Capturing chemical reactions inside biomolecular condensates with reactive Martini simulations.

Biomolecular condensates are phase separated systems that play an important role in the spatio-temporal organisation of cells. Their distinct physico-chemical nature offers a unique environment for chemical reactions to occur. The compartmentalisation of chemical reactions is also believed to be central to the development of early life. To demonstrate how molecular dynamics may be used to capture chemical reactions in condensates, here we perform reactive molecular dynamics simulations using the coarse-grained Martini forcefield. We focus on the formation of rings of benzene-1,3-dithiol inside a synthetic peptide-based condensate, and find that the ring size distribution shifts to larger macrocycles compared to when the reaction takes place in an aqueous environment. Moreover, reaction rates are noticeably increased when the peptides simultaneously undergo phase separation, hinting that condensates may act as chaperones in recruiting molecules to reaction hubs.

Binding of Precursors to Replicator Assemblies Can Improve Replication Fidelity and Mediate Error Correction.

Copying information is vital for life's propagation. Current life forms maintain a low error rate in replication, using complex machinery to prevent and correct errors. However, primitive life had to deal with higher error rates, limiting its ability to evolve. Discovering mechanisms to reduce errors would alleviate this constraint. Here, we introduce a new mechanism that decreases error rates and corrects errors in synthetic self-replicating systems driven by self-assembly. Previous work showed that macrocycle replication occurs through the accumulation of precursor material on the sides of the fibrous replicator assemblies. Stochastic simulations now reveal that selective precursor binding to the fiber surface enhances replication fidelity and error correction. Centrifugation experiments show that replicator fibers can exhibit the necessary selectivity in precursor binding. Our results suggest that synthetic replicator systems are more evolvable than previously thought, encouraging further evolution-focused experiments.

Generalist versus Specialist Self-Replicators.

Darwinian evolution, including the selection of the fittest species under given environmental conditions, is a major milestone in the development of synthetic living systems. In this regard, generalist or specialist behavior (the ability to replicate in a broader or narrower, more specific food environment) are of importance. Here we demonstrate generalist and specialist behavior in dynamic combinatorial libraries composed of a peptide-based and an oligo(ethylene glycol) based building block. Three different sets of macrocyclic replicators could be distinguished based on their supramolecular organization: two prepared from a single building block as well as one prepared from an equimolar mixture of them. Peptide-containing hexamer replicators were found to be generalists, i. e. they could replicate in a broad range of food niches, whereas the octamer peptide-based replicator and hexameric ethyleneoxide-based replicator were proven to be specialists, i. e. they only replicate in very specific food niches that correspond to their composition. However, sequence specificity cannot be demonstrated for either of the generalist replicators. The generalist versus specialist nature of these replicators was linked to their supramolecular organization. Assembly modes that accommodate structurally different building blocks lead to generalist replicators, while assembly modes that are more restrictive yield specialist replicators.

Light-driven eco-evolutionary dynamics in a synthetic replicator system.

Darwinian evolution involves the inheritance and selection of variations in reproducing entities. Selection can be based on, among others, interactions with the environment. Conversely, the replicating entities can also affect their environment generating a reciprocal feedback on evolutionary dynamics. The onset of such eco-evolutionary dynamics marks a stepping stone in the transition from chemistry to biology. Yet the bottom-up creation of a molecular system that exhibits eco-evolutionary dynamics has remained elusive. Here we describe the onset of such dynamics in a minimal system containing two synthetic self-replicators. The replicators are capable of binding and activating a co-factor, enabling them to change the oxidation state of their environment through photoredox catalysis. The replicator distribution adapts to this change and, depending on light intensity, one or the other replicator becomes dominant. This study shows how behaviour analogous to eco-evolutionary dynamics-which until now has been restricted to biology-can be created using an artificial minimal replicator system.

Enantioselective Self-Replicators.

