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The emergence of biopolymer building blocks is a crucial step during the origins of life1–6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.Heat flows through thin, crack-like geo-compartments are shown to purify previously mixed compounds and enhance their reactivity, providing a selective mechanism for separating molecules relevant to the chemical origins of life. [ABSTRACT FROM AUTHOR]

Mechanisms of nucleic acid accumulation were likely critical to life's emergence in the ferruginous oceans of the early Earth. How exactly prebiotic geological settings accumulated nucleic acids from dilute aqueous solutions, is poorly understood. As a possible solution to this concentration problem, we simulated the conditions of prebiotic low‐temperature alkaline hydrothermal vents in co‐precipitation experiments to investigate the potential of ferruginous chemical gardens to accumulate nucleic acids via sorption. The injection of an alkaline solution into an artificial ferruginous solution under anoxic conditions (O2 < 0.01% of present atmospheric levels) and at ambient temperatures, caused the precipitation of amakinite ("white rust"), which quickly converted to chloride‐containing fougerite ("green rust"). RNA was only extractable from the ferruginous solution in the presence of a phosphate buffer, suggesting RNA in solution was bound to Fe2+ ions. During chimney formation, this iron‐bound RNA rapidly accumulated in the white and green rust chimney structure from the surrounding ferruginous solution at the fastest rates in the initial white rust phase and correspondingly slower rates in the following green rust phase. This represents a new mechanism for nucleic acid accumulation in the ferruginous oceans of the early Earth, in addition to wet‐dry cycles and may have helped to concentrate RNA in a dilute prebiotic ocean. [ABSTRACT FROM AUTHOR]

The emergence of functional oligonucleotides on early Earth required a molecular selection mechanism to screen for specific sequences with prebiotic functions. Cyclic processes such as daily temperature oscillations were ubiquitous in this environment and could trigger oligonucleotide phase separation. Here, we propose sequence selection based on phase separation cycles realized through sedimentation in a system subjected to the feeding of oligonucleotides. Using theory and experiments with DNA, we show sequence-specific enrichment in the sedimented dense phase, in particular of short 22-mer DNA sequences. The underlying mechanism selects for complementarity, as it enriches sequences that tightly interact in the dense phase through base-pairing. Our mechanism also enables initially weakly biased pools to enhance their sequence bias or to replace the previously most abundant sequences as the cycles progress. Our findings provide an example of a selection mechanism that may have eased screening for auto-catalytic self-replicating oligonucleotides. [ABSTRACT FROM AUTHOR]

Silicate glasses are ubiquitous on terrestrial planets wherever molten rock - generated by volcanism or impacts - is quenched by air or water. Hence, they provided ready and abundant reactive solid surfaces for prebiotic chemistry on the early Earth. Here, we show that rock glass composition determines basicity, which in turn modulates the outcome of prebiotic synthetic processes such as the polymerization of 3', 5' cyclic guanosine monophosphate. [ABSTRACT FROM AUTHOR]

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. Non-equilibrium conditions at heated water-air interfaces can model Hadean microenvironments. Here, the authors show that such conditions enable one-pot synthesis, strand release and folding of functional RNAs similar to modern biological systems. [ABSTRACT FROM AUTHOR]

Understanding the sequence-dependent DNA damage formation requires probing a complete pool of sequences over a wide dose range of the damage-causing exposure. We used high throughput sequencing to simultaneously obtain the dose dependence and quantum yields for oligonucleotide damages for all possible 4096 DNA sequences with hexamer length. We exposed the DNA to ultraviolet radiation at 266 nm and doses of up to 500 absorbed photons per base. At the dimer level, our results confirm existing literature values of photodamage, whereas we now quantified the susceptibility of sequence motifs to UV irradiation up to previously inaccessible polymer lengths. This revealed the protective effect of the sequence context in preventing the formation of UV-lesions. For example, the rate to form dipyrimidine lesions is strongly reduced by nearby guanine bases. Our results provide a complete picture of the sensitivity of oligonucleotides to UV irradiation and allow us to predict their abundance in high-UV environments. [ABSTRACT FROM AUTHOR]

