Oscillations in the formation and dissolution of molecular assemblies inside living cells are pivotal in orchestrating various cellular functions and processes. However, designing such rhythmic patterns in synthetic cells remains a challenge. Here, we demonstrate the spontaneous emergence of spatio-temporal oscillations in the number of droplets, size, and their spatial distribution within a synthetic cell. The coacervate-based droplets in these synthetic cells sediment and fuse at the cell's bottom. Through a size control mechanism, the sedimented, large droplets shrink by expelling droplet material. The expelled molecules nucleate new droplets at the top of the synthetic cell, which grow and sediment again. These oscillations are sustained by converting chemical fuel into waste and can continue for hundreds of periods without evidence of fatigue. Strikingly, the period of the oscillation is in the minute's regime and tunable. The design of oscillating artificial organelles in synthetic cells brings us closer to creating more life-like materials and de novo life.

In life, molecular architectures, like the cytoskeletal proteins or the nucleolus, catalyze the conversion of chemical fuels to perform their functions. For example, tubulin catalyzes the hydrolysis of GTP to form a dynamic cytoskeletal network. In contrast, myosin uses the energy obtained by catalyzing the hydrolysis of ATP to exert forces. Artificial examples of such beautiful architectures are scarce partly because synthetic chemically fueled reaction cycles are relatively rare. Here, we introduce a new chemical reaction cycle driven by the hydration of a carbodiimide. Unlike other carbodiimide-fueled reaction cycles, the proposed cycle forms a transient 5(4H)-oxazolone. The reaction cycle is efficient in forming the transient product and is robust to operate under a wide range of fuel inputs, pH, and temperatures. The versatility of the precursors is vast, and we demonstrate several molecular designs that yield chemically fueled droplets, fibers, and crystals. We anticipate that the reaction cycle can offer a range of other assemblies and, due to its versatility, can also be incorporated into molecular motors and machines.

Nature uses dynamic, molecular self-assembly to create cellular architectures that adapt to their environment. For example, a guanosine triphosphate (GTP)-driven reaction cycle activates and deactivates tubulin for dynamic assembly into microtubules. Inspired by dynamic self-assembly in biology, recent studies have developed synthetic analogs of assemblies regulated by chemically fueled reaction cycles. A challenge in these studies is to control the interplay between rapid disassembly and kinetic trapping of building blocks known as dynamic instabilities. In this work, we show how molecular design can tune the tendency of molecules to remain trapped in their assembly. We show how that design can alter the dynamic of emerging assemblies. Our work should give design rules for approaching dynamic instabilities in chemically fueled assemblies to create new adaptive nanotechnologies.

Nature uses dynamic, molecular self-assembly to create cellular architectures that adapt to their environment. For example, a guanosine triphosphate (GTP)-driven reaction cycle activates and deactivates tubulin for assembly into microtubules and disassembly. Inspired by dynamic self-assembly in biology, multiple studies have developed synthetic analogs of assemblies regulated by chemical chemically fueled reaction cycles. A challenge in most of these studies is that molecules assemble upon activation but do not disassemble upon deactivation. In other words, they remain kinetically trapped, and the resulting assemblies are not dynamic. In this work, we show how molecular design dictates the tendency of deactivated molecules to remain trapped in the assembled state. We also show how molecular design can be used to tune the dynamics of the reaction cycle. Our work should result in chemically fueled assemblies that are truly dynamic in that dis-assembly immediately follows deactivation.

