Pierre-Alain Monnard

Membrane self-assembly processes: steps toward the first cellular life.

This review addresses the question of the origin of life, with emphasis on plausible boundary structures that may have initially provided cellular compartmentation. Some form of compartmentation is a necessary prerequisite for maintaining the integrity of interdependent molecular systems that are associated with metabolism, and for permitting variations required for speciation. The fact that lipid‐bilayer membranes define boundaries of all contemporary living cells suggests that protocellular compartments were likely to have required similar, self‐assembled boundaries. Amphiphiles such as short‐chain fatty acids, which were presumably available on the early Earth, can self‐assemble into stable vesicles that encapsulate hydrophilic solutes with catalytic activity. Their suspensions in aqueous media have therefore been used to investigate nutrient uptake across simple membranes and encapsulated catalyzed reactions, both of which would be essential processes in protocellular life forms. Anat Rec 268:196–207, 2002.

Influence of Ionic Inorganic Solutes on Self-Assembly and Polymerization Processes Related to Early Forms of Life: Implications for a Prebiotic Aqueous Medium

A commonly accepted view is that life began in a marine environment, which would imply the presence of inorganic ions such as Na+, Cl-, Mg2+, Ca2+, and Fe2+. We have investigated two processes relevant to the origin of life--membrane self-assembly and RNA polymerization--and established that both are adversely affected by ionic solute concentrations much lower than those of contemporary oceans. In particular, monocarboxylic acid vesicles, which are plausible models of primitive membrane systems, are completely disrupted by low concentrations of divalent cations, such as magnesium and calcium, and by high sodium chloride concentrations as well. Similarly, a nonenzymatic, nontemplated polymerization of activated RNA monomers in ice/eutectic phases (in a solution of low initial ionic strength) yields oligomers with > 80% of the original monomers incorporated, but polymerization in initially higher ionic strength aqueous solutions is markedly inhibited. These observations suggest that cellular life may not have begun in a marine environment because the abundance of ionic inorganic solutes would have significantly inhibited the chemical and physical processes that lead to self-assembly of more complex molecular systems.

Entrapment of nucleic acids in liposomes

The entrapment efficiency of three main methods used in the literature for the encapsulation of nucleic acids in liposomes were studied using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. In particular the reverse phase method, the dehydration/rehydration method, and the freeze/thawing method were compared to each other under standardised conditions, i.e. using in every case the same concentration of guest molecules (DNA, tRNA and ATP as low molecular weight analogue) and equally extruded liposomes. The percentage of entrapment strictly referred to the material localized inside the liposomes, i.e. particular care was devoted to ruling out the contribution of the nucleic acid material bound to the outer surface of the liposomes: this was eliminated by extensive enzymatic digestion prior to column chromatography. Depending on the conditions used, the percentage of the entrapped material varied between 10 and 54% of the initial amount. Further, the encapsulation efficiency was markedly affected by the salt concentration, by the size of liposomes, but to a lower degree by the molecular weight of the guest molecules. In general, we observed that the freeze/thawing encapsulation procedure was the most efficient one. In a second part of the work the freeze/thawing method was applied to encapsulate DNA (369 bp and 3368 bp, respectively) using liposomes obtained from POPC mixed with 1-10% charged cosurfactant, i.e. phosphatidylserine (PS) or didodecyldimethylammonium bromide (DDAB), respectively. Whereas PS had no significant effect, the entrapment efficiency went up to 60% in POPC/DDAB (97.5:2.5) liposomes. The large entrapment efficiency of DNA permits spectroscopic investigations of the DNA encapsulated in the water pool of the liposomes. UV absorption and circular dichroism spectra were practically the same as in water, indicating no appreciable perturbation of the electronic transitions or of the conformation of the entrapped biopolymer. This was in contrast to the DNA bound externally to the POPC/DDAB liposomes which showed significant spectral changes with respect to DNA dissolved in water.

Models of primitive cellular life: polymerases and templates in liposomes

Nutrient transport, polymerization and expression of genetic information in cellular compartments are hallmarks of all life today, and must have appeared at some point during the origin and early evolution of life. Because the first cellular life lacked membrane transport systems based on highly evolved proteins, they presumably depended on simpler processes of nutrient uptake. Using a system consisting of an RNA polymerase and DNA template entrapped in submicrometre-sized lipid vesicles (liposomes), we found that the liposome membrane could be made sufficiently permeable to allow access of ionized substrate molecules as large as nucleoside triphosphates (NTPs) to the enzyme. The encapsulated polymerase transcribed the template-specific base sequences of the DNA to the RNA that was synthesized. These experiments demonstrate that units of genetic information can be associated with a functional catalyst in a single compartment, and that transcription of gene-sized DNA fragments can be achieved by relying solely on passive diffusion to supply NTPs substrates.

Enzymatic reactions in liposomes using the detergent-induced liposome loading method

Microcompartmentalization is a crucial step in the origin of life. More than 30 years ago, Oparin et al. proposed models based on biochemical reactions taking place in so-called coacervates. Their intention was to develop systems with which semipermeable microcompartments could be established. In the present work we follow their intuition, but we use well-characterized bilayer structures instead of the poorly characterized coacervates. Liposomes from phospholipids can be used as microreactors but they exhibit only a modest permeability and, therefore, chemical reactions occurring inside these structures are depleted after a relatively short period. Here it is shown that even highly stable liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be used as semipermeable microreactors when treated with sodium cholate. Using this kind of mixed liposomes, we describe a biochemical reaction occurring inside the liposomes while the same reaction is prevented in the external medium. In addition, we show that this cholate-induced permeability of POPC bilayers can even be used to load macromolecules such as enzymes from the outside.

