Search

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR.

Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S -adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.

RNA switches respond to specific ligands to control gene expression. They are widely used in synthetic biology applications and hold potential for future RNA-based therapeutic breakthroughs. However, the crux is their precise design. Here, we will discuss how inverse-RNA-folding could be utilized for the accurate design of RNA switches., Competing Interests: Declaration of interests No interests are declared by the authors., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Basic research and functional analyses of circular RNA (circRNA) have been limited by challenges in circRNA formation of desired length and sequence in adequate yields. Nowadays, circular RNA can be obtained using enzymatic, "ribozymatic," or modulated splice events. However, there are few records for the directed circularization of RNA. Here, we present a proof of principle for an affordable and efficient RNA-based system for the controlled synthesis of circRNA with a physiological 3',5'-phosphodiester conjunction. The engineered hairpin ribozyme variant circular ribozyme 3 (CRZ-3) performs self-cleavage poorly. We designed an activator-polyamine complex to complete cleavage as a prerequisite for subsequent circularization. The developed protocol allows synthesizing circRNA without external enzymatic assistance and adds a controllable way of circularization to the existing methods., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.

OLE RNA is a ~600-nucleotide noncoding RNA present in many Gram-positive bacteria that thrive mostly in extreme environments, including elevated temperature, salt, and pH conditions. The precise biochemical functions of this highly conserved RNA remain unknown, but it forms a ribonucleoprotein (RNP) complex that localizes to cell membranes. Genetic disruption of the RNA or its essential protein partners causes reduced cell growth under various stress conditions. These phenotypes include sensitivity to short-chain alcohols, cold intolerance, reduced growth on sub-optimal carbon sources, and intolerance of even modest concentrations of Mg 2+ . Thus, many bacterial species appear to employ OLE RNA as a component of an intricate RNP apparatus to monitor fundamental cellular processes and make physiological and metabolic adaptations. Herein we hypothesize that the OLE RNP complex is functionally equivalent to the eukaryotic TOR complexes, which integrate signals from various diverse pathways to coordinate processes central to cell growth, replication, and survival., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Site-specific introduction of bioorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post-synthetic bioconjugation reactions. Here, we report a ribozyme-based method for the synthesis of aldehyde-functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site-specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5-amino-4-formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde-reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3-trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme's area of application from a methyltransferase to a tool for site-specific late-stage functionalization of RNA., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

An extremely small proportion of the X-ray crystal structures deposited in the Protein Data Bank are of RNA or RNA-protein complexes. This is due to three main obstacles to the successful determination of RNA structure: (1) low yields of pure, properly folded RNA; (2) difficulty creating crystal contacts due to low sequence diversity; and (3) limited methods for phasing. Various approaches have been developed to address these obstacles, such as native RNA purification, engineered crystallization modules, and incorporation of proteins to assist in phasing. In this review, we will discuss these strategies and provide examples of how they are used in practice.

The rapid development of cryogenic electron microscopy (cryo-EM) enables the structure determination of macromolecules without the need for crystallization. Protein, protein-lipid, and protein-nucleic acid complexes can now be routinely resolved by cryo-EM single-particle analysis (SPA) to near-atomic or atomic resolution. Here we describe the structure determination of pure RNAs by SPA, from cryo-specimen preparation to data collection and 3D reconstruction. This protocol is useful to yield many cryo-EM structures of RNA, here exemplified by the Tetrahymena L-21 ScaI ribozyme at 3.1-Å resolution., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600 nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA. Disruption of the genes for either OLE RNA, OapA, or OapB result in the inability of cells to overcome cold, alcohol, or Mg 2+ stresses. In the current study, we used in vivo crosslinking followed by OLE RNA isolation to identify the protein YbxF as a potential additional partner in the OLE RNP complex. Notably, a mutation in the gene for this same protein was also reported to be present in a strain wherein the complex is nonfunctional. The B. halodurans YbxF (herein renamed OapC) is homologous to a bacterial protein earlier demonstrated to bind kink turn (k-turn) RNA structural motifs. In vitro RNA-protein binding assays reveal that OLE RNA forms a previously unrecognized k-turn that serves as the natural binding site for YbxF/OapC. Moreover, B. halodurans cells carrying OLE RNAs with disruptive mutations in the k-turn exhibit phenotypes identical to cells lacking functional OLE RNP complexes. These findings reveal that the YbxF/OapC protein of B. halodurans is important for the formation of a functional OLE RNP complex., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In vitro transcription (IVT) using T7 RNA polymerase has become the most common method to synthesize RNAs for use in basic research and pharmaceutical applications. To solve the heterogeneity issue associated with the system, cis-acting ribozymes have been exploited to direct co-transcriptional processing to yield target RNAs with precisely defined ends. However, traditionally used ribozymes have many limitations, such as low efficiency and special sequence requirements of target RNAs. In addition, the introduction of ribozymes in IVT complicates the downstream purification of target RNAs. Here we describe a new cassette of engineered ribozymes (HHV-Kt and Twister-Kt) that can work in concert to produce RNA with defined 5' and 3' ends. The pair of ribozymes displayed reliably high activity when working with RNA of various lengths and structures. The engineered ribozymes also carry a K-turn RNA motif that enables fast post-IVT clearance of cleaved ribozymes and uncleaved precursors using K-turn affinity resins. Finally, we demonstrated the scalability of our system for the rapid production of large quantities of homogeneous RNA samples.

