Generating Artificial Ribozymes Using Sparse Coevolutionary Models.

RNA ribozyme (Walter Engelke, Biologist (London, England) 49:199-203, 2002) datasets typically contain from a few hundred to a few thousand naturally occurring sequences. However, the potential sequence space of RNA is huge. For example, the number of possible RNA sequences of length 150 nucleotides is approximately 1 0 90 , a figure that far surpasses the estimated number of atoms in the known universe, which is around 1 0 80 . This disparity highlights a vast realm of sequence variability that remains unexplored by natural evolution. In this context, generative models emerge as a powerful tool. Learning from existing natural instances, these models can create artificial variants that extend beyond the currently known sequences. In this chapter, we will go through the use of a generative model based on direct coupling analysis (DCA) (Russ et al., Science 369:440-445, 2020; Trinquier et al., Nat Commun 12:5800, 2021; Calvanese et al., Nucleic Acids Res 52(10):5465-5477, 2024) applied to the twister ribozyme RNA family with three key applications: generating artificial twister ribozymes, designing potentially functional mutations of a natural wild type, and predicting mutational effects.

Origin & influence of autocatalytic reaction networks at the advent of the RNA world.

Research on the origin of life investigates the transition from abiotic chemistry to the emergence of biology, with the 'RNA world hypothesis' as the leading theory. RNA's dual role in storage and catalysis suggests its importance in this narrative. The discovery of natural ribozymes emphasizes RNA's catalytic capabilities in prebiotic environments, supporting the plausibility of an RNA world and prompting exploration of precellular evolution. Collective autocatalytic sets (CASs) mark a crucial milestone in this transition, fostering complexity through autocatalysis. While modern biology emphasizes sequence-specific polymerases, remnants of CASs persist in primary metabolism highlighting their significance. Autocatalysis, driven by CASs, promotes complexity through mutually interdependent catalytic sets. Yet, the transition from ribonucleotides to complex RNA oligomers remains puzzling. Questions persist about the genesis of the first self-replicating RNA molecule, RNA's stability in prebiotic conditions, and the shift to complex molecular reproduction. This review delves into diverse facets of the RNA world's emergence, addressing critical bottlenecks and scientific advances. Integrating insights from simulation and in vitro evolution research, we illuminate the multistep biogenesis of catalytic RNA from the abiotic world. Through this exploration, we aim to elucidate the journey from the primordial soup to the dawn of life, emphasizing the interplay between chemistry and biology in understanding life's origins.

Prebiotic chiral transfer from self-aminoacylating ribozymes may favor either handedness.

Modern life is essentially homochiral, containing D-sugars in nucleic acid backbones and L-amino acids in proteins. Since coded proteins are theorized to have developed from a prebiotic RNA World, the homochirality of L-amino acids observed in all known life presumably resulted from chiral transfer from a homochiral D-RNA World. This transfer would have been mediated by aminoacyl-RNAs defining the genetic code. Previous work on aminoacyl transfer using tRNA mimics has suggested that aminoacylation using D-RNA may be inherently biased toward reactivity with L-amino acids, implying a deterministic path from a D-RNA World to L-proteins. Using a model system of self-aminoacylating D-ribozymes and epimerizable activated amino acid analogs, we test the chiral selectivity of 15 ribozymes derived from an exhaustive search of sequence space. All of the ribozymes exhibit detectable selectivity, and a substantial fraction react preferentially to produce the D-enantiomer of the product. Furthermore, chiral preference is conserved within sequence families. These results are consistent with the transfer of chiral information from RNA to proteins but do not support an intrinsic bias of D-RNA for L-amino acids. Different aminoacylation structures result in different directions of chiral selectivity, such that L-proteins need not emerge from a D-RNA World.

In Vitro Self-Circularization Methods Based on Self-Splicing Ribozyme.

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.

Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation.

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis.

Evaluating Performance of Different RNA Secondary Structure Prediction Programs Using Self-cleaving Ribozymes.