Life is based on informational polymers such as DNA or RNA. For their polymerization, high concentrations of complex monomer building blocks are required. Therefore, the dilution by diffusion poses a major problem before early life could establish a non-equilibrium of compartmentalization. Here, we explored a natural non-equilibrium habitat to polymerize RNA and DNA. A heat flux across thin rock cracks is shown to accumulate and maintain nucleotides. This boosts the polymerization to RNA and DNA inside the crack. Moreover, the polymers remain localized, aiding both the creation of longer polymers and fostering downstream evolutionary steps. In a closed system, we found single nucleotides concentrate 104-fold at the bottom of the crack compared to the top after 24 hours. We detected enhanced polymerization for 2 different activation chemistries: aminoimidazole-activated DNA nucleotides and 2′,3′-cyclic RNA nucleotides. The copolymerization of 2′,3′-cGMP and 2′,3′-cCMP in the thermal pore showed an increased heterogeneity in sequence composition compared to isothermal drying. Finite element models unravelled the combined polymerization and accumulation kinetics and indicated that the escape of the nucleotides from such a crack is negligible over a time span of years. The thermal non-equilibrium habitat establishes a cell-like compartment that actively accumulates nucleotides for polymerization and traps the resulting oligomers. We argue that the setting creates a pre-cellular non-equilibrium steady state for the first steps of molecular evolution. [ABSTRACT FROM AUTHOR]

Spatial proton gradients create energy in biological systems and are likely a driving force for prebiotic systems. Due to the fast diffusion of protons, they are however difficult to create as steady state, unless driven by other non-equilibria such as thermal gradients. Here, we quantitatively predict the heat-flux driven formation of pH gradients for the case of a simple acid-base reaction system. To this end, we (i) establish a theoretical framework that describes the spatial interplay of chemical reactions with thermal convection, thermophoresis, and electrostatic forces by a separation of timescales, and (ii) report quantitative measurements in a purpose-built microfluidic device. We show experimentally that the slope of such pH gradients undergoes pronounced amplitude changes in a concentration-dependent manner and can even be inverted. The predictions of the theoretical framework fully reflect these features and establish an understanding of how naturally occurring non-equilibrium environmental conditions can drive pH gradients. Cells use pH gradients to drive the synthesis of adenosine triphosphate (ATP), but the physicochemical mechanisms that can produce pH gradients in non-equilibrium settings are poorly understood. The authors here theoretically and experimentally investigate the formation of a pH gradient in an acid-base reaction system, driven by a heat flow, providing insights on how crude non-equilibrium systems can feed chemical gradients exploitable by life. [ABSTRACT FROM AUTHOR]

For the emergence of life, the abiotic synthesis of RNA from its monomers is a central step. We found that in alkaline, drying conditions in bulk and at heated air‐water interfaces, 2′,3′‐cyclic nucleotides oligomerised without additional catalyst, forming up to 10‐mers within a day. The oligomerisation proceeded at a pH range of 7–12, at temperatures between 40–80 °C and was marginally enhanced by K+ ions. Among the canonical ribonucleotides, cGMP oligomerised most efficiently. Quantification was performed using HPLC coupled to ESI‐TOF by fitting the isotope distribution to the mass spectra. Our study suggests a oligomerisation mechanism where cGMP aids the incorporation of the relatively unreactive nucleotides C, A and U. The 2′,3′‐cyclic ribonucleotides are byproducts of prebiotic phosphorylation, nucleotide syntheses and RNA hydrolysis, indicating direct recycling pathways. The simple reaction condition offers a plausible entry point for RNA to the evolution of life on early Earth. [ABSTRACT FROM AUTHOR]

