Search

The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs., Angew. Chem., Int. Ed. Engl.;60(50)

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin of life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into extremely long chains. Our results show that formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype – phenotype linkage.

The replication of nucleic acids is a prerequisite for the survival and evolution of living systems. Before the advent of protein synthesis, genetic information was most likely stored on and replicated by RNA. However, experimental systems for sustained RNA-dependent RNA-replication are difficult to realise due to the high thermodynamic stability of duplex products and the low chemical stability of catalytic polynucleotides. Using a derivative of a group I intron as a model for an RNA replicase, we show that heated air-water interfaces exposed to a plausible CO2-rich atmosphere drive complete cycles of plus and minus strand replication. The reaction is driven by autonomous oscillations in salt concentrations and pH that transiently destabilise RNA duplexes. Newly synthesised strands can dissociate from their templates and adopt catalytically active conformations. Our results suggest that an abundant Hadean microenvironment may have promoted the replication of RNAs while maintaining the activity of the synthesised ribozymes.

Growth and division of biological cells is based on the complex orchestration of spatiotemporally controlled reactions driven by highly evolved proteins. In contrast, it remains unknown how their primordial predecessors could achieve a stable inheritance of cytosolic components before the advent of translation. An attractive scenario assumes that periodic changes of environmental conditions acted as pacemakers for the proliferation of early protocells. Using catalytic RNA (ribozymes) as models for primitive biocatalytic molecules, we demonstrate that the repeated freezing and thawing of aqueous solutions enables the assembly of active ribozymes from inactive precursors encapsulated in separate lipid vesicle populations. Furthermore, we show that encapsulated ligase ribozymes can overcome freezing-induced content loss and successive dilution by freeze-thaw driven propagation in feedstock vesicles. Thus, cyclic freezing and melting of aqueous solvents – a plausible physicochemical driver likely present on early Earth – provides a simple scenario that uncouples compartment growth and division from nucleic acid self-replication, while maintaining the propagation of these replicators inside new vesicle populations.

Catalytic nucleic acids, such as ribozymes, are central to a variety of origin-of-life scenarios. Typically, they require elevated magnesium concentrations for folding and activity, but their function can be inhibited by high concentrations of monovalent salts. Here we show that geologically plausible high-sodium, low-magnesium solutions derived from leaching basalt (rock and remelted glass) inhibit ribozyme catalysis, but that this activity can be rescued by selective magnesium up-concentration by heat flow across rock fissures. In contrast to up-concentration by dehydration or freezing, this system is so far from equilibrium that it can actively alter the Mg:Na salt ratio to an extent that enables key ribozyme activities, such as self-replication and RNA extension, in otherwise challenging solution conditions. The principle demonstrated here is applicable to a broad range of salt concentrations and compositions, and, as such, highly relevant to various origin-of-life scenarios.

RNA‐catalyzed RNA ligation is widely believed to be a key reaction for primordial biology. However, since typical chemical routes towards activating RNA substrates are incompatible with ribozyme catalysis, it remains unclear how prebiotic systems generated and sustained pools of activated building blocks needed to form increasingly larger and complex RNA. Herein, we demonstrate in situ activation of RNA substrates under reaction conditions amenable to catalysis by the hairpin ribozyme. We found that diamidophosphate (DAP) and imidazole drive the formation of 2′,3′‐cyclic phosphate RNA mono‐ and oligonucleotides from monophosphorylated precursors in frozen water‐ice. This long‐lived activation enables iterative enzymatic assembly of long RNAs. Our results provide a plausible scenario for the generation of higher‐energy substrates required to fuel ribozyme‐catalyzed RNA synthesis in the absence of a highly evolved metabolism., A plausible scenario for the prebiotic generation of activated RNA substrates under mild aqueous conditions is presented. Using water‐soluble diamidophosphate, in situ production of 2′,3′‐cyclic phosphate‐activated oligoribonucleotides and their subsequent ligation by a ribozyme can be achieved.

The ability to produce single-stranded DNA on a preparative scale from low amounts of starting templates is necessary for most research involving deoxyribozymes, but is particularly important for performing in vitro selections. While the production of single-stranded RNA is straightforward by means of in vitro transcription, the enzymatic production of single-stranded DNA (ssDNA) on a preparative scale is often difficult. Nevertheless, several methods for the production of ssDNA have been published over the years. Here, we present two PCR methods that we find to be particularly effective, fast, and affordable, which we have adapted for our own needs.

RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.

The RNA world hypothesis is probably the most extensively studied model for the emergence of life on Earth. Despite a large body of evidence supporting the idea that RNA is capable of kick-starting autocatalytic self-replication and thus initiating the emergence of life, seemingly insurmountable weaknesses in the theory have also been highlighted. These problems could be overcome by novel experimental approaches, including out-of-equilibrium environments, and the exploration of an early co-evolution of RNA and other key biomolecules such as peptides and DNA, which might be necessary to mitigate the shortcomings of RNA-only systems.