Accurate identification of the correct, biologically relevant RNA structures is critical to understanding various aspects of RNA biology since proper folding represents the key to the functionality of all types of RNA molecules and plays pivotal roles in many essential biological processes. Thus, a plethora of approaches have been developed to predict, identify, or solve RNA structures based on various computational, molecular, genetic, chemical, or physicochemical strategies. Purely computational approaches hold distinct advantages over all other strategies in terms of the ease of implementation, time, speed, cost, and throughput, but they strongly underperform in terms of accuracy that significantly limits their broader application. Nonetheless, the advantages of these methods led to a steady development of multiple in silico RNA secondary structure prediction approaches including recent deep learning-based programs. Here, we compared the accuracy of predictions of biologically relevant secondary structures of dozens of self-cleaving ribozyme sequences using seven in silico RNA folding prediction tools with tasks of varying complexity. We found that while many programs performed well in relatively simple tasks, their performance varied significantly in more complex RNA folding problems. However, in general, a modern deep learning method outperformed the other programs in the complex tasks in predicting the RNA secondary structures, at least based on the specific class of sequences tested, suggesting that it may represent the future of RNA structure prediction algorithms.

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

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

Nicotinamide Riboside: What It Takes to Incorporate It into RNA.

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.

A potential role for RNA aminoacylation prior to its role in peptide synthesis.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis.

Engineering of RNase P Ribozymes for Therapy against Human Cytomegalovirus Infection.

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

The structure and catalytic mechanism of a pseudoknot-containing hammerhead ribozyme.

We have determined the crystal structure of a pseudoknot (PK)-containing hammerhead ribozyme that closely resembles the pistol ribozyme, with essentially identical secondary structure and connectivity. The activity is more sensitive to deletion of the G8 2'OH than to the absence of magnesium ions, indicating that the catalytic mechanism is the same as the extended hammerhead, and distinct from the pistol ribozyme. Here we show that nucleophilic attack is almost perfectly in-line, and the G8 2'OH is well positioned to act as general acid, being directed towards the O5' leaving group, and 2.9 Å away from it. Despite the similarity in overall structure to the pistol ribozyme, the local structure close to the cleavage site differs, and the PK hammerhead retains its unique mechanistic identity and demonstrates enhanced activity over other hammerhead ribozymes under standard conditions.

Efficient circularization of protein-encoding RNAs via a novel cis-splicing system.

Circular RNAs (circRNAs) have emerged as a promising alternative to linear mRNA, owing to their unique properties and potential therapeutic applications, driving the development of novel approaches for their production. This study introduces a cis-splicing system that efficiently produces circRNAs by incorporating a ribozyme core at one end of the precursor, thereby eliminating the need for additional spacer elements between the ribozyme and the gene of interest (GOI). In this cis-splicing system, sequences resembling homologous arms at both ends of the precursor are crucial for forming the P9.0 duplex, which in turn facilitates effective self-splicing and circularization. We demonstrate that the precise recognition of the second transesterification site depends more on the structural characteristics of P9.0 adjacent to the ωG position than on the nucleotide composition of the P9.0-ωG itself. Further optimization of structural elements, like P10 and P1-ex, significantly improves circularization efficiency. The circRNAs generated through the cis-splicing system exhibit prolonged protein expression and minimal activation of the innate immune response. This study provides a comprehensive exploration of circRNA generation via a novel strategy and offers valuable insights into the structural engineering of RNA, paving the way for future advancements in circRNA-based applications.

A cis-acting ligase ribozyme generates circular RNA in vitro for ectopic protein functioning.

Delivering synthetic protein-coding RNA bypassing the DNA stage for ectopic protein functioning is a novel therapeutic strategy. Joining the linear RNA head-to-tail covalently could be a state-of-the-art strategy for functioning longer. Here we enroll a cis-acting ligase ribozyme (RzL) to generate circular RNA (circRNA) in vitro for ectopic protein expression. The RNA circularization is confirmed by masking the 5' phosphate group, resisting exonuclease RNase R digestion, failing for further tailing, and sequencing the RT-PCR products of the joined region. Interestingly, one internal ribosome entry site (IRES) renders circRNA translation competent, but two IRES in cis, not trans, hamper the translation. The circRNA with highly potent in translation is conferred for antiviral functioning. Accompanying specific guided RNA, a circRNA expressing ribonuclease Cas13 shows excellent potential against the corresponding RNA virus, further extending circRNA functioning in its growing list of applications.