The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3',5'‐cyclic guanosine monophosphate (3',5'‐cGMP) has been proposed as a candidate to create oligonucleotides of 15–20 units in length. However, the reported sensitivity of the reaction to the presence of cations raised questions of whether this chemistry could be relevant in a geological context. The experiments in this study show that the presence of cations is not restrictive as long as the reaction is conducted in an acidic environment, in contrast to previous reports that suggested optimal conditions at pH 9. [ABSTRACT FROM AUTHOR]

Previous studies on the polymerization of 3',5' cyclic guanosine monophosphate (cGMP) demonstrated the potential of the compound in the abiotic generation of the first oligonucleotide sequences on the early Earth. These experiments were conducted under idealized laboratory conditions, which logically raises the question whether the same chemistry could take place in the harsh environment present on our planet in its earliest days. In the current study, we focus on the mineralogical context of this chemistry and show that numerous, but not all, common minerals assumed to be present on the early Earth could host the polymerization of H‐form 3',5' cGMP. In particular, we have found that quartz varieties are especially suitable for this purpose, similar to andalusite, amphibole or micas. On the contrary, olivine, calcite, and serpentine‐group minerals interfere with the studied polymerization chemistry. Our results show that crystallization on mineral surfaces, which is mainly a diffusion controlled process, determines the ability of 3',5' cGMP to polymerize. The observation that numerous amorphous and crystalline SiO2 forms are compatible with the oligomerization chemistry suggests that the process could commonly occur in a wide range of primordial environments allowing for crystallization of the cyclic monomers from a dropping solution. [ABSTRACT FROM AUTHOR]

Microbial fossils preserved by early diagenetic chert provide a window into the Proterozoic biosphere, but seawater chemistry, microbial processes, and the interactions between microbes and the environment that contributed to this preservation are not well constrained. Here, we use fossilization experiments to explore the processes that preserve marine cyanobacterial biofilms by the precipitation of amorphous silica in a seawater medium that is analogous to Proterozoic seawater. These experiments demonstrate that the exceptional silicification of benthic marine cyanobacteria analogous to the oldest diagnostic cyanobacterial fossils requires interactions among extracellular polymeric substances (EPS), photosynthetically induced pH changes, magnesium cations (Mg2+), and >70 ppm silica. [ABSTRACT FROM AUTHOR]

Microscale thermophoresis (MST) is a versatile technique to measure binding affinities of binder–ligand systems, based on the directional movement of molecules in a temperature gradient. We extended MST to measure binding kinetics as well as binding affinity in a single experiment by increasing the thermal dissipation of the sample. The kinetic relaxation fingerprints were derived from the fluorescence changes during thermodynamic re‐equilibration of the sample after local heating. Using this method, we measured DNA hybridization on‐rates and off‐rates in the range 104–106 m−1 s−1 and 10−4–10−1 s−1, respectively. We observed the expected exponential dependence of the DNA hybridization off‐rates on salt concentration, strand length and inverse temperature. The measured on‐rates showed a linear dependence on salt concentration and weak dependence on strand length and temperature. For biomolecular interactions with large enthalpic contributions, the kinetic MST technique offers a robust, cost‐effective and immobilization‐free determination of kinetic rates and binding affinity simultaneously, even in crowded solutions. [ABSTRACT FROM AUTHOR]

Microscale thermophoresis (MST) is a versatile technique to measure binding affinities of binder–ligand systems, based on the directional movement of molecules in a temperature gradient. We extended MST to measure binding kinetics as well as binding affinity in a single experiment by increasing the thermal dissipation of the sample. The kinetic relaxation fingerprints were derived from the fluorescence changes during thermodynamic re‐equilibration of the sample after local heating. Using this method, we measured DNA hybridization on‐rates and off‐rates in the range 104–106 m−1 s−1 and 10−4–10−1 s−1, respectively. We observed the expected exponential dependence of the DNA hybridization off‐rates on salt concentration, strand length and inverse temperature. The measured on‐rates showed a linear dependence on salt concentration and weak dependence on strand length and temperature. For biomolecular interactions with large enthalpic contributions, the kinetic MST technique offers a robust, cost‐effective and immobilization‐free determination of kinetic rates and binding affinity simultaneously, even in crowded solutions. [ABSTRACT FROM AUTHOR]