The coliphage MS2 is a well-established model organism that has helped to reveal a variety of fundamental concepts for RNA-based translational control and viral RNA packaging. Despite the comprehensive characterisation of the MS2 life cycle, the macromolecular composition of its RNA replicase remained obscure. Here, we sought to identify the missing host proteins required for the assembly of the active replicase complex. By combining a purified, inactive MS2 replicase sub-complex with selected modules of an in vitro translation system, we confirmed that the three suspected host factors EF-Ts, EF-Tu and ribosomal protein S1 form an active core-enzyme with the viral replicase subunit. Unexpectedly, we also found that the translation initiation factors IF1 and IF3 directly modulate MS2 replicase activity. While IF1 enhances replicase activity in a template-independent manner, IF3 acts as an inhibitor that prevents polymerase initiation and / or elongation. Both observations suggest a previously unknown role of these host proteins during the phage life cycle. Finally, we demonstrate the in vitro formation of small RNAs that contain minimal motifs required for MS2 replicase-dependent amplification. Our work sheds new light on the architecture of the MS2 replication machinery while also providing the basis for a new cell-free evolution platform.

Membraneless organelles like stress granules are active liquid-liquid phase-separated droplets that are involved in many intracellular processes. Their active and dynamic behavior is often regulated by ATP-dependent reactions. However, how exactly membraneless organelles control their dynamic composition remains poorly understood. Herein, we present a model for membraneless organelles based on RNA-containing active coacervate droplets regulated by a fuel-driven reaction cycle. These droplets emerge when fuel is present, but decay without. Moreover, we find these droplets can transiently up-concentrate functional RNA, and that this up-take is accelerated by the chemical reaction cycle. Finally, we show that in their pathway towards decay, these droplets self-divide asymmetrically. Self-division combined with emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of life, and we believe that our work could be a stepping stone towards its synthesis.

The bottom-up construction of bio-inspired systems capable of self-maintenance and reproduction is a central goal in systems chemistry and synthetic biology. A particular challenge in such systems is the continuous regeneration of key proteins required for macromolecular synthesis. Here, we probe self-maintenance of a reconstituted in vitro translation system challenged by serial transfer of selected key proteins. We find that the system can simultaneously regenerate multiple essential polypeptides, which then contribute to the maintenance of protein expression after serial transfer. The presented strategy offers a robust methodology for probing and optimizing continuous self-regeneration of proteins in cell-free environments.

Ein großes deutsches Max‐Planck‐Forschungskonsortium (“MaxSynBio”) untersucht lebende Systeme aus fundamentaler Perspektive. Das Forschungsprogramm von MaxSynBio verfolgt dabei ausschließlich einen “Bottom‐up”‐Ansatz in der synthetischen Biologie: Es konzentriert sich auf die detaillierte Analyse und das Verständnis essentieller Lebensprozesse, indem es diese Prozesse als elementare Module in minimalen synthetischen Systemen rekonstituiert. Das ultimative Ziel von MaxSynBio ist es, eine lebende Zelle komplett aus nicht lebenden Komponenten zu konstruieren. Die grundlegenden Erkenntnisse aus den Aktivitäten in MaxSynBio können auf lange Sicht zur Etablierung neuer biotechnologischer Verfahren führen, die auf synthetischen Zellkonstrukten basieren. Langfristig könnten dadurch Organismen ersetzt werden, die derzeit in der konventionellen Biotechnologie Verwendung finden. This is the German version of Angewandte Chemie.

Understanding the prebiotic origins of the nucleic acids is a long-standing challenge. The latest experiments support the idea that the first nucleic acid encoded information using a mixed ‘alphabet’ of RNA and DNA subunits. RNA and DNA nucleosides might have emerged together on prebiotic Earth.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA–peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

Catalysis by nucleic acids is indispensable for extant cellular life, and it is widely accepted that nucleic acid enzymes were crucial for the emergence of primitive life 3.5‐4 billion years ago. However, geochemical conditions on early Earth must have differed greatly from the constant internal milieus of today's cells. In order to explore plausible scenarios for early molecular evolution, it is therefore essential to understand how different physicochemical parameters, such as temperature, pH, and ionic composition, influence nucleic acid catalysis and to explore to what extent nucleic acid enzymes can adapt to non‐physiological conditions. In this article, we give an overview of the research on catalysis of nucleic acids, in particular catalytic RNAs (ribozymes) and DNAs (deoxyribozymes), under extreme and/or unusual conditions that may relate to prebiotic environments., Many different physicochemical parameters influence nucleic acid catalysis, including temperature, pH, pressure, dehydration and metal ion composition. In order to explore plausible scenarios for early molecular evolution and investigate to what extent nucleic acid enzymes can adapt to non‐physiological conditions, it is essential to understand how different environmental conditions both inhibit and potentiate nucleic acid catalysis. This review demonstrates that nucleic acid catalysts can tolerate a wide range of challenging environments, and in many cases are enhanced by environmental factors.

