Nonenzymatic template-directed RNA synthesis inside model protocells.
ABSTRACT: Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg(2+) on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg(2+) ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg(2+)-catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.
Project description:The chemical replication of RNA inside fatty acid vesicles is a plausible step in the emergence of cellular life. On the primitive Earth, simple protocells with the ability to import nucleotides and short oligomers from their environment could potentially have replicated and retained larger genomic RNA oligonucleotides within a spatially defined compartment. We have previously shown that short 5'-phosphoroimidazolide-activated "helper" RNA oligomers enable the nonenzymatic copying of mixed-sequence templates in solution, using 5'-phosphoroimidazolide-activated mononucleotides. Here, we report that citrate-chelated Mg2+, a catalyst of nonenzymatic primer extension, enhances fatty acid membrane permeability to such short RNA oligomers up to the size of tetramers, without disrupting vesicle membranes. In addition, selective permeability of short, but not long, oligomers can be further enhanced by elevating the temperature. The ability to increase the permeability of fatty acid membranes to short oligonucleotides allows for the nonenzymatic copying of RNA templates containing all four nucleotides inside vesicles, bringing us one step closer to the goal of building a protocell capable of Darwinian evolution.
Project description:The advent of Darwinian evolution required the emergence of molecular mechanisms for the heritable variation of fitness. One model for such a system involves competing protocell populations, each consisting of a replicating genetic polymer within a replicating vesicle. In this model, each genetic polymer imparts a selective advantage to its protocell by, for example, coding for a catalyst that generates a useful metabolite. Here, we report a partial model of such nascent evolutionary traits in a system that consists of fatty-acid vesicles containing a dipeptide catalyst, which catalyses the formation of a second dipeptide. The newly formed dipeptide binds to vesicle membranes, which imparts enhanced affinity for fatty acids and thus promotes vesicle growth. The catalysed dipeptide synthesis proceeds with higher efficiency in vesicles than in free solution, which further enhances fitness. Our observations suggest that, in a replicating protocell with an RNA genome, ribozyme-catalysed peptide synthesis might have been sufficient to initiate Darwinian evolution.
Project description:Contemporary phospholipid-based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross dimyristoyl phosphatidylcholine membranes through defects formed at the gel-to-liquid transition temperature, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles that retain encapsulated oligonucleotides and are capable of growth and division. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery; that is, they could have been obligate heterotrophs.
Project description:The development of a sequence-general nucleic acid copying system is an essential step in the assembly of a synthetic protocell, an autonomously replicating spatially localized chemical system capable of spontaneous Darwinian evolution. Previously described nonenzymatic template-copying experiments have validated the concept of nonenzymatic replication, but have not yet achieved robust, sequence-general polynucleotide replication. The 5'-phosphorimidazolides of the 2'-amino-2',3'-dideoxyribonucleotides are attractive as potential monomers for such a system because they polymerize by forming 2'-->5' linkages, which are favored in nonenzymatic polymerization reactions using similarly activated ribonucleotides on RNA templates. Furthermore, the 5'-activated 2'-amino nucleotides do not cyclize. We recently described the rapid and efficient nonenzymatic copying of a DNA homopolymer template (dC(15)) encapsulated within fatty acid vesicles using 2'-amino-2',3'-dideoxyguanosine-5'-phosphorimidazolide as the activated monomer. However, to realize a true Darwinian system, the template-copying chemistry must be able to copy most sequences and their complements to allow for the transmission of information from generation to generation. Here, we describe the copying of a series of nucleic acid templates using 2'-amino-2',3'-dideoxynucleotide-5'-phosphorimidazolides. Polymerization reactions proceed rapidly to completion on short homopolymer RNA and LNA templates, which favor an A-type duplex geometry. We show that more efficiently copied sequences are generated by replacing the adenine nucleobase with diaminopurine, and uracil with C5-(1-propynyl)uracil. Finally, we explore the copying of longer, mixed-sequence RNA templates to assess the sequence-general copying ability of 2'-amino-2',3'-dideoxynucleoside-5'-phosphorimidazolides. Our results are a significant step forward in the realization of a self-replicating genetic polymer compatible with protocell template copying and suggest that N2'-->P5'-phosphoramidate DNA may have the potential to function as a self-replicating system.