The central question in the origin of life is to understand how structure can emerge from randomness. The Eigen theory of replication states, for sequences that are copied one base at a time, that the replication fidelity has to surpass an error threshold to avoid that replicated specific sequences become random because of the incorporated replication errors [M. Eigen, Naturwissenschaften 58 (10), 465-523 (1971)]. Here,we showed that linking short oligomers from a random sequence pool in a templated ligation reaction reduced the sequence space of product strands. We started from 12-mer oligonucleotides with two bases in all possible combinations and triggered enzymatic ligation under temperature cycles. Surprisingly, we found the robust creation of long, highly structured sequences with low entropy. At the ligation site, complementary and alternating sequence patterns developed. However, between the ligation sites, we found either an A-rich or a T-rich sequence within a single oligonucleotide. Our modeling suggests that avoidance of hairpins was the likely cause for these two complementary sequence pools. What emerged was a network of complementary sequences that acted both as templates and substrates of the reaction. This self-selecting ligation reaction could be restarted by only a few majority sequences. The findings showed that replication by random templated ligation from a random sequence input will lead to a highly structured, long, and nonrandom sequence pool. This is a favorable starting point for a subsequent Darwinian evolution searching for higher catalytic functions in an RNA world scenario. [ABSTRACT FROM AUTHOR]

All life on Earth is built of organic molecules, so the primordial sources of reduced carbon remain a major open question in studies of the origin of life. A variant of the alkaline-hydrothermal-vent theory for life's emergence suggests that organics could have been produced by the reduction of CO2 via H2 oxidation, facilitated by geologically sustained pH gradients. The process would be an abiotic analog-and proposed evolutionary predecessor-of the Wood-Ljungdahl acetyl-CoA pathway of modern archaea and bacteria. The first energetic bottleneck of the pathway involves the endergonic reduction of CO2 with H2 to formate (HCOO-), which has proven elusive in mild abiotic settings. Here we show the reduction of CO2 with H2 at room temperature under moderate pressures (1.5 bar), driven by microfluidic pH gradients across inorganic Fe(Ni)S precipitates. Isotopic labeling with 13C confirmed formate production. Separately, deuterium (2H) labeling indicated that electron transfer to CO2 does not occur via direct hydrogenation with H2 but instead, freshly deposited Fe(Ni)S precipitates appear to facilitate electron transfer in an electrochemical-cell mechanism with two distinct half-reactions. Decreasing the pH gradient significantly, removing H2, or eliminating the precipitate yielded no detectable product. Our work demonstrates the feasibility of spatially separated yet electrically coupled geochemical reactions as drivers of otherwise endergonic processes. Beyond corroborating the ability of early-Earth alkaline hydrothermal systems to couple carbon reduction to hydrogen oxidation through biologically relevant mechanisms, these results may also be of significance for industrial and environmental applications, where other redox reactions could be facilitated using similarly mild approaches. [ABSTRACT FROM AUTHOR]

Unilamellar lipid vesicles can serve as model for protocells. We present a vesicle fission mechanism in a thermal gradient under flow in a convection chamber, where vesicles cycle cold and hot regions periodically. Crucial to obtain fission of the vesicles in this scenario is a temperature-induced membrane phase transition that vesicles experience multiple times. We model the temperature gradient of the chamber with a capillary to study single vesicles on their way through the temperature gradient in an external field of shear forces. Starting in the gel-like phase the spherical vesicles are heated above their main melting temperature resulting in a dumbbell-deformation. Further downstream a temperature drop below the transition temperature induces splitting of the vesicles without further physical or chemical intervention. This mechanism also holds for less cooperative systems, as shown here for a lipid alloy with a broad transition temperature width of 8 K. We find a critical tether length that can be understood from the transition width and the locally applied temperature gradient. This combination of a temperature-induced membrane phase transition and realistic flow scenarios as given e.g. in a white smoker enable a fission mechanism that can contribute to the understanding of more advanced protocell cycles. [ABSTRACT FROM AUTHOR]