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Circular at the very beginning: on the initial genomes in the RNA world.

It is likely that an RNA world existed in early life, when RNA played both the roles of the genome and functional molecules, thereby undergoing Darwinian evolution. However, even with only one type of polymer, it seems quite necessary to introduce a labour division concerning these two roles because folding is required for functional molecules (ribozymes) but unfavourable for the genome (as a template in replication). Notably, while ribozymes tend to have adopted a linear form for folding without constraints, a circular form, which might have been topologically hindered in folding, seems more suitable for an RNA template. Another advantage of involving a circular genome could have been to resist RNA's end-degradation. Here, we explore the scenario of a circular RNA genome plus linear ribozyme(s) at the precellular stage of the RNA world through computer modelling. The results suggest that a one-gene scene could have been 'maintained', albeit with rather a low efficiency for the circular genome to produce the ribozyme, which required precise chain-break or chain-synthesis. This strict requirement may have been relieved by introducing a 'noncoding' sequence into the genome, which had the potential to derive a second gene through mutation. A two-gene scene may have 'run well' with the two corresponding ribozymes promoting the replication of the circular genome from different respects. Circular genomes with more genes might have arisen later in RNA-based protocells. Therefore, circular genomes, which are common in the modern living world, may have had their 'root' at the very beginning of life.

Simulation-Guided Conformational Space Exploration to Assess Reactive Conformations of a Ribozyme.

Self-splicing ribozymes are small ribonucleic acid (RNA) enzymes that catalyze their own cleavage through a transphosphoesterification reaction. While this process is involved in some specific steps of viral RNA replication and splicing, it is also of importance in the context of the (putative) first autocatalytic RNA-based systems that could have preceded the emergence of modern life. The uncatalyzed phosphoester bond formation is thermodynamically very unfavorable, and many experimental studies have focused on understanding the molecular features of catalysis in these ribozymes. However, chemical reaction paths are short-lived and not easily characterized by experimental approaches, so molecular simulation approaches appear as an ideal tool to unveil the molecular details of the reaction. Here, we focus on the model hairpin ribozyme. We show that identifying a relevant initial conformation for reactivity studies, which is frequently overlooked in mixed quantum-classical studies that predominantly concentrate on the chemical reaction itself, can be highly challenging. These challenges stem from limitations in both available experimental structures (which are chemically altered to prevent self-cleavage) and the accuracy of force fields, together with the necessity for comprehensive sampling. We show that molecular dynamics simulations, combined with extensive conformational phase space exploration with Hamiltonian replica-exchange simulations, enable us to characterize the relevant conformational basins of the minimal hairpin ribozyme in the ligated state prior to self-cleavage. We find that what is usually considered a canonical reactive conformation with active site geometries and hydrogen-bond patterns that are optimal for the addition-elimination reaction with general acid/general base catalysis is metastable and only marginally populated. The thermodynamically stable conformation appears to be consistent with the expectations of a mechanism that does not require the direct participation of ribozyme residues in the reaction. While these observations may suffer from forcefield inaccuracies, all investigated forcefields lead to the same conclusions upon proper sampling, contrasting with previous investigations on shorter timescales suggesting that at least one reparametrization of the Amber99 forcefield allowed to stabilize aligned active site conformations. Our study demonstrates that identifying the most pertinent reactant state conformation holds equal importance alongside the accurate determination of the thermodynamics and kinetics of the chemical steps of the reaction.

Fitness Landscapes and Evolution of Catalytic RNA.

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.

Gas-sensing riboceptors.

