δ), and in-line nucleophilic attack (α). In addition, we uncover conservation of two catalytic devices, each of which centers on the activation of the 2′OH nucleophile by a guanine: one to acidify the 2′OH by hydrogen bond donation to it (γ′) and one to acidify the 2′OH by releasing it from non-productive interactions by competitive hydrogen bonding (γ′′). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies, and help explain absence of U at ribozyme active sites. The identified γ′ and γ′′ catalytic strategies help unify the catalytic strategies shared amongst catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.

Some RNA classes require folding into the proper higher-order structures to exert their functions. Hammerhead ribozyme (HHR) requires a folding conformation stabilized by tertiary interaction for full activity. A rationally engineered HHR was developed that was inactive, but could be activated by a synthetic RNA-binding ligand, naphthyridine carbamate tetramer with Z-stilbene linker (Z-NCTS). Binding of Z-NCTS could induce the formation of an active folding structure and thereby restore ribozyme activity, where Z-NCTS acts as a molecular glue to bring two isolated RNA loops into contact with each other. Next, we designed a Z-NCTS-responsive genetic switch using the HHR sequence inserted into the 3' untranslated region as a cis-acting element. We demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z-NCTS and was functional in mammalian cells.

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

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

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

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

RNA is a promising biomaterial for self-assembly of nano-sized structures with a wide range of applications in nanotechnology and synthetic biology. Several RNA-based nanostructures have been reported, but most are unrelated to intracellular RNA, which possesses modular structures that are sufficiently large and complex to serve as catalysts to promote sophisticated chemical reactions. In this study, we designed dimeric RNA structures based on the Tetrahymena group I ribozyme. The resulting dimeric RNAs (tecto group I ribozyme; tecto-GIRz) exhibit catalytic ability that depended on controlled dimerization, by which a pair of ribozymes can be activated to perform cleavage and splicing reactions of two distinct substrates. Modular redesign of complex RNA structures affords large ribozymes for use as modules in RNA nanotechnology and RNA synthetic biology.

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

We have engineered a hairpin ribozyme that in a two-step reaction supports RNA recombination by catalysing the cleavage of two non-functional RNA precursors (first step) followed by the recombination of two of the cleavage fragments into a functional RNA (second step). The recombination product (yield: 76%) is a fully functional hammerhead ribozyme.

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

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

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

Evidence has been growing that an RNA world existed before DNA- and protein-based life. In this regard, a longstanding research goal has been to develop functional RNAs capable of catalyzing numerous chemical reactions. Much effort has been undertaken in finding RNA enzymes that catalyze the replication of RNA molecules, including the enzymes themselves. This, however, is far from being resolved. Apart from replication, other functionalities that contribute to higher genetic complexity and extended functional space are of high relevance to RNA-world scenarios. We have engineered a variety of hairpin ribozyme descendants possessing activity for RNA recombination, circularization, and oligomerization. Furthermore, we have developed ribozymes that can be regulated by external cofactors such as flavine mononucleotide (FMN), and we have designed a system for RNA-catalyzed RNA aminoacylation as a possible path toward a transition from the RNA world to modern life.

© 2014 by the authors; licensee MDPI, Basel, Switzerland. The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme’s possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.

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

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

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions.

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

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

In the transition from the RNA world to the modern DNA/protein world, RNA-catalyzed aminoacylation might have been a key step towards early translation. A number of ribozymes capable of aminoacylating their own 3' termini have been developed by in vitro selection. However, all of those catalysts require a previously activated amino acid-typically an aminoacyl-AMP-as substrate. Here we present two ribozymes connected by intermolecular base pairing and carrying out the two steps of aminoacylation: ribozyme 1 loads nonactivated phenylalanine onto its phosphorylated 5' terminus, thereby forming a high-energy mixed anhydride. Thereafter, a complex of ribozymes 1 and 2 is formed by intermolecular base pairing, and the "activated" phenylalanine is transferred from the 5' terminus of ribozyme 1 to the 3' terminus of ribozyme 2. This kind of simple RNA aminoacylase complex was engineered from previously selected ribozymes possessing the two required activities. RNA aminoacylation with a nonactivated amino acid as described here is advantageous to RNA world scenarios because initial amino acid activation by an additional reagent (in most cases, ATP) and an additional ribozyme would not be necessary.

