Visualizing ribonuclease digestion of RNA-like polymers produced by hot wet-dry cycles.

Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.

Computational Models of Polymer Synthesis Driven by Dehydration/Rehydration Cycles: Repurination in Simulated Hydrothermal Fields.

Cycles of biologically relevant reactions are an alternative to an origin of life emerging from a steady state away from equilibrium. The cycles involve a rate at which polymers are synthesized and accumulate in microscopic compartments called protocells, and two rates in which monomers and polymers are chemically degraded by hydrolytic reactions. Recent experiments have demonstrated that polymers are synthesized from mononucleotides and accumulate during cycles of hydration and dehydration, which means that the rate of polymer synthesis during the dehydrated phase of the cycle is balanced (but not dominated) by the rate of polymer hydrolysis during the hydrated phase of the cycle. Furthermore, depurination must be balanced by the reverse process of repurination. Here we describe a computational model that was inspired by experimental results, can be generalized to accommodate other reaction parameters, and has qualitative predictive power.

Conjecture and hypothesis: The importance of reality checks.

In origins of life research, it is important to understand the difference between conjecture and hypothesis. This commentary explores the difference and recommends alternative hypotheses as a way to advance our understanding of how life can begin on the Earth and other habitable planets. As an example of how this approach can be used, two conditions have been proposed for sites conducive to the origin of life: hydrothermal vents in salty seawater, and fresh water hydrothermal fields associated with volcanic landmasses. These are considered as alternative hypotheses and the accumulating weight of evidence for each site is described and analyzed.

Membranes and the Origin of Life: A Century of Conjecture.

Cells are the units of all life today, and are defined by their membranous boundaries. The membranes have multiple functions; the most obvious being that, in the absence of a boundary, the systems of functional macromolecular components of the cytosol would spill into the environment and disperse. Membranes also contain the pigments essential for photosynthesis, electron transport enzymes that pump and maintain proton gradients, the ATP synthase that uses proton gradients to produce energy for the cell, and enzymes that use ATP to maintain ion gradients essential for life. But what about the function of membranes in the first forms of cellular life? Could life have begun in the absence of membranous boundaries? In order to answer that question, this review presents a history of the key research observations that began over a century ago.

Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile, providing a viable mechanism for the emergence of protocells.

Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells.

Salt-promoted synthesis of RNA-like molecules in simulated hydrothermal conditions.

A fundamental problem in origins of life research is how the first polymers with the properties of nucleic acids were synthesized and incorporated into living systems on the prebiotic Earth. Here, we show that RNA-like polymers can be synthesized non-enzymatically from 5'-phosphate mononucleosides in salty environments. The polymers were identified and analyzed by gel electrophoresis, nanopore analysis, UV spectra, and action of RNases. The synthesis of phosphodiester bonds is driven by the chemical potential made available in the fluctuating hydrated and anhydrous conditions of hydrothermal fields associated with volcanic land masses.

Correlated electrical and optical analysis of single nanoparticles and biomolecules on a nanopore-gated optofluidic chip.

The analysis of individual biological nanoparticles has significantly advanced our understanding of fundamental biological processes but is also rapidly becoming relevant for molecular diagnostic applications in the emerging field of personalized medicine. Both optical and electrical methods for the detection and analysis of single biomolecules have been developed, but they are generally not used in concert and in suitably integrated form to allow for multimodal analysis with high throughput. Here we report on a dual-mode electrical and optical single-nanoparticle sensing device with capabilities that would not be available with each technique individually. The new method is based on an optofluidic chip with an integrated nanopore that serves as a smart gate to control the delivery of individual nanoparticles to an optical excitation region for ensemble-free optical analysis in rapid succession. We demonstrate electro-optofluidic size discrimination of fluorescent nanobeads, electro-optical detection of single fluorescently labeled influenza viruses, and the identification of single viruses within a mixture of equally sized fluorescent nanoparticles with up to 100% fidelity.

Generation of oligonucleotides under hydrothermal conditions by non-enzymatic polymerization.

