Small-molecule autocatalysis drives compartment growth, competition and reproduction.

Sustained autocatalysis coupled to compartment growth and division is a key step in the origin of life, but an experimental demonstration of this phenomenon in an artificial system has previously proven elusive. We show that autocatalytic reactions within compartments-when autocatalysis, and reactant and solvent exchange outpace product exchange-drive osmosis and diffusion, resulting in compartment growth. We demonstrate, using the formose reaction compartmentalized in aqueous droplets in an emulsion, that compartment volume can more than double. Competition for a common reactant (formaldehyde) causes variation in droplet growth rate based on the composition of the surrounding droplets. These growth rate variations are partially transmitted after selective division of the largest droplets by shearing, which converts growth-rate differences into differences in droplet frequency. This shows how a combination of properties of living systems (growth, division, variation, competition, rudimentary heredity and selection) can arise from simple physical-chemical processes and may have paved the way for the emergence of evolution by natural selection.

Environmental control programs the emergence of distinct functional ensembles from unconstrained chemical reactions.

Many approaches to the origin of life focus on how the molecules found in biology might be made in the absence of biological processes, from the simplest plausible starting materials. Another approach could be to view the emergence of the chemistry of biology as process whereby the environment effectively directs "primordial soups" toward structure, function, and genetic systems over time. This does not require the molecules found in biology today to be made initially, and leads to the hypothesis that environment can direct chemical soups toward order, and eventually living systems. Herein, we show how unconstrained condensation reactions can be steered by changes in the reaction environment, such as order of reactant addition, and addition of salts or minerals. Using omics techniques to survey the resulting chemical ensembles we demonstrate there are distinct, significant, and reproducible differences between the product mixtures. Furthermore, we observe that these differences in composition have consequences, manifested in clearly different structural and functional properties. We demonstrate that simple variations in environmental parameters lead to differentiation of distinct chemical ensembles from both amino acid mixtures and a primordial soup model. We show that the synthetic complexity emerging from such unconstrained reactions is not as intractable as often suggested, when viewed through a chemically agnostic lens. An open approach to complexity can generate compositional, structural, and functional diversity from fixed sets of simple starting materials, suggesting that differentiation of chemical ensembles can occur in the wider environment without the need for biological machinery.

Approach to classify, separate, and enrich objects in groups using ensemble sorting.

The sorting of objects into groups is a fundamental operation, critical in the preparation and purification of populations of cells, crystals, beads, or droplets, necessary for research and applications in biology, chemistry, and materials science. Most of the efforts exploring such purification have focused on two areas: the degree of separation and the measurement precision required for effective separation. Conventionally, achieving good separation ultimately requires that the objects are considered one by one (which can be both slow and expensive), and the ability to measure the sorted objects by increasing sensitivity as well as reducing sorting errors. Here we present an approach to sorting that addresses both critical limitations with a scheme that allows us to approach the theoretical limit for the accuracy of sorting decisions. Rather than sorting individual objects, we sort the objects in ensembles, via a set of registers which are then in turn sorted themselves into a second symmetric set of registers in a lossless manner. By repeating this process, we can arrive at high sorting purity with a low set of constraints. We demonstrate both the theory behind this idea and identify the critical parameters (ensemble population and sorting time), and show the utility and robustness of our method with simulations and experimental systems spanning several orders of scale, sorting populations of macroscopic beads and microfluidic droplets. Our method is general in nature and simplifies the sorting process, and thus stands to enhance many different areas of science, such as purification, enrichment of rare objects, and separation of dynamic populations.

A recursive microfluidic platform to explore the emergence of chemical evolution.

We propose that a chemically agnostic approach to explore the origin of life, using an automated recursive platform based on droplet microfluidics, could be used to induce artificial chemical evolution by iterations of growth, speciation, selection, and propagation. To explore this, we set about designing an open source prototype of a fully automated evolution machine, comprising seven modules. These modules are a droplet generator, droplet transfer, passive and active size sorting, splitter, incubation chamber, reservoir, and injectors, all run together via a LabVIEWTM program integration system. Together we aim for the system to be used to drive cycles of droplet birth, selection, fusion, and propagation. As a proof of principle, in addition to the working individual modules, we present data showing the osmotic exchange of glycylglycine containing and pure aqueous droplets, showing that the fittest droplets exhibit higher osomolarity relative to their neighbours, and increase in size compared to their neighbours. This demonstrates the ability of our platform to explore some different physicochemical conditions, combining the efficiency and unbiased nature of automation with our ability to select droplets as functional units based on simple criteria.

Comprehensive experimental fitness landscape and evolutionary network for small RNA.

The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world.

Cascade of reduced speed and accuracy after errors in enzyme-free copying of nucleic acid sequences.

Nonenzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5'-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5'-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time), and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. On the basis of these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to 2 orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large "leaps" through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.

The plausibility of RNA-templated peptides: simultaneous RNA affinity for adjacent peptide side chains.

According to the RNA world hypothesis, coded peptide synthesis (translation) must have been first catalyzed by RNAs. Here, we show that small RNA sequences can simultaneously bind the dissimilar amino acids His and Phe in peptide linkage. We used in vitro counterselection/selection to isolate a pool of RNAs that bind the dipeptide NH(2)-His-Phe-COOH with K (D) ranging from 36 to 480 µM. These sites contact both side chains, usually including the protonated imidazole of His, but bind-free L: -His and L: -Phe with much lower, sometimes undetectable, affinities. The most frequent His-Phe sites do not usually contain previously isolated sites for individual amino acids, and are only ≈35 % larger than previously known separate His and Phe sites. Nonetheless, His-Phe sites appear enriched in His anticodons, as previous L: -His sites also were. Accordingly, these data add to existing experimental evidence for a stereochemical genetic code. In these peptide sites, bound amino acids approach each other to a proximity that allows a covalent peptide linkage. Isolation of several RNAs embracing two amino acids with a linking peptide bond supports the idea that a direct-RNA-template could encode primordial peptides, though crucial experiments remain.

Phototrophic phylotypes dominate mesothermal microbial mats associated with hot springs in Yellowstone National Park.

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.