Prebiotically relevant mixed fatty acid vesicles support anionic solute encapsulation and photochemically catalyzed trans-membrane charge transport

The spontaneous assembly of amphiphile-based compartments in aqueous solution is widely viewed as a key step in models for the abiotic formation of primitive cell-like structures. Proposed organic components for such systems consist of mixed short chain fatty acids (FA) and polycyclic aromatic hydrocarbon (PAH) species, the composition of which have been modeled after organic extracts of carbonaceous meteorites. Self-assembly of amphiphiles from these extracts into aqueous suspensions of bilayer structures was long ago demonstrated, although little has since been reported concerning the stability and potential functionality of these complex mixtures. This work explores the thermodynamic and kinetic stability of vesicles prepared from complex mixtures of short chain FA species (CH3COOH–C9H19COOH) with membrane solubilized PAH species. Critical vesicle concentration measurements and ultrafiltration analyses of decanoic acid in the presence of other shorter chain FA species indicate the formation of mixed component vesicle phases composed mainly of C10–C8 FA components. An electrostatic barrier to trans-membrane diffusion of negative charges allows observation of stably encapsulated poly-anionic solutes inside these vesicles. As a model for primitive energy transduction, trans-membrane electron transfer between EDTA and encapsulated ferricyanide was demonstrated, driven catalytically via PAH photochemistry without substantial decomposition of the chromophores or vesicles. These results indicate a plausible role for compartmentalization and catalysis by short chain fatty acids and PAH species in prebiotic vesicle-encapsulated systems.

Preparation of Vesicles from Nonphospholipid Amphiphiles

Publisher Summary This chapter describes the properties and preparation of lipid vesicles from the simplest kinds of amphiphilic compounds. It addresses issues of stability, permeability, and encapsulation of macromolecules and shows how such vesicles can be used to encapsulate functional enzymes. The chapter also describes how these vesicles are related to naturally occurring substances, particularly the vesicular membranes that are produced from ancient organic compounds present in carbonaceous meteorites. Self-reproduction is an essential property of living systems. When a primitive cell was able to replicate its encapsulated macromolecular components, an increase of its membrane area was necessary to accommodate the internal growth. To model this evolutionary step, fatty acid vesicle systems have been designed successfully, using anhydride derivatives as a source of additional membrane amphiphiles. The prebiotic availability of fatty acids or their precursors and their properties of encapsulation, high permeability, and membrane growth make them ideal model systems for investigating primitive forms of life.

Formation of RNA Phosphodiester Bond by Histidine-Containing Dipeptides

A new scenario for prebiotic formation of nucleic acid oligomers is presented. Peptide catalysis is applied to achieve condensation of activated RNA monomers into short RNA chains. As catalysts, L‐dipeptides containing a histidine residue, primarily Ser‐His, were used. Reactions were carried out in selforganised environment, a water‐ice eutectic phase, with low concentrations of reactants. Incubation periods up to 30 days resulted in the formation of short oligomers of RNA. During the oligomerisation, an active intermediate (dipeptide–mononucleotide) is produced, which is the reactive species. Details of the mechanism and kinetics, which were elucidated with a set of control experiments, further establish that the imidazole side chain of a histidine at the carboxyl end of the dipeptide plays a crucial role in the catalysis. These results suggest that this oligomerisation catalysis occurs by a transamination mechanism. Because peptides are much more likely products of spontaneous condensation than nucleotide chains, their potential as catalysts for the formation of RNA is interesting from the origin‐of‐life perspective. Finally, the formation of the dipeptide–mononucleotide intermediate and its significance for catalysis might also be viewed as the tell‐tale signs of a new example of organocatalysis.

Eutectic phase in water-ice: a self-assembled environment conducive to metal-catalyzed non-enzymatic RNA polymerization.

Information and catalytic polymers play an essential role in contemporary cellular life, and their emergence must have been crucial during the complex processes that led to the assembly of the first living systems. Polymerization reactions producing these molecules would have had to occur in aqueous medium, which is known to disfavor such reactions. Thus, it was proposed early on that these polymerizations had to be supported by particular environments, such as mineral surfaces and eutectic phases in water‐ice, which would have led to the concentration of the monomers out of the bulk aqueous medium and their condensation. This review presents the work conducted to understand how the eutectic phases in water‐ice might have promoted RNA polymerization, thereby presumably contributing to the emergence of the ancient information and catalytic system envisioned by the ‘RNA‐World’ hypothesis.

Interactions between Catalysts and Amphiphilic Structures and their Implications for a Protocell Model

One of the essential elements of any cell, including primitive ancestors, is a structural component that protects and confines the metabolism and genes while allowing access to essential nutrients. For the targeted protocell model, bilayers of decanoic acid, a single-chain fatty acid amphiphile, are used as the container. These bilayers interact with a ruthenium-nucleobase complex, the metabolic complex, to convert amphiphile precursors into more amphiphiles. These interactions are dependent on non-covalent bonding. The initial rate of conversion of an oily precursor molecule into fatty acid was examined as a function of these interactions. It is shown that the precursor molecule associates strongly with decanoic acid structures. This results in a high dependence of conversion rates on the interaction of the catalyst with the self-assembled structures. The observed rate logically increases when a tight interaction between catalyst complex and container exists. A strong association between the metabolic complex and the container was achieved by bonding a sufficiently long hydrocarbon tail to the complex. Surprisingly, the rate enhancement was nearly as strong when the ruthenium and nucleobase elements of the complex were each given their own hydrocarbon tail and existed as separate molecules, as when the two elements were covalently bonded to each other and the resulting molecule was given a hydrocarbon tail. These results provide insights into the possibilities and constraints of such a reaction system in relation to building the ultimate protocell.