The generation of a chemical system capable of replication and evolution is a key objective of synthetic biology. This could be achieved by in vitro reconstitution of a minimal self-sustaining central dogma consisting of DNA replication, transcription and translation. Here, we present an in vitro translation system, which enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions. In particular, we demonstrate self-replication of a multipartite genome of more than 116 kb encompassing the full set of Escherichia coli translation factors, all three ribosomal RNAs, an energy regeneration system, as well as RNA and DNA polymerases. Parallel to DNA replication, our system enables synthesis of at least 30 encoded translation factors, half of which are expressed in amounts equal to or greater than their respective input levels. Our optimized cell-free expression platform could provide a chassis for the generation of a partially self-replicating in vitro translation system., A main objective of synthetic biology is the creation of chemical systems capable of replication and evolution. Here, the authors demonstrate combined self-replication and expression of multipartite genomes in vitro.

Recombination, the exchange of information between different genetic polymer strands, is of fundamental importance in biology for genome maintenance and genetic diversification and is mediated by dedicated recombinase enzymes. Here, we describe an innate capacity for non-enzymatic recombination (and ligation) in random-sequence genetic oligomer pools. Specifically, we examine random and semi-random eicosamer (N20) pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids). While DNA, ANA and HNA pools proved inert, RNA (and to a lesser extent AtNA) pools displayed diverse modes of spontaneous intermolecular recombination, connecting recombination mechanistically to the vicinal ring cis-diol configuration shared by RNA and AtNA. Thus, the chemical constitution that renders both susceptible to hydrolysis emerges as the fundamental determinant of an innate capacity for recombination, which is shown to promote a concomitant increase in compositional, informational and structural pool complexity and hence evolutionary potential. ispartof: ELIFE vol:7 ispartof: location:England status: published

A large German research consortium mainly within the Max Planck Society (“MaxSynBio”) was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom‐up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non‐living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

Non-equilibrium conditions must have been crucial for the assembly of the first informational polymers of early life, but supporting their formation and continuous enrichment in a long-lasting environment. Here, we explore how gas bubbles in water subjected to a thermal gradient, a likely scenario within crustal mafic rocks on the early Earth, drive a complex, continuous enrichment of prebiotic molecules. NRA precursors, monomers, active ribozymes, oligonucleotides and lipids are shown to (1) cycle between dry and wet states, enabling the central step of RNA phosphorylation, (2) accumulate at the gas-water interface to drastically increase ribozymatic activity, (3) condense into hydrogels, (4) form pure crystals and (5) encapsulate into protecting vesicle aggregates that subsequently undergo fission. These effects occur within less than 30 min. The findings unite, in one location, the physical conditions that were crucial for the chemical emergence of biopolymers. They suggest that heated microbubbles could have hosted the first cycles of molecular evolution., Comment: 27 pages, 7 Figures

The spontaneous emergence of function from prebiotic pools of informational polymers is a central conjecture of current origin of life scenarios. However, the innate functional capacity of random genetic polymer pools is unknown. Here, we have examined the ab initio activity of random and semi-random eicosamer pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids) with respect to a simple functional test: the capacity for intermolecular ligation and recombination. While DNA, ANA and HNA pools proved inert, naïve RNA and AtNA pools displayed diverse modes of intermolecular recombination in eutectic ice phases. Recombination appears linked to the vicinal ring cis-diol shared by RNA and AtNA. Thus, the chemical configuration that renders both susceptible to hydrolysis also enables substantial spontaneous intrapool recombination in the absence of activation chemistry with a concomitant increase in the compositional and structural complexity of recombined pools.

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalization via coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides., Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via coacervation. Here authors demonstrate that a coacervate microenvironment supports RNA catalysis whilst selectively sequestering RNA based on length.

The creation of self-replicating minimal cells is a major goal in synthetic biology and origin of life research. In order to be considered alive, a minimal cell must have several characteristics such as the ability to reproduce and adapt to its environment. Our research focuses on genetic replication and evolution in such minimal systems.

Bottom-up synthetic biology uses both biological and artificial chemical building blocks to create biomimetic systems, including artificial cells. Existing and new technologies such as microfluidics are being developed and applied to the assembly processes. In this special issue, experts present and review the latest progress in this rapidly expanding and diverse field.