Project description:The generation of synthetic forms of cellular life requires solutions to the problem of how biological processes such as cyclic growth and division could emerge from purely physical and chemical systems. Small unilamellar fatty acid vesicles grow when fed with fatty acid micelles and can be forced to divide by extrusion, but this artificial division process results in significant loss of protocell contents during each division cycle. Here we describe a simple and efficient pathway for model protocell membrane growth and division. The growth of large multilamellar fatty acid vesicles fed with fatty acid micelles, in a solution where solute permeation across the membranes is slow, results in the transformation of initially spherical vesicles into long thread-like vesicles, a process driven by the transient imbalance between surface area and volume growth. Modest shear forces are then sufficient to cause the thread-like vesicles to divide into multiple daughter vesicles without loss of internal contents. In an environment of gentle shear, protocell growth and division are thus coupled processes. We show that model protocells can proceed through multiple cycles of reproduction. Encapsulated RNA molecules, representing a primitive genome, are distributed to the daughter vesicles. Our observations bring us closer to the laboratory synthesis of a complete protocell consisting of a self-replicating genome and a self-replicating membrane compartment. In addition, the robustness and simplicity of this pathway suggests that similar processes might have occurred under the prebiotic conditions of the early Earth.
Project description:Here we develop a computational model that examines one of the first major biological innovations-the origin of heredity in simple protocells. The model assumes that the earliest protocells were autotrophic, producing organic matter from CO2 and H2 Carbon fixation was facilitated by geologically sustained proton gradients across fatty acid membranes, via iron-sulfur nanocrystals lodged within the membranes. Thermodynamic models suggest that organics formed this way should include amino acids and fatty acids. We assume that fatty acids partition to the membrane. Some hydrophobic amino acids chelate FeS nanocrystals, producing three positive feedbacks: (i) an increase in catalytic surface area; (ii) partitioning of FeS nanocrystals to the membrane; and (iii) a proton-motive active site for carbon fixing that mimics the enzyme Ech. These positive feedbacks enable the fastest-growing protocells to dominate the early ecosystem through a simple form of heredity. We propose that as new organics are produced inside the protocells, the localized high-energy environment is more likely to form ribonucleotides, linking RNA replication to its ability to drive protocell growth from the beginning. Our novel conceptualization sets out conditions under which protocell heredity and competition could arise, and points to where crucial experimental work is required.This article is part of the themed issue 'Process and pattern in innovations from cells to societies'.
Project description:Protocell multilevel selection models have been proposed to study the evolutionary dynamics of vesicles encapsulating a set of replicating, competing and mutating sequences. The frequency of the different sequence types determines protocell survival through a fitness function. One of the defining features of these models is the genetic load generated when the protocell divides and its sequences are assorted between the offspring vesicles. However, these stochastic assortment effects disappear when the redundancy of each sequence type is sufficiently high. The fitness dependence of the vesicle with its sequence content is usually defined without considering a realistic account on how the lower level dynamics would specify the protocell fitness. Here, we present a protocell model with a fitness function determined by the output flux of a simple metabolic network with the aim of understanding how the evolution of both kinetic and topological features of metabolism would have been constrained by the particularities of the protocell evolutionary dynamics. In our model, the sequences inside the vesicle are both the carriers of information and Michaelis-Menten catalysts exhibiting saturation. We found that the saturation of the catalysts controlling the metabolic fluxes, achievable by modifying the kinetic or stoichiometric parameters, provides a mechanism to ameliorate the assortment load by increasing the redundancy of the catalytic sequences required to achieve the maximum flux. Regarding the network architecture, we conclude that combinations of parallel network motifs and bimolecular catalysts are a robust way to increase the complexity of the metabolism enclosed by the protocell.
Project description:One widely held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components. Here we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg(2+), which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg(2+) environments. The synthetic transformation requires both the catalyst and vesicles that solubilize the water-insoluble precursor lipid. We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells.
Project description:The development of programmable microscale materials with cell-like functions, dynamics and collective behaviour is an important milestone in systems chemistry, soft matter bioengineering and synthetic protobiology. Here, polymer/nucleotide coacervate micro-droplets are reconfigured into membrane-bounded polyoxometalate coacervate vesicles (PCVs) in the presence of a bio-inspired Ru-based polyoxometalate catalyst to produce synzyme protocells (Ru4PCVs) with catalase-like activity. We exploit the synthetic protocells for the implementation of multi-compartmentalized cell-like models capable of collective synzyme-mediated buoyancy, parallel catalytic processing in individual horseradish peroxidase-containing Ru4PCVs, and chemical signalling in distributed or encapsulated multi-catalytic protocell communities. Our results highlight a new type of catalytic micro-compartment with multi-functional activity and provide a step towards the development of protocell reaction networks.
Project description:The transition from independent molecular entities to cellular structures with integrated behaviors was a crucial aspect of the origin of life. We show that simple physical principles can mediate a coordinated interaction between genome and compartment boundary, independent of any genomic functions beyond self-replication. RNA, encapsulated in fatty acid vesicles, exerts an osmotic pressure on the vesicle membrane that drives the uptake of additional membrane components, leading to membrane growth at the expense of relaxed vesicles, which shrink. Thus, more efficient RNA replication could cause faster cell growth, leading to the emergence of Darwinian evolution at the cellular level.