Using molecular self-assembly, supramolecular chemists can create Gigadalton-structures with angstrom precision held together by non-covalent interactions. However, despite relying on the same molecular toolbox for self-assembly, these synthetic structures lack the complexity and sophistication of biological assemblies. Those assemblies are non-equilibrium structures that rely on the constant consumption of energy transduced from the hydrolysis of chemical fuels like ATP and GTP, which endows them with dynamic properties, e.g., temporal and spatial control and self-healing ability. Thus, to synthesize life-like materials, we have to find a reaction cycle that converts chemical energy to regulate self-assembly. We and others recently found that this can be done by a reaction cycle that hydrates carbodiimides. This feature article aims to provide an overview of how the energy transduced from carbodiimide hydration can alter the function of molecules and regulate molecular assemblies. The goal is to offer the reader design considerations for carbodiimide-driven reaction cycles to create a desired morphology or function of the assembly and ultimately to push chemically fueled self-assembly further towards the bottom-up synthesis of life.

In chemically fueled self-assembly, a reaction cycle activates and deactivates molecules for self-assembly. The resulting assembly is dynamic and should be endowed with unique behavior in this kinetically controlled regime. Recent works have mainly focused on design rules for the activation of molecules for self-assembly, thereby assuming that disassembly upon deactivation inherently follows. However, that is not always the case. This work shows a family of peptides that assemble into colloids regulated through a chemical reaction cycle. Despite their similarity in assembly, we find that they follow a different disassembly pathway upon deactivation. The colloids from several peptides completely disassemble as fuel depletes while others transition into fibers. Our findings demonstrate that assembly and disassembly should be taken into account in chemically fueled self-assembly.

Chemically fueled emulsions are solutions with droplets made of phase-separated molecules that are activated and deactivated by a chemical reaction cycle. These emulsions play a crucial role in biology as a class of membrane-less organelles. Moreover, theoretical studies show that droplets in these emulsions can evolve to the same size or spontaneously self-divide when fuel is abundant. All of these exciting properties, i. e., emergence, decay, collective behavior, and self-division, are pivotal to the functioning of life. However, these theoretical predictions lack experimental systems to test them quantitively. Here, we describe the synthesis of synthetic emulsions formed by a fuel-driven chemical cycle, and we find a surprising new behavior, i. e., the dynamics of droplet growth is regulated by the kinetics of the fuel-driven reaction cycle. Consequently, the average volume of these droplets grows orders of magnitude faster compared to Ostwald ripening. Combining experiments and theory, we elucidate the underlying mechanism.

We investigate active droplets that form at the expense of a chemical fuel in aqueous buffer and vanish autonomously. Dynamic light scattering reveals the scattered intensity, the hydrodynamic radius, and the width of the size distribution with high precision as well as high temporal and spatial resolutions. Comparing the resulting time-dependent behavior of the droplet characteristics with the time-dependent concentration of the anhydrides, the roles of the chemical reaction cycle and of colloidal growth processes are elucidated. The droplet sizes and lifetimes depend strongly on the hydrophobicity of the precursor, and the growth rate is found to correlate with the deactivation rate of the product.

Materials that release drugs with a constant, zero-order rate are of great importance to improve therapeutic efficacy and reduce toxic side effects.

Membraneless organelles like stress granules are active liquid-liquid phase-separated droplets that are involved in many intracellular processes. Their active and dynamic behavior is often regulated by ATP-dependent reactions. However, how exactly membraneless organelles control their dynamic composition remains poorly understood. Herein, we present a model for membraneless organelles based on RNA-containing active coacervate droplets regulated by a fuel-driven reaction cycle. These droplets emerge when fuel is present, but decay without. Moreover, we find these droplets can transiently up-concentrate functional RNA, and that this up-take is accelerated by the chemical reaction cycle. Finally, we show that in their pathway towards decay, these droplets self-divide asymmetrically. Self-division combined with emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of life, and we believe that our work could be a stepping stone towards its synthesis.

Membraneless organelles like stress granules are active liquid-liquid phase-separated droplets that are involved in many intracellular processes. Their active and dynamic behavior is often regulated by ATP-dependent reactions. However, how exactly membraneless organelles control their dynamic composition remains poorly understood. Herein, we present a model for membraneless organelles based on RNA-containing active coacervate droplets regulated by a fuel-driven reaction cycle. These droplets emerge when fuel is present, but decay without. Moreover, we find these droplets can transiently up-concentrate functional RNA, and that this up-take is accelerated by the chemical reaction cycle. Finally, we show that in their pathway towards decay, these droplets self-divide asymmetrically. Self-division combined with emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of life, and we believe that our work could be a stepping stone towards its synthesis.

