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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]

It is generally expected that the kinetics of reactions inside living cells differs from the situation in bulk solutions. Macromolecular crowding and specific binding interactions could change the diffusion properties and the availability of free molecules. Their impact on reaction kinetics in the relevant context of living cells is still elusive, mainly because the difficulty of capturing fast kinetics in vivo. This article shows spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET-labeled dsDNA probe by lipofection. We characterized the hybridization reaction kinetics with a kinetic range of 10 [micro]s to 1 s by a combination of laser-driven temperature oscillations and stroboscopic fluorescence imaging. The time constant of the hybridization depended on DNA concentration within individual cells and between cells. A quantitative analysis of the concentration dependence revealed several-fold accelerated kinetics as compared with free solution for a 16-bp probe and decelerated kinetics for a 12-bp probe. We did not find significant effects of crowding agents on the hybridization kinetics in vitro. Our results suggest that the reaction rates in vivo are specifically modulated by binding interactions for the two probes, possibly triggered by their different lengths. In general, the presented imaging modality of temperature oscillation optical lock-in microscopy allows to probe biomolecular interactions in different cell compartments in living cells for systems biology. DNA | in vivo | molecular crowding | temperature oscillation | optical lock-in microscopy doi/10.1073/pnas.0901313106

We simulate molecular transport in elongated hydrothermal pore systems influenced by a thermal gradient. We find extreme accumulation of molecules in a wide variety of plugged pores. The mechanism is able to provide highly concentrated single nucleotides, suitable for operations of an RNA world at the origin of life. It is driven solely by the thermal gradient across a pore. On the one hand, the fluid is shuttled by thermal convection along the pore, whereas on the other hand, the molecules drift across the pore, driven by thermodiffusion. As a result, millimeter-sized pores accumulate even single nucleotides more than [10.sup.8]-fold into micrometer-sized regions. The enhanced concentration of molecules is found in the bulk water near the closed bottom end of the pore. Because the accumulation depends exponentially on the pore length and temperature difference, it is considerably robust with respect to changes in the cleft geometry and the molecular dimensions. Whereas thin pores can concentrate only long polynucleotides, thicker pores accumulate short and long polynucleotidesequally well and allow various molecular compositions. This setting also provides a temperature oscillation, shown previously to exponentially replicate DNA in the protein-assisted PCR. Our results indicate that, for life to evolve, complicated active membrane transport is not required for the initial steps. We find that interlinked mineral pores in a thermal gradient provide a compelling high-concentration starting point for the molecular evolution of life. concentration problem | hydrothermal vents | molecular evolution | origin of life problem | RNA world

Molecules drift along temperature gradients, an effect called thermophoresis, the Soret effect, or thermodiffusion. In liquids, its theoretical foundation is the subject of a long-standing debate. By using an all-optical microfluidic fluorescence method, we present experimental results for DNA and polystyrene beads over a large range of particle sizes, salt concentrations, and temperatures. The data support a unifying theory based on solvation entropy. Stated in simple terms, the Soret coefficient is given by the negative solvation entropy, divided by kT. The theory predicts the thermo-diffusion of polystyrene beads and DNA without any free parameters. We assume a local thermodynamic equilibrium of the solvent molecules around the molecule. This assumption is fulfilled for moderate temperature gradients below a fluctuation criterion. For both DNA and polystyrene beads, thermophoretic motion changes sign at lower temperatures. This thermophilicity toward lower temperatures is attributed to an increasing positive entropy of hydration, whereas the generally dominating thermophobicity is explained by the negative entropy of ionic shielding. The understanding of thermodiffusion sets the stage for detailed probing of solvation properties of colloids and biomolecules. For example, we successfully determine the effective charge of DNA and beads over a size range that is not accessible with electrophoresis. DNA | fluorescence | microfluidic | Sorer | thermodiffusion

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]

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]

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]

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 CO 2 via H 2 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 CO 2 with H 2 to formate (HCOO - ), which has proven elusive in mild abiotic settings. Here we show the reduction of CO 2 with H 2 at room temperature under moderate pressures (1.5 bar), driven by microfluidic pH gradients across inorganic Fe(Ni)S precipitates. Isotopic labeling with 13 C confirmed formate production. Separately, deuterium ( 2 H) labeling indicated that electron transfer to CO 2 does not occur via direct hydrogenation with H 2 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 H 2 , 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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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.

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.