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The origin of genetic systems is the central problem in the study of the origin of life for which various explanatory hypotheses have been presented. One model suggests that both ancestral transfer ribonucleic acid (tRNA) molecules and primitive ribosomes were originally involved in RNA replication (Campbell 1991). According to this model the early tRNA molecules catalyzed their own self-loading with a trinucleotide complementary to their anticodon triplet, while the primordial ribosome (protoribosome) catalyzed the transfer of these terminal trinucleotides from one tRNA to another tRNA harboring the growing RNA polymer at the 3´-end.Here we present the notion that the anticodon-codon-like pairs presumably located in the acceptor stem of primordial tRNAs (Rodin et al. 1996) (thus being and remaining, after the code and translation origins, the major contributor to the RNA operational code (Schimmel et al. 1993)) might have originally been used for RNA replication rather than translation; these anticodon and acceptor stem triplets would have been involved in accurately loading the 3'-end of tRNAs with a trinucleotide complementary to their anticodon triplet, thus allowing the accurate repair of tRNAs for their use by the protoribosome during RNA replication.We propose that tRNAs could have catalyzed their own trinucleotide self-loading by forming catalytic tRNA dimers which would have had polymerase activity. Therefore, the loading mechanism and its evolution may have been a basic step in the emergence of new genetic mechanisms such as genetic translation. The evolutionary implications of this proposed loading mechanism are also discussed., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Liquid-liquid phase separation (LLPS) has been known to drive formation of biomolecular compartments, which can encapsulate RNA and proteins among other cosolutes. Such compartments, which lack a lipid membrane, have been implicated in origins of life scenarios as they can easily uptake and concentrate biomolecules, similar to intracellular condensates. Indeed, chemical interactions that drive LLPS in vitro have also been shown to lead to similar sub-cellular compartments in vivo. Here we describe methods to prepare compartments formed by complex coacervates, which are driven by LLPS of oppositely-charged polyions, and to probe the structures and functions of RNAs in them. These methods can be adapted to study RNA biochemistry in compartments formed by diverse artificial and biological macromolecules., (© 2021 Elsevier Inc. All rights reserved.)

As a mental framework for the transition of self-replicating biological forms, the RNA world concept stipulates a dual function of RNAs as genetic substance and catalyst. The chaperoning function is found intrinsic to ribozymes involved in protein synthesis and tRNA maturation, enriching the primordial RNA world with proteins of biological relevance. The ribozyme-resident protein folding activity, even before the advent of protein-based molecular chaperone, must have expedited the transition of the RNA world into the present protein theatre.