Self-replicating molecules provide a simple approach for investigating fundamental processes in scenarios of the emergence of life. Although homochirality is an important aspect of life and of how it emerged, the effects of chirality on self-replicators have received only little attention so far. Here, we report several self-assembled self-replicators with enantioselectivity that emerge spontaneously and grow only from enantiopure material. These require a relatively small number of chiral units in the replicators (down to eight) and in the precursors (down to a single chiral unit), compared to the only other enantioselective replicator reported previously. One replicator was found to incorporate material of its own handedness with high fidelity when provided with a racemic mixture of precursors, thus sorting (L)- and (D)-precursors into (L)- and (D)-replicators. Systematic studies reveal that the presence or absence of enantioselectivity depends on structural features (ring size of the replicator) that appear to impose constraints on its supramolecular organization. This work reveals new aspects of the little researched interplay between chirality and self-replication and represents another step toward the de novo synthesis of life.

Tailorable and Biocompatible Supramolecular-Based Hydrogels Featuring two Dynamic Covalent Chemistries.

Dynamic covalent chemistry (DCC) has proven to be a valuable tool in creating fascinating molecules, structures, and emergent properties in fully synthetic systems. Here we report a system that uses two dynamic covalent bonds in tandem, namely disulfides and hydrazones, for the formation of hydrogels containing biologically relevant ligands. The reversibility of disulfide bonds allows fiber formation upon oxidation of dithiol-peptide building block, while the reaction between NH-NH2 functionalized C-terminus and aldehyde cross-linkers results in a gel. The same bond-forming reaction was exploited for the "decoration" of the supramolecular assemblies by cell-adhesion-promoting sequences (RGD and LDV). Fast triggered gelation, cytocompatibility and ability to "on-demand" chemically customize fibrillar scaffold offer potential for applying these systems as a bioactive platform for cell culture and tissue engineering.

(Re-)Directing Oligomerization of a Single Building Block into Two Specific Dynamic Covalent Foldamers through pH.

Dynamic foldamers are synthetic folded molecules which can change their conformation in response to an external stimulus and are currently at the forefront of foldamer chemistry. However, constitutionally dynamic foldamers, which can change not only their conformation but also their molecular constitution in response to their environment, are without precedent. We now report a size- and shape-switching small dynamic covalent foldamer network which responds to changes in pH. Specifically, acidic conditions direct the oligomerization of a dipeptide-based building block into a 16-subunit macrocycle with well-defined conformation and with high selectivity. At higher pH the same building block yields another cyclic foldamer with a smaller ring size (9mer). The two foldamers readily and repeatedly interconvert upon adjustment of the pH of the solution. We have previously shown that addition of a template can direct oligomerization of the same building block to yet other rings sizes (including a 12mer and a 13mer, accompanied by a minor amount of 14mer). This brings the total number of discrete foldamers that can be accessed from a single building block to five. For a single building block system to exhibit such highly diverse structure space is unique and sets this system of foldamers apart from proteins. Furthermore, the emergence of constitutional dynamicity opens up new avenues to foldamers with adaptive behavior.

Selection of diverse polymorphic structures from a small dynamic molecular network controlled by the environment.

The complex interplay between systems and their environment plays an important role in processes ranging from self-assembly to evolution. Polymorphism, where, from the same ingredients different products can be formed, is likely to be an important enabler for evolutionary adaptation. Environmental pressures may induce polymorphic behaviour, where different pressures result in different structural organisation. Here we show that by combining covalent and non-covalent bond formation three distinct polymorphs can emerge from the same small dynamic molecular network: vesicular aggregates, self-replicating fibres and nanoribbons, depending on the nature of the solvent environment. Additionally, a particular set of conditions allows the transient co-existence of both vesicles and fibres.

Teaching an Old Compound New Tricks: Reversible Transamidation in Maleamic Acids.