We show how fluid can be moved by a laser scanning microscope. Selected parts of a fluid film are pumped along the path of a moving warm spot which is generated by the repetitive motion of an infrared laser focus. With this technique, we remotely drive arbitrary two-dimensional fluid flow patterns with a resolution of 2 μm. Pump speeds of 150 μm/s are reached in water with a maximal temperature increase in the local spot of 10 K. Various experiments confirm that the fluid motion results from the dynamic thermal expansion in a gradient of viscosity. The viscosity in the spot is reduced by its enhanced temperature. This leads to a broken symmetry between thermal expansion and thermal contraction in the front and the wake of the spot. As result the fluid moves opposite to the spot direction due to both the asymmetric thermal expansion in the spot front and the asymmetric thermal contraction in its wake. We derive an analytical expression for the fluid speed from the Navier–Stokes equations. Its predictions are experimentally confirmed without fitting parameters under a number of different conditions. In water, this nonlinearity leads to a fluid step of <100 nm for each passage of the spot. Since the spot movement can be repeated in the kilohertz regime, fluid speeds can exceed 100 μm/s. Using this technique, we pump nanoparticles over millimeters through a gel. An all-optical creation of a dilution series of DNA and biomolecules by aliquotation and mixing is demonstrated for fluids sandwiched between untreated and unstructured, disposable microscope cover slips. The shown optical remote control of fluid flow expands the microfluidic paradigm into previously inaccessible regimes of tiny volumes, closed flow paths, fast switching between flow patterns, and remote fluid control under extreme fluid conditions. [ABSTRACT FROM AUTHOR]

To understand the emergence of life, a better understanding of the physical chemistry of primordial non‐equilibrium conditions is essential. Significant salt concentrations are required for the catalytic function of RNA. The separation of oligonucleotides into single strands is a difficult problem as the hydrolysis of RNA becomes a limiting factor at high temperatures. Salt concentrations modulate the melting of DNA or RNA, and its periodic modulation would enable melting and annealing cycles at low temperatures. In our experiments, a moderate temperature difference created a miniaturized water cycle, resulting in fluctuations in salt concentration, leading to melting of oligonucleotides at temperatures 20 °C below the melting temperature. This would enable the reshuffling of duplex oligonucleotides, necessary for ligation chain replication. The findings suggest an autonomous route to overcome the strand‐separation problem of non‐enzymatic replication in early evolution. [ABSTRACT FROM AUTHOR]

To understand the emergence of life, a better understanding of the physical chemistry of primordial non‐equilibrium conditions is essential. Significant salt concentrations are required for the catalytic function of RNA. The separation of oligonucleotides into single strands is a difficult problem as the hydrolysis of RNA becomes a limiting factor at high temperatures. Salt concentrations modulate the melting of DNA or RNA, and its periodic modulation would enable melting and annealing cycles at low temperatures. In our experiments, a moderate temperature difference created a miniaturized water cycle, resulting in fluctuations in salt concentration, leading to melting of oligonucleotides at temperatures 20 °C below the melting temperature. This would enable the reshuffling of duplex oligonucleotides, necessary for ligation chain replication. The findings suggest an autonomous route to overcome the strand‐separation problem of non‐enzymatic replication in early evolution. [ABSTRACT FROM AUTHOR]

