Heat flows enrich prebiotic building blocks and enhance their reactivity.

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.

Replication elongates short DNA, reduces sequence bias and develops trimer structure.

The origin of molecular evolution required the replication of short oligonucleotides to form longer polymers. Prebiotically plausible oligonucleotide pools tend to contain more of some nucleobases than others. It has been unclear whether this initial bias persists and how it affects replication. To investigate this, we examined the evolution of 12-mer biased short DNA pools using an enzymatic model system. This allowed us to study the long timescales involved in evolution, since it is not yet possible with currently investigated prebiotic replication chemistries. Our analysis using next-generation sequencing from different time points revealed that the initial nucleotide bias of the pool disappeared in the elongated pool after isothermal replication. In contrast, the nucleotide composition at each position in the elongated sequences remained biased and varied with both position and initial bias. Furthermore, we observed the emergence of highly periodic dimer and trimer motifs in the rapidly elongated sequences. This shift in nucleotide composition and the emergence of structure through templated replication could help explain how biased prebiotic pools could undergo molecular evolution and lead to complex functional nucleic acids.

Sequence self-selection by cyclic phase separation.

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.

High-Fidelity RNA Copying via 2',3'-Cyclic Phosphate Ligation.

Templated ligation offers an efficient approach to replicate long strands in an RNA world. The 2',3'-cyclic phosphate (>P) is a prebiotically available activation that also forms during RNA hydrolysis. Using gel electrophoresis and high-performance liquid chromatography, we found that the templated ligation of RNA with >P proceeds in simple low-salt aqueous solutions with 1 mM MgCl2 under alkaline pH ranging from 9 to 11 and temperatures from -20 to 25 °C. No additional catalysts were required. In contrast to previous reports, we found an increase in the number of canonical linkages to 50%. The reaction proceeds in a sequence-specific manner, with an experimentally determined ligation fidelity of 82% at the 3' end and 91% at the 5' end of the ligation site. With splinted oligomers, five ligations created a 96-mer strand, demonstrating a pathway for the ribozyme assembly. Due to the low salt requirements, the ligation conditions will be compatible with strand separation. Templated ligation mediated by 2',3'-cyclic phosphate in alkaline conditions therefore offers a performant replication and elongation reaction for RNA on early Earth.

Structured sequences emerge from random pool when replicated by templated ligation.

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.

Thermophoresis beyond Local Thermodynamic Equilibrium.

We measure the thermophoresis of polysterene beads over a wide range of temperature gradients and find a pronounced nonlinear phoretic characteristic. The transition to the nonlinear behavior is marked by a drastic slowing down of thermophoretic motion and is characterized by a Péclet number of order unity as corroborated for different particle sizes and salt concentrations. The data follow a single master curve covering the entire nonlinear regime for all system parameters upon proper rescaling of the temperature gradients with the Péclet number. For low thermal gradients, the thermal drift velocity follows a theoretical linear model relying on the local-equilibrium assumption, while linear theoretical approaches based on hydrodynamic stresses, ignoring fluctuations, predict significantly slower thermophoretic motion for steeper thermal gradients. Our findings suggest that thermophoresis is fluctuation dominated for small gradients and crosses over to a drift-dominated regime for larger Péclet numbers in striking contrast to electrophoresis.

A heated rock crack captures and polymerizes primordial DNA and RNA.

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.

CO2 reduction driven by a pH gradient.

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.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.

Prebiotic Foam Environments to Oligomerize and Accumulate RNA.

When water interacts with porous rocks, its wetting and surface tension properties create air bubbles in large number. To probe their relevance as a setting for the emergence of life, we microfluidically created foams that were stabilized with lipids. A persistent non-equilibrium setting was provided by a thermal gradient. The foam's large surface area triggers capillary flows and wet-dry reactions that accumulate, aggregate and oligomerize RNA, offering a compelling habitat for RNA-based early life as it offers both wet and dry conditions in direct neighborhood. Lipids were screened to stabilize the foams. The prebiotically more probable myristic acid stabilized foams over many hours. The capillary flow created by the evaporation at the water-air interface provided an attractive force for molecule localization and selection for molecule size. For example, self-binding oligonucleotide sequences accumulated and formed micrometer-sized aggregates which were shuttled between gas bubbles. The wet-dry cycles at the foam bubble interfaces triggered a non-enzymatic RNA oligomerization from 2',3'-cyclic CMP and GMP which despite the small dry reaction volume was superior to the corresponding dry reaction. The found characteristics make heated foams an interesting, localized setting for early molecular evolution.