Artificial RNA switches are an emerging class of genetic controllers suitable for synthetic biology applications. Aptazymes are fusions composed of an aptamer domain and a self-cleaving ribozyme. The utilization of aptazymes for conditional gene expression displays several advantages over employing conventional transcription factor-based techniques as aptazymes require minimal genomic space, fulfill their function without the need of protein cofactors and most importantly are reprogrammable with respect to ligand selectivity and the RNA function to be regulated. Technologies that enable the generation of aptazymes to defined input ligands are of interest for the construction of biocomputing devices and biosensing applications. In this chapter we present a method that facilitates the in vivo screening of randomized pools of aptazymes in mammalian cells.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools., (© 2020 Miao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus ( Ta.it. I1). We show that Ta.it. I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it. I1 group IIC intron coordinates the progression of the forward splicing reaction through a π-π' interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the π-π' interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the π-π' interaction., (© 2020 Smathers and Robart; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Most scenarios for the origin of life assume that RNA played a key role in both catalysis and information storage. The A, U, G, and C nucleobases in modern RNA all participate in secondary structure formation and replication. However, the rapid deamination of C to U and the absence of C in meteorite samples suggest that prebiotic RNA may have been deficient in cytosine. Here, we assess the ability of RNA sequences formed from a three-letter AUG alphabet to perform both structural and genetic roles in comparison to sequences formed from the AUGC alphabet. Despite forming less thermodynamically stable helices, the AUG alphabet can find a broad range of structures and thus appears sufficient for catalysis in the RNA World. However, in the AUG case, longer sequences are required to form structures with an equivalent complexity. Replication in the AUG alphabet requires GU pairing. Sequence fidelity in the AUG alphabet is low whenever G's are present in the sequence. We find that AUG sequences evolve to AU sequences if GU pairing is rare, and to RU sequences if GU pairing is common (R denotes A or G). It is not possible to conserve a G at a specific site in either case. These problems do not rule out the possibility of an RNA World based on AUG, but they show that it wouldbe significantly more difficult than with a four-base alphabet., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a maturase protein, which promotes splicing through an unknown mechanism. The mechanism of splice site exchange within the RNA active site during catalysis also remains unclear. We determined two cryo-EM structures at 3.6-Å resolution of a group II intron reverse splicing into DNA. These structures reveal that the branch-site domain VI helix swings 90°, enabling substrate exchange during DNA integration. The maturase assists catalysis through a transient RNA-protein contact with domain VI that positions the branch-site adenosine for lariat formation during forward splicing. These findings provide the first direct evidence of the role the maturase plays during group II intron catalysis. The domain VI dynamics closely parallel spliceosomal branch-site helix movement and provide strong evidence for a retroelement origin of the spliceosome., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inherited retinal diseases (IRD) encompass a wide spectrum of hereditary blindness with significant genetic heterogeneity. Therapeutics regulating gene expression on an RNA level have significant promise for treating IRD. In this review, we review the molecular basis of oligonucleotide therapeutics such as ribozymes, RNA interference (RNAi), antisense oligonucleotides (ASO), CRISPRi/a, and their applications to treatments of IRD.

An artificially evolved ribozyme can catalyse the synthesis of RNA by using trinucleotide triphosphates as building blocks., Competing Interests: AC, TC No competing interests declared, (© 2018, Crisp et al.)

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox, we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit '+' and '-' strands in segments and assemble them into a new active ribozyme., Competing Interests: JA, AR, AM, EG, PH No competing interests declared, (© 2018, Attwater et al.)

Circular DNAs are frequent genomic molecules, especially among the simplest life beings, whereas circular RNAs have been regarded as weird nucleic acids in biology. Now we know that eukaryotes are able to express circRNAs, mostly derived from backsplicing mechanisms, and playing different biological roles such as regulation of RNA splicing and transcription, among others. However, a second natural and highly efficient pathway for the expression in vivo of circRNAs has been recently reported, which allows the accumulation of abundant small (100-1000 nt) non-coding RNA circles through the participation of small self-cleaving RNAs or ribozymes called hammerhead ribozymes. These genome-encoded circRNAs with ribozymes seem to be a new family of small and nonautonomous retrotransposable elements of plants and animals (so-called retrozymes), which will offer functional clues to the biology and evolution of circular RNA molecules as well as new biotechnological tools in this emerging field.