Dynamic combinatorial chemistry is a method widely used for generating responsive libraries of compounds, with applications ranging from chemical biology to materials science. It relies on dynamic covalent bonds that are able to form in a reversible manner in mild conditions, and therefore requires the discovery of new types of these bonds in order to progress. Amides, due to their high stability, have been scarcely used in this field and typically require an external catalyst or harsh conditions for exchange. Compounds able to undergo uncatalysed transamidation at room temperature are still rare exceptions. In this work, we describe reversible amide formation and transamidation in a class of compounds known as maleamic acids. Due to the presence of a carboxylic acid in ß-position, these compounds are in equilibrium with their anhydride and amine precursors in organic solvents at room temperature. First, we show that this equilibrium is responsive to external stimuli: by alternating the additions of a Brønsted acid and a base, we can switch between amide and anhydride several times without side-reactions. Next, we prove that this equilibrium provides a pathway for reversible transamidation without any added catalyst, leading to thermodynamic distributions of amides at room temperature. Lastly, we use different preparation conditions and concentrations of Brønsted acid to access different library distributions, easily controlling the transition between kinetic and thermodynamic regimes. Our results show that maleamic acids can undergo transamidation in mild conditions in a reversible and tunable way, establishing them as a new addition to the toolbox of dynamic combinatorial chemistry.

Stochastic Emergence of Two Distinct Self-Replicators from a Dynamic Combinatorial Library.

Unraveling how chemistry can give rise to biology is one of the greatest challenges of contemporary science. Achieving life-like properties in chemical systems is therefore a popular topic of research. Synthetic chemical systems are usually deterministic: the outcome is determined by the experimental conditions. In contrast, many phenomena that occur in nature are not deterministic but caused by random fluctuations (stochastic). Here, we report on how, from a mixture of two synthetic molecules, two different self-replicators emerge in a stochastic fashion. Under the same experimental conditions, the two self-replicators are formed in various ratios over several repeats of the experiment. We show that this variation is caused by a stochastic nucleation process and that this stochasticity is more pronounced close to a phase boundary. While stochastic nucleation processes are common in crystal growth and chiral symmetry breaking, it is unprecedented for systems of synthetic self-replicators.

An Optical Probe for Real-Time Monitoring of Self-Replicator Emergence and Distinguishing between Replicators.

Self-replicating systems play an important role in research on the synthesis and origin of life. Monitoring of these systems has mostly relied on techniques such as NMR or chromatography, which are limited in throughput and demanding when monitoring replication in real time. To circumvent these problems, we now developed a pattern-generating fluorescent molecular probe (an ID-probe) capable of discriminating replicators of different chemical composition and monitoring the process of replicator formation in real time, giving distinct signatures for starting materials, intermediates, and final products. Optical monitoring of replicators dramatically reduces the analysis time and sample quantities compared to most currently used methods and opens the door for future high-throughput experimentation in protocell environments.

Out-of-Equilibrium Self-Replication Allows Selection for Dynamic Kinetic Stability in a System of Competing Replicators.

Among the key characteristics of living systems are their ability to self-replicate and the fact that they exist in an open system away from equilibrium. Herein, we show how the outcome of the competition between two self-replicators, differing in size and building block composition, is different depending on whether the experiments are conducted in a closed vial or in an open and out-of-equilibrium replication-destruction regime. In the closed system, the slower replicator eventually prevails over the faster competitor. In a replication-destruction regime, implemented through a flow system, the outcome of the competition is reversed and the faster replicator dominates. The interpretation of the experimental observations is supported by a mass-action-kinetics model. These results represent one of the few experimental manifestations of selection among competing self-replicators based on dynamic kinetic stability and pave the way towards Darwinian evolution of abiotic systems.

An Approach to the De Novo Synthesis of Life.

