Earth’s crust and primordial ocean formed more than 4 billion years ago and life is believed to have originated on earth at least 3.6 billion years ago. This suggests that primitive cellular life must have evolved from non-living matter during that period of several hundred million years. To study the transition from chemistry to biology, a simple vesicular system called a protocell is an ideal model that is self-organized and contains informational or metabolic materials. This thesis starts by exploring the replication of a model genetic material under plausible prebiotic conditions. The non-enzymatic copying of RNA was found to be catalyzed by Fe2+, which used to be abundant in aqueous environments on the early anoxic earth. Fe2+ was found to be a better catalyst of non-enzymatic RNA copying and ligation in slightly acidic to neutral pH conditions than Mg2+, the divalent cation used to catalyze these reactions in previous studies. This finding suggests that ferrous iron could have facilitated the replication and evolution of RNA on the prebiotic earth. To gain a better understanding of the properties of protocell membranes, the impact of membrane composition and multi-bilayer structure on non-enzymatic and enzymatic biochemical reactions was studied. A fatty acid/phospholipid hybrid membrane system was proposed as a potential intermediate state in protocellular evolution. This membrane composition was investigated for its stability and permeability, two fundamental features of functional protocells. The system proved stable in the presence of divalent cations and retained permeability to small building block molecule. Vesicles with this composition were shown to host faster non-enzymatic RNA copying, and to enable enzymatic protein synthesis. To study the effects of multi-lamellarity, giant multilamellar vesicles (GMVs) were prepared by an extrusion-dialysis method. Compared with small unilamellar vesicles (SUVs), GMVs show slightly better ability to retain encapsulated RNA, while maintaining good permeability for small charged molecules. The multilamellar structure also promotes non-enzymatic RNA copying, providing preliminary evidence that membranes could also mediate catalytic functions as well as acting as a compartment. / 2020-07-02T00:00:00Z

Cells use lipid membranes to organize and define their chemical environments. All cell membranes are based on a common structure: bilayers composed of phospholipids with two hydrocarbon chains. How did biology converge on this particular solution for cellular encapsulation? The first cell membranes are proposed to have assembled from simple, single-chain lipids, such as fatty acids and their derivatives, which would have been available in the prebiotic environment. Here we argue that the physical properties of fatty acid membranes would have made them well suited for a role as primitive cell membranes and predisposed their evolution to modern, phospholipid-based membranes. We first considered models for primitive membrane self-assembly, which faces significant concentration barriers due to the entropic cost of aggregation and the solubility of single-chain lipids. We therefore identified two physical mechanisms by which fatty acid membrane assembly can proceed from dilute solutions. Thermal diffusion columns, a proposed prebiotic concentration method, drive the formation of fatty acid vesicles by concentrating an initially isotropic solution past the critical concentration necessary for aggregation. Alternatively, mixtures of fatty acids with varying chain lengths, the expected products of abiotic lipid synthesis, intrinsically reduce the concentration barrier to aggregation through their polydispersity. These results motivated us to better understand the phase behavior of fatty acids in solutions. We found that the composition of fatty acid aggregates, whether vesicles or micelles, is also determined by concentration. Fatty acid vesicles feature significant amounts of coexisting micelles, whose abundance is enriched in low concentration solutions. We utilized this micelle-vesicle equilibrium to drive the growth of pre-existing fatty acid vesicles by changing amphiphile concentration. We next considered the evolution of phospholipid membranes, which was a critical and necessary step for the early evolution of cells. We found that the incorporation of even small amounts of phospholipids drives the growth of fatty acid vesicles by competition for monomers with neighboring vesicles lacking phospholipids. This competitive growth would have provided a strong selective advantage for primitive cells to evolve the catalytic machinery needed to synthesize phospholipids from their single-chain precursors. Growth is caused by any relative difference in phospholipid content, suggesting an evolutionary arms race among primitive cells for increasingly phospholipid membranes. What would have been the consequences for early cells of such a transition in membrane composition? We found that increasing phospholipid content inhibits the permeability of fatty acid membranes through changes in bilayer fluidity. For early heterotrophic cells, the emergence of increasingly phospholipid membranes would have therefore imposed new selective pressures for the evolution of membrane transport machinery and metabolism. Our model for early membrane evolution led us to develop prebiotic models for phospholipid chemistry. The assembly of phospholipids from single-chain substrates requires a single reaction: the acyltransfer of an activated fatty acid onto a glycerol monoester or lysophospholipid. We developed a synthetic model for this reaction that incorporates a copper-catalyzed azide-alkyne cycloaddition and showed that it drives de novo vesicle assembly.

All known living cells contain a complex set of molecular machinery to support their growth and replication. However, the earliest cells must have been much simpler, consisting of a compartment and a genetic material to allow for Darwinian evolution. To study these intermediates, plausible model `protocells' must be synthesized in the laboratory since no fossils remain. Recent work has shown that fatty acids can self-assemble into vesicles that are able to grow and divide through simple mechanisms. However, a self-replicating protocell genome has not yet been developed. Here we discuss studies of systems that allow for the copying of nucleic acid templates without enzymes and how they could be developed into a genetic material.