We previously reported that 5'-mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of hydration-dehydration (HD) cycles. However, hydrolysis of oligomers can occur during hydration, and it is important to better understand the steady state in which ester bond synthesis is balanced by hydrolysis. In order to study condensation products of mononucleotides and hydrolysis of their polymers, we established a simulation of HD cycles that would occur on the early Earth when volcanic land masses emerged from the ocean over 4 billion years ago. At this stage on early Earth, precipitation produced hydrothermal fields characterized by small aqueous pools undergoing evaporation and refilling at elevated temperatures. Here, we confirm that under these conditions, the chemical potential made available by cycles of hydration and dehydration is sufficient to drive synthesis of ester bonds. If 5'-mononucleotides are in solution at millimolar concentrations, then oligomers resembling RNA are synthesized and exist in a steady state with their monomers. Furthermore, if the mononucleotides can form complementary base pairs, then some of the products have properties suggesting that secondary structures are present, including duplex species stabilized by hydrogen bonds.

Modelling energy-harvesting processes in primitive cells: Proton transport across bilayers driven by the oxidation of sulfite.

A frequently debated topic related to the origin of life centers around the question of how complex forms of life on today's Earth may have evolved over time from simpler predecessors. For example, the question of how proton concentration gradients across cellular membranes developed in ancestral protocells remains unanswered. This process, which is indispensable for the generation of chemical energy in modern organisms, is driven by energy derived from redox processes in the respiratory chain. Since it is highly unlikely that the complex machinery of the respiratory chain was available on early Earth, we provide an example of how proton gradients can be established in less complex systems. Utilizing liposomes as models of primitive cells, we were able to generate proton gradients of about two pH units across the liposome bilayers using redox reactions as the driving force. Electrons were transferred from sodium sulfite present on the outside of the liposomes to ferricyanide, which was trapped on the inside. A lipid-soluble phenazine derivative served as a shuttle that transferred both electrons and protons across the lipid bilayer. Because sulfite would have been an abundant reduced solute available to the earliest cells, we propose that it may have been a primary source of redox energy for primitive chemiosmotic energy transduction.

Could Life Have Started on Mars? Planetary Conditions That Assemble and Destroy Protocells.

Early Mars was likely habitable, but could life actually have started there? While cellular life emerged from prebiotic chemistry through a pre-Darwinian selection process relevant to both Earth and Mars, each planet posed unique selection 'hurdles' to this process. We focus on drivers of selection in prebiotic chemistry generic to Earth-like worlds and specific to Mars, such as an iron-rich surface. Iron, calcium, and magnesium cations are abundant in hydrothermal settings on Earth and Mars, a promising environment for an origin of life. We investigated the impact of cations on the stability and disruption of different primitive cell membranes under different pH conditions. The relative destabilizing effect of cations on membranes observed in this study is Ca2+ > Fe2+ > Mg2+. Cation concentrations in Earth systems today are too low to disrupt primitive membranes, but on Mars concentrations could have been elevated enough to disrupt membranes during surface dehydration. Membranes and RNA interact during dehydration-rehydration cycles to mutually stabilize each other in cation-rich solutions, and optimal membrane composition can be 'selected' by environmental factors such as pH and cation concentrations. We introduce an approach that considers how life may have evolved differently under the Martian planetary conditions and selective pressures.

Liquid crystalline nanostructures: organizing matrices for non-enzymatic nucleic acid polymerization.

When a mixture of amphiphilic compounds and solute molecules undergoes dehydration, a liquid crystalline multilamellar matrix is produced that organizes and concentrates solute molecules between lamellae. If the solutes are nucleotides, RNA-like molecules can be synthesized by a condensation reaction when the system is exposed to one or more cycles of dehydration at elevated temperatures, followed by rehydration. The chemical potential driving the reaction is supplied by the anhydrous conditions in which water becomes a leaving group, with heat providing activation energy. Liquid crystalline matrices composed of amphiphilic compounds could have promoted polymerization reactions in prebiotic conditions, giving rise to the molecular complexity required for the origin of cellular life.

Template-Directed Replication and Chiral Resolution during Wet-Dry Cycling in Hydrothermal Pools.