As the remit of chemistry expands beyond molecules to systems, new synthetic targets appear on the horizon. Among these, life represents perhaps the ultimate synthetic challenge. Building on an increasingly detailed understanding of the inner workings of living systems and advances in organic synthesis and supramolecular chemistry, the de novo synthesis of life (i.e., the construction of a new form of life based on completely synthetic components) is coming within reach. This Account presents our first steps in the journey toward this long-term goal. The synthesis of life requires the functional integration of different subsystems that harbor the different characteristics that are deemed essential to life. The most important of these are self-replication, metabolism, and compartmentalization. Integrating these features into a single system, maintaining this system out of equilibrium, and allowing it to undergo Darwinian evolution should ideally result in the emergence of life. Our journey toward de novo life started with the serendipitous discovery of a new mechanism of self-replication. We found that self-assembly in a mixture of interconverting oligomers is a general way of achieving self-replication, where the assembly process drives the synthesis of the very molecules that assemble. Mechanically induced breakage of the growing replicating assemblies resulted in their exponential growth, which is an important enabler for achieving Darwinian evolution. Through this mechanism, the self-replication of compounds containing peptides, nucleobases, and fully synthetic molecules was achieved. Several examples of evolutionary dynamics have been observed in these systems, including the spontaneous diversification of replicators allowing them to specialize on different food sets, history dependence of replicator composition, and the spontaneous emergence of parasitic behavior. Peptide-based replicator assemblies were found to organize their peptide units in space in a manner that, inadvertently, gives rise to microenvironments that are capable of catalysis of chemical reactions or binding-induced activation of cofactors. Among the reactions that can be catalyzed by the replicators are ones that produce the precursors from which these replicators grow, amounting to the first examples of the assimilation of a proto-metabolism. Operating these replicators in a chemically fueled out-of-equilibrium replication-destruction regime was found to promote an increase in their molecular complexity. Fueling counteracts the inherent tendency of replicators to evolve toward lower complexity (caused by the fact that smaller replicators tend to replicate faster). Among the remaining steps on the road to de novo life are now to assimilate compartmentalization and achieve open-ended evolution of the resulting system. Success in the synthesis of de novo life, once obtained, will have far-reaching implications for our understanding of what life is, for the search for extraterrestrial life, for how life may have originated on earth, and for every-day life by opening up new vistas in the form living technology and materials.

Self-Sorting in Dynamic Combinatorial Libraries Leads to the Co-Existence of Foldamers and Self-Replicators.

Nature segregates fundamental tasks such as information storage/transmission and catalysis between two different compound classes (e.g. polynucleotides for replication and folded polyamides for catalysis). This division of labor is likely a product of evolution, raising the question of how simpler systems in which replicators and folded macromolecules co-exist may emerge in the transition from chemistry to biology. In synthetic systems, achieving co-existence of replicators and foldamers in a single molecular network remains an unsolved problem. Previous work on dynamic molecular networks has given rise to either self-replicating fibers or well-defined foldamer structures (or completely un-sorted complex systems). We report a system in which two cross-reactive dithiol (nucleobase- and peptide-based) building blocks self-sort into a replicator fiber and foldamer that both emerge spontaneously and co-exist. The self-sorting behavior remains prevalent across different building block ratios as two phases of emergence occur: replicator growth followed by foldamer formation. This is attributed to the autocatalytic formation of the replicator fiber, followed by enrichment of the system in the remaining building block, which is subsequently incorporated into a foldamer.

Two Sides of the Same Coin: Emergence of Foldamers and Self-Replicators from Dynamic Combinatorial Libraries.

The ability of molecules and systems to make copies of themselves and the ability of molecules to fold into stable, well-defined three-dimensional conformations are of considerable importance in the formation and persistence of life. The question of how, during the emergence of life, oligomerization reactions become selective and channel these reactions toward a small number of specific products remains largely unanswered. Herein, we demonstrate a fully synthetic chemical system where structurally complex foldamers and self-replicating assemblies emerge spontaneously and with high selectivity from pools of oligomers as a result of forming noncovalent interactions. Whether foldamers or replicators form depends on remarkably small differences in building block structures and composition and experimental conditions. We also observed the dynamic transformation of a foldamer into a replicator. These results show that the structural requirements/design criteria for building blocks that lead to foldamers are similar to those that lead to replicators. What determines whether folding or replication takes place is not necessarily the type of noncovalent interaction, but only whether they occur intra- or intermolecularly. This work brings together, for the first time, the fields of replicator and foldamer chemistry.

Chemical Fueling Enables Molecular Complexification of Self-Replicators*.

Unravelling how the complexity of living systems can (have) emerge(d) from simple chemical reactions is one of the grand challenges in contemporary science. Evolving systems of self-replicating molecules may hold the key to this question. Here we show that, when a system of replicators is subjected to a regime where replication competes with replicator destruction, simple and fast replicators can give way to more complex and slower ones. The structurally more complex replicator was found to be functionally more proficient in the catalysis of a model reaction. These results show that chemical fueling can maintain systems of replicators out of equilibrium, populating more complex replicators that are otherwise not readily accessible. Such complexification represents an important requirement for achieving open-ended evolution as it should allow improved and ultimately also new functions to emerge.