Active droplets are made of phase-separated molecules that are activated and deactivated by a metabolic reaction cycle. Such droplets play a crucial role in biology as a class of membrane-less organelles. Moreover, theoretical studies show that active droplets can evolve to the same size or spontaneously self-divide when energy is abundant. All of these exciting properties, i.e., emergence, decay, collective behavior, and self-division, are pivotal to the functioning of life. However, these theoretical predictions lack experimental systems to test them quantitively. Here, we describe the synthesis of synthetic active droplets driven by a metabolic chemical cycle and we find a surprising new behavior, i.e., the dynamics of droplet-growth is regulated by the kinetics of the fuel-driven reaction cycle. Consequently, these droplets ripen orders of magnitude faster compared to Ostwald ripening. Combining experiments and theory, we elucidate the underlying mechanism, which could help better understand how cells regulate the growth of membrane-less organelles.

Self-assembled cationic micelles are an attractive platform for binding biologically-relevant polyanions such as heparin. This has potential applications in coagulation control, where a synthetic heparin rescue agent could be a useful replacement for protamine, which is in current clinical use. However, micelles can have low stability in human serum and unacceptable toxicity profiles. This paper reports the optimisation of self-assembled multivalent (SAMul) arrays of amphiphilic ligands to bind heparin in competitive conditions. Specifically, modification of the hydrophobic unit kinetically stabilises the self-assembled nanostructures, preventing loss of binding ability in the presence of human serum - cholesterol hydrophobic units significantly outperform systems with a simple aliphatic chain. It is demonstrated that serum albumin disrupts the binding thermodynamics of the latter system. Molecular simulation shows aliphatic lipids can more easily be removed from the self-assembled nanostructures than the cholesterol analogues. This agrees with the experimental observation that the cholesterol-based systems undergo slower disassembly and subsequent degradation via ester hydrolysis. Furthermore, by stabilising the SAMul nanostructures, toxicity towards human cells is decreased and biocompatibility enhanced, with markedly improved survival of human hepatoblastoma cells in an MTT assay.

In molecular self-assembly, molecules interact with each other by non-covalent interactions to form larger structures. The process occurs in-equilibrium, which means that molecules can leave the assembly and reassemble elsewhere, but that occurs on average with equal rates. For self-assembly, peptides have proven to be a particularly useful building block, in part because of their versatility. Biology also uses self-assembly to create functional materials. For example, components of the cytoskeleton, like actin filament and microtubules, are self-assembled proteins. In other words, biology uses the same building blocks and design rules as supramolecular chemists to create functional structures. However, biological assemblies are vastly more complex than their synthetic counterparts. The discrepancy is in part because proteins are more complex than the peptides we use as building blocks. Another contributing factor to the difference in complexity is that most assemblies in living systems exist out of equilibrium. To use peptides for complex functions as biology does, we should understand and be able to create peptide assemblies out of equilibrium. In this work, we review recent advances towards the creation of peptide assemblies that exist out of equilibrium driven by an external energy source.

Solutions of silicon nanocrystals (SiNCs) are used in a diverse range of applications because of their tunable photoluminescence, biocompatibility, and the abundance of Si. In dissipative supramolecular materials, self-assembly of molecules or nanoparticles is driven by a chemical reaction network that irreversible consumes fuel. The properties of the emerging structures are controlled by the kinetics of the underlying chemical reaction network. Herein, we demonstrate the dissipative self-assembly of photoluminescent SiNCs driven by a chemical fuel. A chemical reaction induces self-assembly of the water-soluble SiNCs. However, the assemblies are transient, and when the chemical reaction network runs out of fuel, the SiNCs disassemble. The lifetime of the assemblies is controlled by the amount of fuel added. As an application of the transient supramolecular material, we demonstrate that the platform can be used to control the delayed uptake of the nanocrystals by mammalian cells.