We consider a spatial model of replication in the RNA World in which polymerase ribozymes use neighbouring strands as templates. Point mutation errors create parasites that have the same replication rate as the polymerase. We have shown previously that spatial clustering allows survival of the polymerases as long as the error rate is below a critical error threshold. Here, we additionally consider errors where a polymerase prematurely terminates replication before reaching the end of the template, creating shorter parasites that are replicated faster than the functional polymerase. In well-known experiments where Qβ RNA is replicated by an RNA polymerase protein, the virus RNA is rapidly replaced by very short non-functional sequences. If the same thing were to occur when the polymerase is a ribozyme, this would mean that termination errors could potentially destroy the RNA World. In this paper, we show that this is not the case in the RNA replication model studied here. When there is continued generation of parasites of all lengths by termination errors, the system can survive up to a finite error threshold, due to the formation of travelling wave patterns; hence termination errors are important, but they do not lead to the inevitable destruction of the RNA World by short parasites. The simplest assumption is that parasite replication rate is inversely proportional to the strand length. In this worst-case scenario, the error threshold for termination errors is much lower than for point mutations. We also consider a more realistic model in which the time for replication of a strand is the sum of a time for binding of the polymerase, and a time for polymerization. When the binding step is considered, termination errors are less serious than in the worst case. In the limit where the binding time is dominant, replication rates are equal for all lengths, and the error threshold for termination is the same as for point mutations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A new family of non-autonomous retrotransposons with self-cleaving hammerhead ribozymes, the so called retrozymes, has recently been found encoded in diverse plant genomes. These retroelements can be actively transcribed, and their RNAs accumulate in the cells as abundant non-coding circular RNAs (circRNAs) of small size (600-1000 nt). Related circRNAs with self-cleaving ribozymes had already been described in plants, and belong to a group of infectious RNA agents with an uncertain origin: the viroids and viroid-like satellites of plant RNA viruses. These pathogenic circRNAs show many structural similarities with retrozyme circRNAs, and both have been found to occur in flowering plants as heterogeneous RNA molecules of positive and negative polarities. Taking all these data together, we hypothesize that circRNAs encoded by genomic retrozymes could have given origin to infectious circRNAs with self-cleaving ribozymes. Moreover, we propose that retrozymes in time could have evolved from the ancient family of Penelope-like retroelements, which also harbour hammerhead ribozymes. Putative retrozyme sequences with hammerhead ribozymes have been detected as well in metazoan genomes, opening the door to a common occurrence of circRNAs with self-cleaving motifs among eukaryotes.

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

The breaking of RNA strands by 2'-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2'-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending on how the reaction is catalyzed. Because of this complexity and the lack of experimental information on transition-state structure, pinning down the chemical details of enzyme-catalyzed RNA strand cleavage has been difficult. Kinetic isotope effects (KIEs) provide information about changes in bonding as a reaction proceeds from ground state to transition state, and therefore they provide a powerful tool for revealing mechanistic detail. Application of kinetic isotope analyses to RNA 2'-O-transphosphorylation faces three fundamental challenges: synthesis of RNA substrate isotopomers with 18 O substitutions at the 2'-O, 5'-O and nonbridging phosphoryl oxygens; determination of the 18 O/ 16 O ratios in the residual unreacted substrate or product RNAs; and analyzing these data to allow calculation of the KIEs for use in evaluating different mechanistic scenarios. In this chapter, we outline methods for surmounting these challenges for solution RNA 2'-O-transphosphorylation reactions, and we describe their initial application to understand nonenzymatic solution reactions and reactions catalyzed by the enzyme ribonuclease A., (© 2017 Elsevier Inc. All rights reserved.)

Large RNAs often utilize GNRA tetraloops as structural elements to stabilize the overall tertiary fold. These tetraloop-receptor (TR) interactions have a conserved geometry in which the tetraloop docks into the receptor at an angle of ~15° from the helix containing the receptor. Here, we show that the conserved GUAAY pentaloop found in domain III of group IIB1 introns participates in a novel class of RNA tertiary interaction with a geometry and mode of binding that are significantly different from that found in GNRA TR interactions. This pentaloop is highly conserved within the IIB1 class and interacts with the minor groove of the catalytic domain V. The base planes of the loop and receptor nucleotides are not coplanar and greatly deviate from standard A-minor motifs. The helical axis of the GUAAY stem loop diverges ~70° from the angle of insertion found in a typical GNRA TR interaction. Therefore, the loop architecture and insertion orientation are distinctive, with in vitro splicing data indicating that a GNRA tetraloop is incompatible at this position. The GUAAY pentaloop-receptor motif is also found in the structure of the eukaryotic thiamine pyrophosphate riboswitch in the context of a hexanucleotide loop sequence. We therefore propose, based on phylogenetic, structural, and biochemical data, that the GUAAY pentaloop-receptor interaction represents a novel structural motif that is present in multiple structured RNAs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins., Competing Interests: R.R.B. and G.F.J. appeared as coauthors of a paper published as Breaker RR, Joyce GF (2014) The expanding view of RNA and DNA function. Chem Biol 21(9):1059–1065. This paper was a Perspective commemorating the 20th anniversary of the journal.

RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As with any outcome of an evolutionary process, the success of in vitro selection experiments depends critically on the starting population. In vitro selections isolate functional nucleic acids that fold into specific structures and form unique binding and catalytic sites. The selection outcomes therefore depend on the molecular and structural diversity of the initial pools. In addition, the experiments are strongly influenced by the length of the starting pool. Longer randomized regions support the formation of more complex structures and presumably allow formation of more intricate tertiary interactions, but they also tend to misfold and aggregate, whereas shorter pools are sufficient to yield simpler motifs. Furthermore, introducing a sequence bias that promotes secondary structure formation appears to prejudice the population towards more functional macromolecules. We review the literature on the influence of the starting pools on the predicted and actual outcomes of laboratory evolution experiments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Group II introns are large, autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays a major role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns have played a major role in genome evolution, as they are likely progenitors of spliceosomal introns, retroelements, and other machinery that controls genetic variation and stability. The structure and catalytic mechanism of group II introns have recently been elucidated through a combination of genetics, chemical biology, solution biochemistry, and crystallography. These studies reveal a dynamic machine that cycles progressively through multiple conformations as it stimulates the various stages of splicing. A central active site, containing a reactive metal ion cluster, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions about the behavior of RNA machines.

Origin of life: Although current life is homochiral (with D nucleic acids), little is known about how homochirality emerged or even if it was a necessary step. The isolation of cross-chiral nucleic acid ligases demonstrate that an early heterochiral life could have been possible., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2015 New York Academy of Sciences.)

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., (© 2014 McNeil and Zimmerly; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb pKa values and allow it to ionize appreciably under biological conditions, thereby extending its molecular diversity. The goal of this chapter is to enable experimental measurement of pKa's in RNA and DNA. A number of experimental methods for measuring pKa values in RNA and DNA have been developed over the last 10 years, including RNA cleavage kinetics; UV-, fluorescence-, and NMR-detected pH titrations; and Raman crystallography. We begin with general considerations for choosing a pKa assay and then describe experimental conditions, advantages, and disadvantages for these assays. Potential pitfalls in measuring a pKa are provided including the presence of apparent pKa's due to a kinetic pKa or coupled acid- and alkali-promoted RNA unfolding, as well as degradation of RNA, precipitation of metal hydroxides and poor baselines. Use of multiple data fitting procedures and the study of appropriate mutants are described as ways to avoid some of these pitfalls. Application of these experimental methods to RNA and DNA will increase the number of available nucleic acid pKa values in the literature, which should deepen insight into biology and provide benchmarks for pKa calculations. Future directions for measuring pKa's in nucleic acids are discussed.

How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described., (© 2014 Elsevier Inc. All rights reserved.)

We have engineered a self-processing RNA, derived from the hairpin ribozyme that runs through a cascade of cleavage and ligation reactions thereby changing its topology. The first two cleavage events leave the resulting RNA with a 5'-OH group and a 2',3'-cyclic phosphate. Thus, upon refolding, intramolecular ligation delivers a cyclic species. In addition, we demonstrate formation of concatemers resulting from multiple intermolecular ligations. Our results demonstrate the potential of RNA for self-supported topology changes and support the suggestion of 2',3'-cyclic phosphates being suitable activated building blocks for reversible phosphodiester bond formation in the RNA world., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native, functional state without assistance of a protein cofactor in vitro. In the cell, trans-acting factors, such as proteins, are however known to modulate the structure and thus the fate of an RNA. DEAD-box proteins, including Mss116p, were recently found to assist folding of group I and group II introns in vitro and in vivo. The underlying mechanism(s) have been studied extensively to explore the contribution of ATP hydrolysis and duplex unwinding in helicase-stimulated intron splicing. Here we summarize the ongoing efforts to understand the novel role of DEAD-box proteins in RNA folding.