All life on earth depends on the generation and exploitation of ionic and pH gradients across membranes. One theory for the origin of life proposes that geological pH gradients were the prebiotic ancestors of these cellular disequilibria. With an alkaline interior and acidic exterior, alkaline vents match the topology of modern cells, but it remains unknown whether the steep pH gradients persist at the microscopic scale. Herein, we demonstrate the existence of 6 pH-unit gradients across micrometer scales in a microfluidic vent replicate. Precipitation of metal sulfides at the interface strengthens the gradients, but even in the absence of precipitates laminar flow sustains the disequilibria. The gradients drive directed transport at the fluid interface, leading to colloid accumulation or depletion. Our results confirm that alkaline vents can provide an exploitable pH gradient, supporting their potential role at the emergence of chemiosmosis and the origin of life. [ABSTRACT FROM AUTHOR]

All life on earth depends on the generation and exploitation of ionic and pH gradients across membranes. One theory for the origin of life proposes that geological pH gradients were the prebiotic ancestors of these cellular disequilibria. With an alkaline interior and acidic exterior, alkaline vents match the topology of modern cells, but it remains unknown whether the steep pH gradients persist at the microscopic scale. Herein, we demonstrate the existence of 6 pH-unit gradients across micrometer scales in a microfluidic vent replicate. Precipitation of metal sulfides at the interface strengthens the gradients, but even in the absence of precipitates laminar flow sustains the disequilibria. The gradients drive directed transport at the fluid interface, leading to colloid accumulation or depletion. Our results confirm that alkaline vents can provide an exploitable pH gradient, supporting their potential role at the emergence of chemiosmosis and the origin of life. [ABSTRACT FROM AUTHOR]

How life emerged is one of the major questions that remains to be answered. Apart from being of interest for completeness of biology, it is also a very interesting study case from the vantage point of physics. Living organisms are inherently non-equilibrium systems. Since non-equilibrium thermodynamics is still a developing field, the emergence of life is a highly interesting study case. Here we present the progress we have made during the last few years, employing experimental biophysics to capture the mechanisms that could eventually lead to the emergence of life. We show how a simple non-equilibrium system, a thermal gradient, gives rise to a range of relevant phenomena, in particular, polymerization, elongation, and replication of DNA molecules as well as demixing of mixed DNA sequences into sequence-pure hydrogels. 1 Introduction 2 Thermophoresis, the Biased Movement of Molecules in Thermal Gradients 3 The Dynamical Behavior of Accumulated Molecules 4 Overcoming Spiegelman’s Monster 5 Sequence Purification and Oscillations by a Gel-Phase Transition in Thermal Gradients 6 Loose Ends 7 Discussion and Conclusion [ABSTRACT FROM AUTHOR]

How can living matter arise from dead matter? All known living systems are built around information stored in RNA and DNA. To protect this information against molecular degradation and diffusion, the second law of thermodynamics imposes the need for a non-equilibrium driving force. Following a series of successful experiments using thermal gradients, we have shown that heat gradients across sub-millimetre pores can drive accumulation, replication, and selection of ever longer molecules, implementing all the necessary parts for Darwinian evolution. For these lab experiments to proceed with ample speed, however, the temperature gradients have to be quite steep, reaching up to 30 K per 100 μm. Here we use computer simulations based on experimental data to show that 2000-fold shallower temperature gradients – down to 100 K over one metre – can still drive the accumulation of protobiomolecules. This finding opens the door for various environments to potentially host the origins of life: volcanic, water-vapour, or hydrothermal settings. Following the trajectories of single molecules in simulation, we also find that they are subjected to frequent temperature oscillations inside these pores, facilitating e.g. template-directed replication mechanisms. The tilting of the pore configuration is the central strategy to achieve replication in a shallow temperature gradient. Our results suggest that shallow thermal gradients across porous rocks could have facilitated the formation of evolutionary machines, significantly increasing the number of potential sites for the origin of life on young rocky planets. [ABSTRACT FROM AUTHOR]