The commonly supposed template-based format for RNA self-replication requires both duplex assembly and disassembly. This requisite binary provision presents a challenge to the development of a serviceable self-replication model since chemical reactions are thermochemically unidirectional. We submit that a solution to this problem lies in volcanic landmasses that engage in continuous cycles of wetting and drying and thus uniquely provide the twofold state required for self-replication. Moreover, they offer conditions that initiate chain branching, and thus furnish a path to autocatalytic self-replication. The foundations of this dual thermochemical landscape arise from the broad differences in the properties of the bulk water phase on the one hand, and the air/water interfacial regions that emerge in the evaporative stages on the other. With this reaction system as a basis and employing recognized thermochemical and kinetic parameters, we present simulations displaying the spontaneous and autocatalyzed conversion of racemic and unactivated RNA monomers to necessarily homochiral duplex structures over characteristic periods of years.

Polycyclic aromatic hydrocarbons as plausible prebiotic membrane components.

Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C(6)-C(10) fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.

Origins of Life Research: The Conundrum between Laboratory and Field Simulations of Messy Environments.

Most experimental results that guide research related to the origin of life are from laboratory simulations of the early Earth conditions. In the laboratory, emphasis is placed on the purity of reagents and carefully controlled conditions, so there is a natural tendency to reject impurities and lack of control. However, life did not originate in laboratory conditions; therefore, we should take into consideration multiple factors that are likely to have contributed to the environmental complexity of the early Earth. This essay describes eight physical and biophysical factors that spontaneously resolve aqueous dispersions of ionic and organic solutes mixed with mineral particles and thereby promote specific chemical reactions required for life to begin.

Visualizing RNA polymers produced by hot wet-dry cycling.

It is possible that the transition from abiotic systems to life relied on RNA polymers that served as ribozyme-like catalysts and for storing genetic information. The source of such polymers is uncertain, but previous investigations reported that wet-dry cycles simulating prebiotic hot springs provide sufficient energy to drive condensation reactions of mononucleotides to form oligomers and polymers. The aim of the study reported here was to verify this claim and visualize the products prepared from solutions composed of single mononucleotides and 1:1 mixture of two mononucleotides. Therefore, we designed experiments that allowed comparisons of all such mixtures representing six combinations of the four mononucleotides of RNA. We observed irregular stringy patches and crystal strands when wet-dry cycling was performed at room temperature (20 °C). However, when the same solutions were exposed to wet-dry cycles at 80 °C, we observed what appeared to be true polymers. Their thickness was consistent with RNA-like products composed of covalently bonded monomers, while irregular strings and crystal segments of mononucleotides dried or cycled at room temperature were consistent with structures assembled and stabilized by weak hydrogen bonds. In a few instances we observed rings with short polymer attachments. These observations are consistent with previous claims of polymerization during wet-dry cycling. We conclude that RNA-like polymers and rings could have been synthesized non-enzymatically in freshwater hot springs on the prebiotic Earth with sizes sufficient to fold into ribozymes and genetic molecules required for life to begin.

Duplex Formation and the Origins of Homochirality.

The roots of biological homochirality remain unknown despite decades of study. A commonly proposed path includes an initial small enantiomeric excess that was amplified over time, but a satisfactory source of the excess and a plausible amplification process have yet to be described. We propose here a route to oligonucleotide homochirality from unactivated racemic sources based upon the facts that duplex structures are inherently homochiral and their synthesis from strands of complementary string nucleotide subunits is both uncommonly rapid and exergonic. Simulations employing available kinetic and thermochemical data in an iterated sequence of three equilibria in dry/wet cycles running from unactivated and racemic RNA monomers through oligonucleotides to duplex structures have shown that the exceptional association rate distorts the otherwise simple equilibrium string and overcomes the severe kinetic and stoichiometric barriers to the pairing of the statistically scant homochiral fractions. The simulations reveal widespread deracemization and the full conversion of racemic monomers to populations of L- and D-duplexes in a succession of growth in which the initially formed duplexes are replaced over time with increasingly larger descendants. This claim is open to experimental testing.

Urability: A Property of Planetary Bodies That Can Support an Origin of Life.

The concept of habitability is now widely used to describe zones in a solar system in which planets with liquid water can sustain life. Because habitability does not explicitly incorporate the origin of life, this article proposes a new word-urability-which refers to the conditions that allow life to begin. The utility of the word is tested by applying it to combinations of multiple geophysical and geochemical factors that support plausible localized zones that are conducive to the chemical reactions and molecular assembly processes required for the origin of life. The concept of urable worlds, planetary bodies that can sustain an arising of life, is considered for bodies in our own solar system and exoplanets beyond.