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

The RNA world hypothesis describes a stage in the early evolution of life in which RNA served as genome and as the only genome-encoded catalyst. To test whether RNA world organisms could have used cyclic trimetaphosphate as an energy source, we developed an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5′-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, and one ribozyme was analyzed in more detail. The ribozyme was truncated to 96 nt, while retaining full activity. It was converted to a trans-format and reacted with rates of 0.16 min−1 under optimal conditions. The secondary structure appears to contain a four-helical junction motif. This study showed that ribozymes can use trimetaphosphate to triphosphorylate RNA 5′-hydroxyl groups and suggested that RNA world organisms could have used trimetaphosphate as their energy source.

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

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

Protein mutants are studied in a variety of contexts in the life sciences. However, individual mutations need to be generated in order to transcribe and translate the respective protein variants. Here, we introduce a novel strategy for controlling the incorporation of different amino acids in response to an amber stop codon by utilizing switchable designer transfer RNAs in Escherichia coli .

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

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

Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

Origins-of-life research requires searching for a plausible transition from simple chemicals to larger macromolecules that can both hold information and catalyze their own production. We have previously shown that some group I intron ribozymes possess the ability to help synthesize other ribozyme genotypes by recombination reactions in small networks in an autocatalytic fashion. By simplifying these recombination reactions, using fluorescent anisotropy, we quantified the thermodynamic binding strength between two nucleotides of two group I intron RNA fragments for all 16 possible genotype combinations. We provide evidence that the binding strength (KD) between the 3-nucleotide internal guide sequence (IGS) of one ribozyme and its complement in another is correlated to the catalytic ability of the ribozyme. This work demonstrates that one can begin to deconstruct the thermodynamic basis of information in prebiotic RNA systems.

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

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes., Nucleic Acids Research, 44 (10), ISSN:1362-4962, ISSN:0301-5610

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

RNA possesses great potential for expanding the toolbox currently available to synthetic biologists. Here, the modulation of the Hepatitis Delta Virus ribozyme's activity with a series of rationally designed aptamers and effector RNA oligonucleotides is described. The ribozyme was initially fused with an 18-nucleotide hairpin structure that abolished its self-cleaving activity. The binding of a 14-mer oligonucleotide to the hairpin rescued the self-cleavage in a concentration-dependent manner. This modified ribozyme was inserted into the 5' UTR of a reporter gene, and the resulting construct was used to demonstrate that it is possible to modulate the ribozyme activity in cellulo with the oligonucleotide. Subsequently, ribozymes possessing specialized aptamers respecting other logic gates were also successfully designed and found to be functional in vitro. To our knowledge, this is the first example of HDV ribozyme regulation by oligonucleotides, as well as the first allosteric regulation of HDV ribozyme in mammalian cells.

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

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

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

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

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

The sequence corresponding to the first 77 nucleotides of the L1Tc and NARTc non-LTR retrotransposons from Trypanosoma cruzi is an internal promoter (Pr77) that generates abundant, although poorly translatable, un-spliced transcripts. It has been recently described that L1TcRz, an HDV-like ribozyme, resides within the 5′-end of the RNA from the L1Tc and NARTc retrotransposons. Remarkably, the same first 77 nucleotides of L1Tc/NARTc elements comprise both the Pr77 internal promoter and the HDV-like L1TcRz. The L1TcRz cleaves on the 5′-side of the +1 nucleotide of the L1Tc element insuring that the promoter and the ribozyme functions travel with the transposon during retrotransposition. The ribozyme activity would prevent the mobilization of upstream sequences and insure the individuality of the L1Tc/NARTc copies transcribed from associated tandems. The Pr77/L1TcRz sequence is also found in other trypanosomatid’s non-LTR retrotransposons and degenerated retroposons. The possible conservation of the ribozyme...