DNA phase transitions are often induced by the addition of condensation agents or by dry concentration. Herein, we show that the non-equilibrium setting of a moderate heat flow across a water-filled chamber separates and gelates DNA strands with single-base resolution. A dilute mix of DNA with two slightly different gel-forming sequences separates into sequence-pure hydrogels under constant physiological solvent conditions. A single base change in a 36 mer DNA inhibits gelation. Only sequences with the ability to form longer strands are concentrated, further elongated, and finally gelated by length-dependent thermal trapping. No condensation agents, such as multivalent ions, were added. Equilibrium aggregates from dry concentration did not show any sequence separation. RNA is expected to behave identically owing to its equal thermophoretic properties. The highly sequence-specific phase transition points towards new possibilities for non-equilibrium origins of life. [ABSTRACT FROM AUTHOR]

DNA phase transitions are often induced by the addition of condensation agents or by dry concentration. Herein, we show that the non-equilibrium setting of a moderate heat flow across a water-filled chamber separates and gelates DNA strands with single-base resolution. A dilute mix of DNA with two slightly different gel-forming sequences separates into sequence-pure hydrogels under constant physiological solvent conditions. A single base change in a 36 mer DNA inhibits gelation. Only sequences with the ability to form longer strands are concentrated, further elongated, and finally gelated by length-dependent thermal trapping. No condensation agents, such as multivalent ions, were added. Equilibrium aggregates from dry concentration did not show any sequence separation. RNA is expected to behave identically owing to its equal thermophoretic properties. The highly sequence-specific phase transition points towards new possibilities for non-equilibrium origins of life. [ABSTRACT FROM AUTHOR]

Single-phase-to-ground faults in power stations sometimes cause a second phase-to-ground fault in another phase. If the phase-to-ground faults are located on different sides of the generator circuit breaker (CB), one has a double earth fault of a special and so far not analyzed kind with respect to the CB. It may lead to high stresses for the last CB pole to clear. The general expressions for the current, the rate of rise of recovery voltage, and the power frequency recovery voltage across this CB pole have been deduced as function of the sequence values of the short-circuit impedances and surge impedances, respectively. The corresponding results are discussed for typical cases. A detailed case analysis by means of the Electromagnetic Transients Program shows that at rated load, such a double earth fault, produces CB stresses comparable to out-of-phase conditions. A combination of double earth fault and out-of-phase conditions, however, causes a peak value of the transient recovery voltage, which will significantly exceed the values laid down in the standards. [ABSTRACT FROM AUTHOR]

The replication of nucleic acids is central to the origin of life. On the early Earth, suitable non-equilibrium boundary conditions would have been required to surmount the effects of thermodynamic equilibrium such as the dilution and degradation of oligonucleotides. One particularly intractable experimental finding is that short genetic polymers replicate faster and outcompete longer ones, which leads to ever shorter sequences and the loss of genetic information. Here we show that a heat flux across an open pore in submerged rock concentrates replicating oligonucleotides from a constant feeding flow and selects for longer strands. Our experiments utilize the interplay of molecular thermophoresis and laminar convection, the latter driving strand separation and exponential replication. Strands of 75 nucleotides survive whereas strands half as long die out, which inverts the above dilemma of the survival of the shortest. The combined feeding, thermal cycling and positive length selection opens the door for a stable molecular evolution in the long-term microhabitat of heated porous rock. [ABSTRACT FROM AUTHOR]

A combination of thermophoresis and fluid flow can be used to trap molecules and particles. We show that heating by scanning motion of an elongated laser spot creates a strong thermal trap. Additionally, it induces a global fluid flow that feeds the trap. Such "thermal sieve" can accumulate molecules from a large surrounding region within seconds into a 10 lm spot. Numerical modeling gives a quantitative prediction of the effect. Traps can be dynamically created, relocated, and tuned, which can be used for particle sorting. [ABSTRACT FROM AUTHOR]