Emergence of low-symmetry foldamers from single monomers.

Self-assembly is a powerful method to obtain large discrete functional molecular architectures. When using a single building block, self-assembly generally yields symmetrical objects in which all the subunits relate similarly to their neighbours. Here we report the discovery of a family of self-constructing cyclic macromolecules with stable folded conformations of low symmetry, which include some with a prime number (13, 17 and 23) of units, despite being formed from a single component. The formation of these objects amounts to the production of polymers with a perfectly uniform length. Design rules for the spontaneous emergence of such macromolecules include endowing monomers with a strong potential for non-covalent interactions that remain frustrated in competing entropically favoured yet conformationally restrained smaller cycles. The process can also be templated by a guest molecule that itself has an asymmetrical structure, which paves the way to molecular imprinting techniques at the level of single polymer chains.

Caught in the Act: Mechanistic Insight into Supramolecular Polymerization-Driven Self-Replication from Real-Time Visualization.

Self-assembly features prominently in fields ranging from materials science to biophysical chemistry. Assembly pathways, often passing through transient intermediates, can control the outcome of assembly processes. Yet, the mechanisms of self-assembly remain largely obscure due to a lack of experimental tools for probing these pathways at the molecular level. Here, the self-assembly of self-replicators into fibers is visualized in real-time by high-speed atomic force microscopy (HS-AFM). Fiber growth requires the conversion of precursor molecules into six-membered macrocycles, which constitute the fibers. HS-AFM experiments, supported by molecular dynamics simulations, revealed that aggregates of precursor molecules accumulate at the sides of the fibers, which then diffuse to the fiber ends where growth takes place. This mechanism of precursor reservoir formation, followed by one-dimensional diffusion, which guides the precursor molecules to the sites of growth, reduces the entropic penalty associated with colocalizing precursors and growth sites and constitutes a new mechanism for supramolecular polymerization.

Emergence of light-driven protometabolism on recruitment of a photocatalytic cofactor by a self-replicator.

Establishing how life can emerge from inanimate matter is among the grand challenges of contemporary science. Chemical systems that capture life's essential characteristics-replication, metabolism and compartmentalization-offer a route to understanding this momentous process. The synthesis of life, whether based on canonical biomolecules or fully synthetic molecules, requires the functional integration of these three characteristics. Here we show how a system of fully synthetic self-replicating molecules, on recruiting a cofactor, acquires the ability to transform thiols in its environment into disulfide precursors from which the molecules can replicate. The binding of replicator and cofactor enhances the activity of the latter in oxidizing thiols into disulfides through photoredox catalysis and thereby accelerates replication by increasing the availability of the disulfide precursors. This positive feedback marks the emergence of light-driven protometabolism in a system that bears no resemblance to canonical biochemistry and constitutes a major step towards the highly challenging aim of creating a new and completely synthetic form of life.

Spontaneous Emergence of Self-Replicating Molecules Containing Nucleobases and Amino Acids.

The conditions that led to the formation of the first organisms and the ways that life originates from a lifeless chemical soup are poorly understood. The recent hypothesis of "RNA-peptide coevolution" suggests that the current close relationship between amino acids and nucleobases may well have extended to the origin of life. We now show how the interplay between these compound classes can give rise to new self-replicating molecules using a dynamic combinatorial approach. We report two strategies for the fabrication of chimeric amino acid/nucleobase self-replicating macrocycles capable of exponential growth. The first one relies on mixing nucleobase- and peptide-based building blocks, where the ligation of these two gives rise to highly specific chimeric ring structures. The second one starts from peptide nucleic acid (PNA) building blocks in which nucleobases are already linked to amino acids from the start. While previously reported nucleic acid-based self-replicating systems rely on presynthesis of (short) oligonucleotide sequences, self-replication in the present systems start from units containing only a single nucleobase. Self-replication is accompanied by self-assembly, spontaneously giving rise to an ordered one-dimensional arrangement of nucleobase nanostructures.