Dissipative self-assembly is a process in which energy-consuming chemical reaction networks drive the assembly of molecules. Prominent examples from biology include the GTP-fueled microtubule and ATP-driven actin assembly. Pattern formation and oscillatory behavior are some of the unique properties of the emerging assemblies. While artificial counterparts exist, researchers have not observed such complex responses. One reason for the missing complexity is the lack of feedback mechanisms of the assemblies on their chemical reaction network. In this work, we describe the dissipative self-assembly of colloids that protect the hydrolysis of their building blocks. The mechanism of inhibition is generalized and explored for other building blocks. We show that we can tune the level of inhibition by the assemblies. Finally, we show that the robustness of the assemblies towards starvation is affected by the degree of inhibition.

Many biological materials exist in non-equilibrium states driven by the irreversible consumption of high-energy molecules like ATP or GTP. These energy-dissipating structures are governed by kinetics and are thus endowed with unique properties including spatiotemporal control over their presence. Here we show man-made equivalents of materials driven by the consumption of high-energy molecules and explore their unique properties. A chemical reaction network converts dicarboxylates into metastable anhydrides driven by the irreversible consumption of carbodiimide fuels. The anhydrides hydrolyse rapidly to the original dicarboxylates and are designed to assemble into hydrophobic colloids, hydrogels or inks. The spatiotemporal control over the formation and degradation of materials allows for the development of colloids that release hydrophobic contents in a predictable fashion, temporary self-erasing inks and transient hydrogels. Moreover, we show that each material can be re-used for several cycles.

Life is a dissipative nonequilibrium structure that requires constant consumption of energy to sustain itself. How such an unstable state could have selected from an abiotic pool of molecules remains a mystery. Here we show that liquid phase-separation offers a mechanism for the selection of dissipative products from a library of reacting molecules. We bring a set of primitive carboxylic acids out-of-equilibrium by addition of high-energy condensing agents. The resulting anhydrides are transiently present before deactivation via hydrolysis. We find the anhydrides that phase-separate into droplets to protect themselves from hydrolysis and to be more persistent than non-assembling ones. Thus, after several starvation-refueling cycles, the library self-selects the phase-separating anhydrides. We observe that the self-selection mechanism is more effective when the library is brought out-of-equilibrium by periodic addition of batches as opposed to feeding it continuously. Our results suggest that phase-separation offers a selection mechanism for energy dissipating assemblies.

AbstractLösungen von Siliciumnanokristallen (SiNCs) werden z. B. wegen ihrer einstellbaren Photolumineszenz, Biokompatibilität und der Verfügbarkeit von Si in zahlreichen Anwendungen eingesetzt. In dissipativen supramolekularen Materialien wird die Selbstassemblierung von Molekülen oder Nanopartikeln durch ein chemisches Reaktionsnetzwerk angetrieben, das irreversibel Brennstoff verbraucht. Die Eigenschaften der entstehenden Strukturen werden durch die Kinetik des zugrunde liegenden chemischen Reaktionsnetzwerkes gesteuert. Hier wird die dissipative Selbstassemblierung von photolumineszierenden SiNCs demonstriert, die von einem chemischen Brennstoff angetrieben werden. Eine chemische Reaktion setzt die Selbstassemblierung der wasserlöslichen SiNCs in Gang. Allerdings sind die Strukturen transient, und wenn dem chemischen Reaktionsnetzwerk der Brennstoff ausgeht, deassemblieren sich die SiNCs. Die Lebensdauer der Strukturen ist durch die zugegebene Brennstoffmenge steuerbar. Die Plattform kann für die gesteuerte, verzögerte Aufnahme der Nanokristalle durch Säugetierzellen genutzt werden.