Le monoxyde d’azote (NO), un neurotransmetteur important en biologie, a attiré l’attention ces dernières années pour son rôle majeur joué dans l’apparition d’une myriade de maladies telles que certains cancers, diabètes etc. Comprendre les mécanismes biologiques liés à la production du NO pourrait aboutir à la découverte de nouveaux moyens thérapeutiques. Cependant, le fonctionnement de l’enzyme qui produit le monoxyde d’azote, la NO-Synthase, n’est pas complétement élucidé. Dans ce but, des approches biomimétiques peuvent apporter une solution. Les microréacteurs ou proto-cellules, enveloppes imitant sommairement la compartimentation cellulaire sont un outil de choix, permettant de répliquer un environnement contrôlé où les concentrations et distances de réactions sont proches d’une cellule, permettant ainsi d’étudier le fonctionnement de la NO-Synthase. Cette thèse présente trois problématiques qui ont pour but de développer un tel microréacteur encapsulant la NO-Synthase : (1) la libération contrôlé d’espèces réactives déclenchée par un stimulus lumineux, (2) le suivi de l’activité de l’enzyme par des sondes fluorescentes et (3) le contrôle de la réaction enzymatique dans l’espace et dans le temps. Deux systèmes ont été étudiés pour libérer de manière contrôlée des espèces: la première consiste à déstabiliser des nano polymersomes par photo-clivage du copolymère qui le constitue. Le deuxième système est basé sur une rapide augmentation de la pression osmotique par irradiation à l’intérieur des polymersomes, induisant un éclatement de ceux-ci et la libération d’espèces encapsulées. La deuxième problématique abordée est le suivi de l’activité enzymatique au moyen de sondes hydrophobes et hydrophiles fluorescentes qui détectent le monoxyde d’azote à différent endroits du microréacteur. Le dernier point abordé est l’étude des microréacteur et la libération contrôlé en leur sein. / Nitric oxide (NO) has been identified as an important chemical messenger in cells and living organisms. Understanding the mechanism involved in NO production by NO-synthase is of fundamental importance. Mimicking basic cell functions by encapsulating NO-synthase in a controlled and confined cell like environment, could help provide information about the enzyme. Polymersomes resulting from the self-assembly of amphiphilic block copolymers were used as the synthetic cell like microreactor. To this end, three major challenges were addressed in this thesis: (1) controlling species release and concentration inside the microreactor, (2) measuring the enzyme response by NO detection and (3) controlling enzymatic reactions in space and time inside a microreactor. Light was used as the exogenous stimulus to induce release; its application is instantaneous, non-invasive and easy to control spatially and temporally. Two different ways to release species via light excitation were explored. The first strategy involves destabilization of nanopolymersomes by block separation, induced by copolymer photocleavage. The second strategy was to induce fast osmotic pressure increase of the polymersomes internal medium, resulting in bursting and species release. In order to monitor NO production by NO-synthase in different parts of the microreactor, hydrophobic and hydrophilic fluorescent NO probes have been synthesized and studied showing excellent correlation with NO concentration. The release of species inside microreactor was finally achieved in order to control enzymatic reaction.

A synthetic proto-organism could be self-assembled by integrating a lipid proto-container with a proto-metabolic subsystem and a proto-genetic subsystem. This three-component system can use energy and nutrients by means of either redox or photo-chemical reactions, evolve its proto-genome by means of template directed replication, and ultimately die. The evolutionary dynamics of the proto-organism depends crucially on the chemical kinetics of its sub-systems and on their interplay. In this work the template replication kinetics is investigated and it is found that the product inhibition inherent in the ligation-like replication process allows for coexistence of unrelated self-replicating proto-genes in the lipid surface layer. The combined catalytic effects from the proto-genes on the metabolic production rates determine the fate of the strain protocell.

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. The discovery and characterization of an engineered nanopore with diode-like properties is enabling the construction of DIB networks capable of biochemical computing. Moreover, DIB networks might be used as model systems for the study of membrane-based biological phenomena. We develop and experimentally validate an electrical modeling approach for DIB networks. Electrical circuit simulations will be important in guiding the development of increasingly complex DIB networks. In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. Towards this end, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both α-helical and β-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores. Even with the small volumes (~100 nL) that can be used to form DIBs, the separation between two adjacent bilayers in a DIB network is typically still hundreds of microns. In contrast, dual-membrane spanning proteins require the bilayer separation to be much smaller; for example, the bilayer separation for gap junctions must be less than 5 nm. We designed a double bilayer system that consists of two monolayer-coated aqueous spheres brought into contact with each side of a water film submerged in an oil-lipid solution. The spheres could be brought close enough together such that they physically deflected without rupturing the double bilayer. Future work on quantifying the bilayer separation and studying dual-membrane spanning proteins with the double bilayer platform is planned.