The Long Road to a Synthetic Self-Replicating Central Dogma.

The construction of a biochemical system capable of self-replication is a key objective in bottom-up synthetic biology. Throughout the past two decades, a rapid progression in the design of in vitro cell-free systems has provided valuable insight into the requirements for the development of a minimal system capable of self-replication. The main limitations of current systems can be attributed to their macromolecular composition and how the individual macromolecules use the small molecules necessary to drive RNA and protein synthesis. In this Perspective, we discuss the recent steps that have been taken to generate a minimal cell-free system capable of regenerating its own macromolecular components and maintaining the homeostatic balance between macromolecular biogenesis and consumption of primary building blocks. By following the flow of biological information through the central dogma, we compare the current versions of these systems to date and propose potential alterations aimed at designing a model system for self-replicative synthetic cells.

Cell-free expression of RNA encoded genes using MS2 replicase.

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.

Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.

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.

Prebiotically Plausible RNA Activation Compatible with Ribozyme-Catalyzed Ligation.

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.

Special Issue on Bottom-Up Synthetic Biology.

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.

MaxSynBio: Avenues Towards Creating Cells from the Bottom Up.

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.

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2'-5' Linkages.

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.

Non-canonical 3'-5' extension of RNA with prebiotically plausible ribonucleoside 2',3'-cyclic phosphates.

Ribonucleoside 2',3'-cyclic phosphates (N>p's) are generated by multiple prebiotically plausible processes and are credible building blocks for the assembly of early RNA oligomers. While N>p's can be polymerized into short RNAs by non-enzymatic processes with variable efficiency and regioselectivity, no enzymatic route for RNA synthesis had been described. Here we report such a non-canonical 3'-5' nucleotidyl transferase activity. We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-cyclic A-, G-, U-, and CMP) to the 5'-hydroxyl termini of RNA strands with 5' nucleotide addition enhanced in all cases by eutectic ice phase formation at -7 °C. We also observed 5' addition of 2',3'-cyclic phosphate-activated ß-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers. Our results establish a new mode of RNA 3'-5' extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.

A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis.

Most genomes of bacteria contain toxin-antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics.

Design of Novel Synthetic RNA Replicons Based on Emesvirus zinderi.

Self-replicating RNAs (srRNAs) are synthetic molecules designed to mimic the self-replicating ability of viral RNAs. srRNAs hold significant promise for a range of applications, including enhancing protein expression, reprogramming cells into pluripotent stem cells, and creating cell-free systems for experimental evolution. However, the development of srRNAs for use in bacterial systems remains limited. Here, we demonstrate how a srRNA scaffold from Emesvirus zinderi can be engineered into a self-encoding srRNA by incorporating the coding region of the catalytically active replicase subunit. With the help of in vitro replication assays, including an in vitro translation-coupled replication approach, we show that the resulting system enables complete replication cycles of RNA both in cis and trans, including long cargo RNAs such as tethered 5S, 16S, and 23S rRNAs. In summary, our findings suggest that these srRNAs have significant potential for fundamental research, synthetic biology, and general in vitro evolution.

Design principles and applications of synthetic self-replicating RNAs.

With the advent of ever more sophisticated methods for the in vitro synthesis and the in vivo delivery of RNAs, synthetic mRNAs have gained substantial interest both for medical applications, as well as for biotechnology. However, in most biological systems exogeneous mRNAs possess only a limited half-life, especially in fast dividing cells. In contrast, viral RNAs can extend their lifetime by actively replicating inside their host. As such they may serve as scaffolds for the design of synthetic self-replicating RNAs (srRNA), which can be used to increase both the half-life and intracellular concentration of coding RNAs. Synthetic srRNAs may be used to enhance recombinant protein expression or induce the reprogramming of differentiated cells into pluripotent stem cells but also to create cell-free systems for research based on experimental evolution. In this article, we discuss the applications and design principles of srRNAs used for cellular reprogramming, mRNA-based vaccines and tools for synthetic biology. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Periodic temperature changes drive the proliferation of self-replicating RNAs in vesicle populations.

Growth and division of biological cells are 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 ribozyme replicators 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 RNA self-replication, while maintaining the propagation of these replicators inside new vesicle populations.

Ribozyme activity modulates the physical properties of RNA-peptide coacervates.

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 long chains. Our results show that the 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.

Ribozyme-mediated RNA synthesis and replication in a model Hadean microenvironment.

Enzyme-catalyzed replication of nucleic acid sequences is a prerequisite for the survival and evolution of biological entities. Before the advent of protein synthesis, genetic information was most likely stored in and replicated by RNA. However, experimental systems for sustained RNA-dependent RNA-replication are difficult to realise, in part due to the high thermodynamic stability of duplex products and the low chemical stability of catalytic RNAs. Using a derivative of a group I intron as a model for an RNA replicase, we show that heated air-water interfaces that are exposed to a plausible CO2-rich atmosphere enable sense and antisense RNA replication as well as template-dependent synthesis and catalysis of a functional ribozyme in a one-pot reaction. Both reactions are driven by autonomous oscillations in salt concentrations and pH, resulting from precipitation of acidified dew droplets, which transiently destabilise RNA duplexes. Our results suggest that an abundant Hadean microenvironment may have promoted both replication and synthesis of functional RNAs.

PCR Methods for the Generation of Catalytic ssDNA.

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.

Generation of RNA with 2', 3'-Cyclic Phosphates by Deoxyribozyme Cleavage in Frozen Solutions.

The generation of terminal 2', 3'-cyclic phosphates on RNA oligomers is an important process in the study of tRNA splicing and repair, ribozyme catalysis, and RNA circularization. Here, we describe a simple method for producing 2', 3'-cyclic phosphate functionalized RNA by the deoxyribozyme-catalyzed cleavage of a short 3'-RNA overhang in frozen solution. This method avoids the nonspecific modification and degradation of RNA and attached functional groups (e.g., fluorophores) inherent in other methods, and the use of frozen conditions enables cleavage at very low divalent metal ion concentrations, limiting RNA hydrolysis.

In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity.

The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.

Charge-density reduction promotes ribozyme activity in RNA-peptide coacervates via RNA fluidization and magnesium partitioning.

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.

Assembly dynamics and stability of the pneumococcal epsilon zeta antitoxin toxin (PezAT) system from Streptococcus pneumoniae.

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.