Understanding how cells sense gases or gaseous solutes is a fundamental question in biology and is pivotal for the evolution of molecular and organismal life. In numerous organisms, gases can diffuse into cells, be transported, generated, and sensed. Controlling gases in the cellular environment is essential to prevent cellular and molecular damage due to interactions with gas-dependent free radicals. Consequently, the mechanisms governing acute gas sensing are evolutionarily conserved and have been experimentally elucidated in various organisms. However, the scientific literature on direct gas sensing is largely based on hemoprotein-based gasoreceptors (or sensors). As RNA-based G-quadruplex (G4) structures can also bind to heme, I propose that some ribozymes can act as gas-sensing riboceptors (ribonucleic acid receptors). Additionally, I present a few other ideas for non-heme metal ion- or metal cluster-based gas-sensing riboceptors. Studying riboceptors can help understand the evolutionary origins of cellular and gasocrine signaling.

Protocell Effects on RNA Folding, Function, and Evolution.

ConspectusCreating a living system from nonliving matter is a great challenge in chemistry and biophysics. The early history of life can provide inspiration from the idea of the prebiotic "RNA World" established by ribozymes, in which all genetic and catalytic activities were executed by RNA. Such a system could be much simpler than the interdependent central dogma characterizing life today. At the same time, cooperative systems require a mechanism such as cellular compartmentalization in order to survive and evolve. Minimal cells might therefore consist of simple vesicles enclosing a prebiotic RNA metabolism.The internal volume of a vesicle is a distinctive environment due to its closed boundary, which alters diffusion and available volume for macromolecules and changes effective molecular concentrations, among other considerations. These physical effects are mechanistically distinct from chemical interactions, such as electrostatic repulsion, that might also occur between the membrane boundary and encapsulated contents. Both indirect and direct interactions between the membrane and RNA can give rise to nonintuitive, "emergent" behaviors in the model protocell system. We have been examining how encapsulation inside membrane vesicles would affect the folding and activity of entrapped RNA.Using biophysical techniques such as FRET, we characterized ribozyme folding and activity inside vesicles. Encapsulation inside model protocells generally promoted RNA folding, consistent with an excluded volume effect, independently of chemical interactions. This energetic stabilization translated into increased ribozyme activity in two different systems that were studied (hairpin ribozyme and self-aminoacylating RNAs). A particularly intriguing finding was that encapsulation could rescue the activity of mutant ribozymes, suggesting that encapsulation could affect not only folding and activity but also evolution. To study this further, we developed a high-throughput sequencing assay to measure the aminoacylation kinetics of many thousands of ribozyme variants in parallel. The results revealed an unexpected tendency for encapsulation to improve the better ribozyme variants more than worse variants. During evolution, this effect would create a tilted playing field, so to speak, that would give additional fitness gains to already-high-activity variants. According to Fisher's Fundamental Theorem of Natural Selection, the increased variance in fitness should manifest as faster evolutionary adaptation. This prediction was borne out experimentally during in vitro evolution, where we observed that the initially diverse ribozyme population converged more quickly to the most active sequences when they were encapsulated inside vesicles.The studies in this Account have expanded our understanding of emergent protocell behavior, by showing how simply entrapping an RNA inside a vesicle, which could occur spontaneously during vesicle formation, might profoundly affect the evolutionary landscape of the RNA. Because of the exponential dynamics of replication and selection, even small changes to activity and function could lead to major evolutionary consequences. By closely studying the details of minimal yet surprisingly complex protocells, we might one day trace a pathway from encapsulated RNA to a living system.

Two RNA Folds from One Sequence: A Ribozyme with Versatile Substrate Processing Abilities.

We report the design of a single RNA sequence capable of adopting one of two ribozyme folds and catalyzing the cleavage and/or ligation of the respective substrates. The RNA is able to change its conformation in response to its environment, hence it is called chameleon ribozyme (CHR). Efficient RNA cleavage of two different substrates as well as RNA ligation by CHR is demonstrated in separate experiments and in a one pot reaction. Our study shows that sequence variants of the hairpin ribozyme intersect with the hammerhead ribozyme and that rather short RNA molecules can have comprehensive conformational flexibility, which is an important feature for the emergence of new functional folds in early evolution.