A combination of thermophoresis and fluid flow can be used to trap molecules and particles. We show that heating by scanning motion of an elongated laser spot creates a strong thermal trap. Additionally, it induces a global fluid flow that feeds the trap. Such "thermal sieve" can accumulate molecules from a large surrounding region within seconds into a 10 μm spot. Numerical modeling gives a quantitative prediction of the effect. Traps can be dynamically created, relocated, and tuned, which can be used for particle sorting. [ABSTRACT FROM AUTHOR]

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil-surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP-aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536-well plate format make the method high-throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label-free drug discovery screening. [ABSTRACT FROM AUTHOR]

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For the emergence of early life, the formation of biopolymers such as RNA is essential. However, the addition of nucleotide monomers to existing oligonucleotides requires millimolar concentrations. Even in such optimistic settings, no polymerization of RNA longer than about 20 bases could be demonstrated. How then could self-replicating ribozymes appear, for which recent experiments suggest a minimal length of 200 nt? Here, we demonstrate a mechanism to bridge this gap: the escalated polymerization of nucleotides by a spatially confined thermal gradient. The gradient accumulates monomers by thermophoresis and convection while retaining longer polymers exponentially better. Polymerization and accumulation become mutually self-enhancing and result in a hyperexponential escalation of polymer length. We describe this escalation theoretically under the conservative assumption of reversible polymerization. Taking into account the separately measured thermophoretic properties of RNA, we extrapolate the results for primordial RNA polymerization inside a temperature gradient in pores or fissures of rocks. With a dilute, nanomolar concentration of monomers the model predicts that a pore length of 5 cm and a temperature difference of 10 K suffice to polymerize 200-mers of RNA in micromolar concentrations. The probability to generate these long RNAs is raised by a factor of >10600 compared with polymerization in a physical equilibrium. We experimentally validate the theory with the reversible polymerization of DNA blocks in a laser-driven thermal trap. The results confirm that a thermal gradient can significantly enlarge the available sequence space for the emergence of catalytically active polymers. [ABSTRACT FROM AUTHOR]

The key requirement to solve the origin of life puzzle are disequilibrium conditions. Early molecular evolution cannot be explained by initial high concentrations of energetic chemicals since they would just react towards their chemical equilibrium allowing no further development. We argue here that persistent disequilibria are needed to increase complexity during molecular evolution. We propose thermal gradients as the disequilibrium setting which drove Darwinian molecular evolution. On the one hand the thermal gradient gives rise to laminar thermal convection flow with highly regular temperature oscillations that allow melting and replication of DNA. On the other hand molecules move along the thermal gradient, a mechanism termed Soret effect or thermophoresis. Inside a long chamber a combination of the convection flow and thermophoresis leads to a very efficient accumulation of molecules. Short DNA is concentrated thousand-fold, whereas longer DNA is exponentially better accumulated. We demonstrated both scenarios in the same micrometer-sized setting. Forthcoming experiments will reveal how replication and accumulation of DNA in a system, driven only by a thermal gradient, could create a Darwinian process of replication and selection. [ABSTRACT FROM AUTHOR]

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins. [ABSTRACT FROM AUTHOR]

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins. [ABSTRACT FROM AUTHOR]

Two major bottlenecks in elucidating the structure and function of membrane proteins are the difficulty of producing large quantities of functional receptors, and stabilizing them for a sufficient period of time. Selecting the right surfactant is thus crucial. Here we report using peptide surfactants in commercial Escherichia coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). These include the human formyl peptide receptor, human trace amine-associated receptor, and two olfactory receptors. The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. Microscale thermophoresis measurements showed that one olfactory receptor expressed using peptide surfactants bound its known ligand heptanal (molecular weight 114.18). These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs. or even other membrane proteins, for structure and function studies. [ABSTRACT FROM AUTHOR]