The relationship between genotype and phenotype, or the fitness landscape, is the foundation of genetic engineering and evolution. However, mapping fitness landscapes poses a major technical challenge due to the amount of quantifiable data that is required. Catalytic RNA is a special topic in the study of fitness landscapes due to its relatively small sequence space combined with its importance in synthetic biology. The combination of in vitro selection and high-throughput sequencing has recently provided empirical maps of both complete and local RNA fitness landscapes, but the astronomical size of sequence space limits purely experimental investigations. Next steps are likely to involve data-driven interpolation and extrapolation over sequence space using various machine learning techniques. We discuss recent progress in understanding RNA fitness landscapes, particularly with respect to protocells and machine representations of RNA. The confluence of technical advances may significantly impact synthetic biology in the near future.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozymes self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

In vitro circular RNA (circRNA) preparation methods have been gaining a lot of attention recently as several reports suggest that circRNAs are more stable, with better performances in cells and in vivo, than linear RNAs in various biomedical applications. Self-splicing ribozymes are considered a major in vitro circRNA generation method for biomedical applications due to their simplicity and efficiency in the circularization of the gene of interest. This review summarizes, updates, and discusses the recently developed self-circularization methods based on the self-splicing ribozyme, such as group I and II intron ribozymes, and the pros and cons of each method in preparing circRNA in vitro. [ABSTRACT FROM AUTHOR]

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3′ fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila , with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes. [ABSTRACT FROM AUTHOR]

The identification of catalytic RNAs is typically achieved through primarily experimental means. However, only a small fraction of sequence space can be analyzed even with high-throughput techniques. Methods to extrapolate from a limited data set to predict additional ribozyme sequences, particularly in a human-interpretable fashion, could be useful both for designing new functional RNAs and for generating greater understanding about a ribozyme fitness landscape. Using information theory, we express the effects of epistasis (i.e., deviations from additivity) on a ribozyme. This representation was incorporated into a simple model of the epistatic fitness landscape, which identified potentially exploitable combinations of mutations. We used this model to theoretically predict mutants of high activity for a self-aminoacylating ribozyme, identifying potentially active triple and quadruple mutants beyond the experimental data set of single and double mutants. The predictions were validated experimentally, with nine out of nine sequences being accurately predicted to have high activity. This set of sequences included mutants that form a previously unknown evolutionary bridge between two ribozyme families that share a common motif. Individual steps in the method could be examined, understood, and guided by a human, combining interpretability and performance in a simple model to predict ribozyme sequences by extrapolation.

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR.

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) -including a variant that uses trinucleotide triphosphates (triplets) as substrates -have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs. [ABSTRACT FROM AUTHOR]

During the origin of life, encapsulation of RNA inside vesicles is believed to have been a defining feature of the earliest cells (protocells). The confined biophysical environment provided by membrane encapsulation differs from that of bulk solution and has been shown to increase activity as well as evolutionary rate for functional RNA. However, the structural basis of the effect on RNA has not been clear. Here, we studied how encapsulation of the hairpin ribozyme inside model protocells affects ribozyme kinetics, ribozyme folding into the active conformation, and cleavage and ligation activities. We further examined the effect of encapsulation on the folding of a stem-loop RNA structure and on the formation of a triplex structure in a pH-sensitive DNA switch. The results indicate that encapsulation promotes RNA-RNA association, both intermolecular and intramolecular, and also stabilizes tertiary folding, including the docked conformation characteristic of the active hairpin ribozyme and the triplex structure. The effects of encapsulation were sufficient to rescue the activity of folding-deficient mutants of the hairpin ribozyme. Stabilization of multiple modes of nucleic acid folding and interaction thus enhanced the activity of encapsulated nucleic acids. Increased association between RNA molecules may facilitate the formation of more complex structures and cooperative interactions. These effects could promote the emergence of biological functions in an "RNA world" and may have utility in the construction of minimal synthetic cells.