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.

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.

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.

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.

Computational Design of Allosteric Ribozymes via Genetic Algorithms.

In vitro selection of allosteric ribozymes has many challenges, such as complex and time-consuming experimental procedures, uncertain results, and the unwanted functionality of the enriched sequences. The precise computational design of allosteric ribozymes is achievable using RNA secondary structure folding principles. The computational design of allosteric ribozymes is based on experimentally validated EAs, random search algorithms, and a partition function for RNA folding. The in silico design achieves an accuracy exceeding 90%. Various algorithms with different logic gates have been automated via computer programs that can quickly create many allosteric sequences. This can eliminate the need for in vitro selection of allosteric ribozymes, thus vastly reducing the time and cost required.

A tribute to Sidney Altman, one of the architects of modern RNA biology.

This special issue of JBC pays tribute to Sidney Altman, whose discovery of a catalytic role for RNA, a breakthrough made independently by Thomas Cech, overturned the long-held dogma that only proteins can serve as catalysts in biological systems. The discovery of RNA catalysis galvanized biologists to think expansively in new directions and has given rise to a remarkable RNAissance in science and medicine. The collection of articles begins with the story of the discovery of RNase P and builds up to the emerging picture of an unexpectedly vast repertoire of RNase P variants in the three domains of life, including insights derived from recent high-resolution structures on how RNAs, ribonucleoproteins, or protein scaffolds can be used variably to generate an active site for catalyzing the same RNA processing reaction. The series of articles ends with a discussion of more recently discovered endonucleases (Argonautes, Cas), whose parallels with RNase P underscore recurring themes in diverse biological contexts.

Primordial aminoacyl-tRNA synthetases preferred minihelices to full-length tRNA.

Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.

The discovery of a catalytic RNA within RNase P and its legacy.

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

How Natural Enzymes and Synthetic Ribozymes Generate Methylated Nucleotides in RNA.

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.

Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

From Enigma to Revelation: Unravelling Biological Functions of Ubiquitous Small Ribozymes.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

New Insights into the Dependence of CPEB3 Ribozyme Cleavage on Mn2+ and Mg2.

CPEB3 ribozyme is a self-cleaving RNA that occurs naturally in mammals and requires divalent metal ions for efficient activity. Ribozymes exhibit preferences for specific metal ions, but the exact differences in the catalytic mechanisms of various metal ions on the CPEB3 ribozyme remain unclear. Our findings reveal that Mn2+ functions as a more effective cofactor for CPEB3 ribozyme catalysis compared to Mg2+, as confirmed by its stronger binding affinity to CPEB3 by EPR. Cleavage assays of CPEB3 mutants and molecular docking analyses further showed that excessive Mn2+ ions can bind to a second binding site near the catalytic site, hindering CPEB3 catalytic efficiency and contributing to the Mn2+ bell-shaped curve. These results implicate a pivotal role for the local nucleobase-Mn2+ interactions in facilitating RNA folding and modulating the directed attack of nucleophilic reagents. Our study provides new insights and experimental evidence for exploring the divalent cation dependent cleavage mechanism of the CPEB3 ribozyme.

Multiple structural flavors of RNase P in precursor tRNA processing.

The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

High-Fidelity RNA Copying via 2',3'-Cyclic Phosphate Ligation.

Templated ligation offers an efficient approach to replicate long strands in an RNA world. The 2',3'-cyclic phosphate (>P) is a prebiotically available activation that also forms during RNA hydrolysis. Using gel electrophoresis and high-performance liquid chromatography, we found that the templated ligation of RNA with >P proceeds in simple low-salt aqueous solutions with 1 mM MgCl2 under alkaline pH ranging from 9 to 11 and temperatures from -20 to 25 °C. No additional catalysts were required. In contrast to previous reports, we found an increase in the number of canonical linkages to 50%. The reaction proceeds in a sequence-specific manner, with an experimentally determined ligation fidelity of 82% at the 3' end and 91% at the 5' end of the ligation site. With splinted oligomers, five ligations created a 96-mer strand, demonstrating a pathway for the ribozyme assembly. Due to the low salt requirements, the ligation conditions will be compatible with strand separation. Templated ligation mediated by 2',3'-cyclic phosphate in alkaline conditions therefore offers a performant replication and elongation reaction for RNA on early Earth.

Chemically Cross-Linked Hammerhead Ribozyme as an Efficient RNA Interference Tool.

RNA-cleaving ribozymes are promising candidates as general tools of RNA interference (RNAi) in gene manipulation. However, compared with other RNA systems, such as siRNA and CRISPR technologies, the ribozyme tools are still far from broad applications on RNAi due to their poor performance in the cellular context. In this work, we report an efficient RNAi tool based on chemically modified hammerhead ribozyme (HHR). By the introduction of an intramolecular linkage into the minimal HHR to reconstruct the distal interaction within the tertiary ribozyme structure, this cross-linked HHR exhibits efficient RNA substrate cleavage activities with almost no sequence constraint. Cellular experiments suggest that both exogenous and endogenous RNA expression can be dramatically knocked down by this HHR tool with levels comparable to those of siRNA. Unlike the widely applied protein-recruiting RNA systems (siRNA and CRISPR), this ribozyme tool functions solely on RNA itself with great simplicity, which may provide a new approach for gene manipulation in both fundamental and translational studies.

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying.

2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

Deep generative design of RNA family sequences.

RNA engineering has immense potential to drive innovation in biotechnology and medicine. Despite its importance, a versatile platform for the automated design of functional RNA is still lacking. Here, we propose RNA family sequence generator (RfamGen), a deep generative model that designs RNA family sequences in a data-efficient manner by explicitly incorporating alignment and consensus secondary structure information. RfamGen can generate novel and functional RNA family sequences by sampling points from a semantically rich and continuous representation. We have experimentally demonstrated the versatility of RfamGen using diverse RNA families. Furthermore, we confirmed the high success rate of RfamGen in designing functional ribozymes through a quantitative massively parallel assay. Notably, RfamGen successfully generates artificial sequences with higher activity than natural sequences. Overall, RfamGen significantly improves our ability to design functional RNA and opens up new potential for generative RNA engineering in synthetic biology.

An RNA Motif That Enables Optozyme Control and Light-Dependent Gene Expression in Bacteria and Mammalian Cells.

The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.