Acid-Catalyzed RNA-Oligomerization from 3',5'-cGMP.

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.

Kinetic Microscale Thermophoresis for Simultaneous Measurement of Binding Affinity and Kinetics.

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.

Thermal Habitat for RNA Amplification and Accumulation.

The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.

Common coding variant in SERPINA1 increases the risk for large artery stroke.

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3'-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis.

Periodic Melting of Oligonucleotides by Oscillating Salt Concentrations Triggered by Microscale Water Cycles Inside Heated Rock Pores.

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.

Probing the Cooperativity of Binding Networks with High-Throughput Thermophoresis.

The formation of supramolecular complexes is found in many natural systems and is the basis for cooperative behavior. Here, we report on the development of a high-throughput platform to measure the complex binding behavior in 500 nL volumes and 1 536-well plates. The platform enabled us to elucidate the thermodynamic properties of a heterotrimeric DNA complex that portrays the structure of a biological relevant three-way junction. In a complementing set of cooperative networks, binding constants from ∼0.1 nM to ∼10 µM were measured by sampling a high-dimensional concentration space. Each intermediate binding state was probed simultaneously with only a single fluorescent label. Through systematic base pair variations, we observed the influence of the cooperative effect on single base pair mismatches. We further found coupled binding between seemingly independent binding sites through the complex structure of the three-way junction that could not have been observed without the measurement of the entire network. These results promote automated high-throughput thermophoresis to characterize arbitrary binding networks.

Reversible Switching of Cooperating Replicators.

How can molecules with short lifetimes preserve their information over millions of years? For evolution to occur, information-carrying molecules have to replicate before they degrade. Our experiments reveal a robust, reversible cooperation mechanism in oligonucleotide replication. Two inherently slow replicating hairpin molecules can transfer their information to fast crossbreed replicators that outgrow the hairpins. The reverse is also possible. When one replication initiation site is missing, single hairpins reemerge from the crossbreed. With this mechanism, interacting replicators can switch between the hairpin and crossbreed mode, revealing a flexible adaptation to different boundary conditions.

Steep pH Gradients and Directed Colloid Transport in a Microfluidic Alkaline Hydrothermal Pore.

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.

Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy.

In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 µm(2)/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 µm(2)/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms.

Photochemical Microscale Electrophoresis Allows Fast Quantification of Biomolecule Binding.

Intricate spatiotemporal patterns emerge when chemical reactions couple to physical transport. We induce electrophoretic transport by a confined photochemical reaction and use it to infer the binding strength of a second, biomolecular binding reaction under physiological conditions. To this end, we use the photoactive compound 2-nitrobenzaldehyde, which releases a proton upon 375 nm irradiation. The charged photoproducts locally perturb electroneutrality due to differential diffusion, giving rise to an electric potential Φ in the 100 µV range on the micrometer scale. Electrophoresis of biomolecules in this field is counterbalanced by back-diffusion within seconds. The biomolecule concentration is measured by fluorescence and settles proportionally to exp(-µ/D Φ). Typically, binding alters either the diffusion coefficient D or the electrophoretic mobility µ. Hence, the local biomolecule fluorescence directly reflects the binding state. A fit to the law of mass action reveals the dissociation constant of the binding reaction. We apply this approach to quantify the binding of the aptamer TBA15 to its protein target human-α-thrombin and to probe the hybridization of DNA. Dissociation constants in the nanomolar regime were determined and match both results in literature and in control experiments using microscale thermophoresis. As our approach is all-optical, isothermal and requires only nanoliter volumes at nanomolar concentrations, it will allow for the fast screening of biomolecule binding in low volume multiwell formats.