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

The glmS ribozyme-riboswitch is the first known example of a naturally occurring catalytic RNA that employs a small molecule as a coenzyme. Binding of glucosamine-6-phosphate (GlcN6P) activates self-cleavage of the bacterial ribozyme, which is part of the mRNA encoding the metabolic enzyme GlcN6P-synthetase. Cleavage leads to negative feedback regulation. GlcN6P binds in the active site of the ribozyme, where its amine could function as a general acid and electrostatic catalyst. The ribozyme is pre-folded but inactive in the absence of GlcN6P, demonstrating it has evolved strict dependence on the exogenous small molecule. The ribozyme showcases the ability of RNA to co-opt non-covalently bound small molecules to expand its chemical repertoire. Analogue studies demonstrate that some molecules other than GlcN6P, such as l -serine (but not d -serine), can function as weak activators. This suggests how coenzyme use by RNA world ribozymes may have led to evolution of proteins. Primordial cofactor-dependent ribozymes may have evolved to bind their cofactors covalently. If amino acids were used as cofactors, this could have driven the evolution of RNA aminoacylation. The ability to make covalently bound peptide coenzymes may have further increased the fitness of such primordial ribozymes, providing a selective pressure for the invention of translation.

The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.

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

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

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

The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK(a) shifted2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg(2+) ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg(2+) ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg(2+) ion is also found within the active site where it interacts with a newly resolved G.U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R(P) oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg(2+) ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

Self-cleaving RNAs have recently been identified in mammalian genomes. A small ribozyme related in structure to the hepatitis delta virus (HDV) ribozyme occurs in a number of mammals, including chimpanzees and humans, within an intron of the CPEB3 gene. The catalytic mechanisms for the CPEB3 and HDV ribozymes appear to be similar, generating cleavage products with 5'-hydroxyl and 2',3'-cyclic phosphate termini; nonetheless, the cleavage rate reported for the CPEB3 ribozyme is more than 6000-fold slower than for the fastest HDV ribozyme. Herein, we use full-length RNA and cotranscriptional self-cleavage assays to compare reaction rates among human CPEB3, chimp CPEB3, and HDV ribozymes. Our data reveal that a single base change of the upstream flanking sequence, which sequesters an intrinsically weak P1.1 pairing in a misfold, increases the rate of the wild-type human CPEB3 ribozyme by approximately 250-fold; thus, the human ribozyme is intrinsically fast-reacting. Secondary structure determination and native gel analyses reveal that the cleaved population of the CPEB3 ribozyme has a single, secondary structure that closely resembles the HDV ribozyme. In contrast, the precleavage population of the CPEB3 ribozyme appears to have a more diverse secondary structure, possibly reflecting misfolding with the upstream sequence and dynamics intrinsic to the ribozyme. Prior identification of expressed sequence tags (ESTs) in human cells indicated that cleavage activity of the human ribozyme is tissue-specific. It is therefore possible that cellular factors interact with regions upstream of the CPEB3 ribozyme to unmask its high intrinsic reactivity.

The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

A fundamental challenge in gene therapy is to develop approaches for delivering nucleic acid-based gene interfering agents, such as small interfering RNAs and ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. We constructed a functional RNase P ribozyme (M1GS RNA) that targets the overlapping mRNA region of two HCMV capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in human differentiated macrophages that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87–90% in viral CSP expression and a reduction of about 5,000-fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with those carrying the sequence of a control ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that ribozymes expressed following targeted gene transfer with Salmonella -based vectors are highly active and specific in blocking viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella -mediated gene transfer of RNase P ribozymes as an effective approach for gene-targeting applications.