Informationentragende Nukleinsauren sind universell uber 3′-5′-Bindungen verknupft. Regioisomere 2′-5′-Verknupfungen bilden sich dagegen unregelmasig wahrend der nichtenzymatischen RNA-Synthese und waren womoglich hilfreich fur die prabiotische RNA-Replikation. Hier berichten wir uber die enzymatische Synthese von DNA und RNA mit ortsspezifischen 2′-5′-Verknupfungen durch ein gezielt verandertes Polymerase-Enzym, das 3′-Desoxy- oder 3′-O-Methyl-NTPs als Substrat verwendet. Zusatzlich vermelden wir die reverse Transkription der so hergestellten, modifizierten Nukleinsauren zuruck in 3′-5′-verknupfte DNA mit guter Fidelitat. Somit ergibt sich eine schnelle und einfache Methode zur “strukturellen Mutagenese” durch positionsselektives Einfugen von 2′-5′-Verknupfungen, durch die, ohne Anderung der Basensequenz, die Struktur und Funktion von Nukleinsauren anhand lokaler Deformationen durch regioisomere Bindungen untersucht werden kann. Beispielhaft wenden wir diese Methode auf das 10-23-RNA-Endonuklease-DNAzym an.

Information‐bearing nucleic acids display universal 3′‐5′ linkages, but regioisomeric 2′‐5′ linkages occur sporadically in non‐enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site‐specific 2′‐5′ linkages by an engineered polymerase using 3′‐deoxy‐ or 3′‐O‐methyl‐NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′‐5′ linked DNA with good fidelity. This enables a fast and simple method for “structural mutagenesis” by the position‐selective incorporation of 2′‐5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild‐type base sequence as we demonstrate for the 10–23 RNA endonuclease DNAzyme.

A key characteristic of living systems is the storage and replication of information, and as such the development of self-replicating systems capable of heredity is of great importance to the fields of synthetic biology and origin of life research. In this review, the design and implementation of self-replicating systems in the context of bottom-up synthetic biology is discussed, with a particular focus on nucleic acid-based replication including nonenzymatic systems, ribozyme-based systems, and complex in vitro translation coupled RNA and DNA replication. The current state and remaining challenges of the respective fields are discussed, and the potential of individual replicators for synthetic biology applications such as the creation of artificial life capable of Darwinian evolution is also summarized.

RNA polymerase II (Pol II) in Saccharomyces cerevisiae can terminate transcription via several pathways. To study how a mechanism is chosen, we analyzed recruitment of Nrd1, which cooperates with Nab3 and Sen1 to terminate small nucleolar RNAs and other short RNAs. Budding yeast contains three C-terminal domain (CTD) interaction domain (CID) proteins, which bind the CTD of the Pol II largest subunit. Rtt103 and Pcf11 act in mRNA termination, and both preferentially interact with CTD phosphorylated at Ser2. The crystal structure of the Nrd1 CID shows a fold similar to that of Pcf11, but Nrd1 preferentially binds to CTD phosphorylated at Ser5, the form found proximal to promoters. This indicates why Nrd1 cross-links near 5' ends of genes and why the Nrd1-Nab3-Sen1 termination pathway acts specifically at short Pol II-transcribed genes. Nrd1 recruitment to genes involves a combination of interactions with CTD and Nab3.

Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2'-5' linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23 RNA endonuclease DNAzyme.

Cell death in bacteria can be triggered by activation of self-inflicted molecular mechanisms. Pathogenic bacteria often make use of suicide mechanisms in which the death of individual cells benefits survival of the population. Important elements for programmed cell death in bacteria are proteinaceous toxin–antitoxin systems. While the toxin generally resides dormant in the bacterial cytosol in complex with its antitoxin, conditions such as impaired de novo synthesis of the antitoxin or nutritional stress lead to antitoxin degradation and toxin activation. A widespread toxin–antitoxin family consists of the ε/ζ systems, which are distributed over plasmids and chromosomes of various pathogenic bacteria. In its inactive state, the bacteriotoxic ζ toxin protein is inhibited by its cognate antitoxin ε. Upon degradation of ε, the ζ toxin is released allowing this enzyme to poison bacterial cell wall synthesis, which eventually triggers autolysis. ε/ζ systems ensure stable plasmid inheritance by inducing death in plasmid-deprived offspring cells. In contrast, chromosomally encoded ε/ζ systems were reported to contribute to virulence of pathogenic bacteria, possibly by inducing autolysis in individual cells under stressful conditions. The capability of toxin–antitoxin systems to kill bacteria has made them potential targets for new therapeutic compounds. Toxin activation could be hijacked to induce suicide of bacteria. Likewise, the unique mechanism of ζ toxins could serve as template for new drugs. Contrarily, inhibition of virulence-associated ζ toxins might attenuate infections. Here we provide an overview of ε/ζ toxin–antitoxin family and its potential role in the development of new therapeutic approaches in microbial defense.