Job Boekhoven

Mechanically interlocked molecules (MIMs) offer attractive opportunities to modularly integrate bioactivity, molecular recognition, and optical readout within a single supramolecular architecture. Here, we report a fluorescent [2]rotaxane platform based on a cationic organometallic pillarplex ring and fluorescein stoppers as intrinsic imaging labels. The pillarplex ring promotes interactions with biorelevant polyanions such as nucleic acids, while the fluorescein stoppers ensure optical traceability by confocal microscopy. Cellular uptake by A549 cancer cells is demonstrated, showing that the fluorescent rotaxane remains predominantly localized in the cytoplasm. The rotaxane can furthermore be integrated into multicomponent nanoassemblies, such as coacervates and biopharmaceutical-relevant polyplexes. For the latter, in the presence of siRNA and cationic polymers, the rotaxane forms assemblies that even enhance cellular uptake and imaging. Collectively, these findings demonstrate that dye incorporation provides a powerful strategy for modulating pillarplex rotaxanes, thereby enabling controlled uptake, localization within the cell by fluorescence labeling and co-assembly.

The bottom-up synthesis of life aims to replicate the characteristics of natural living systems from basic abiotic building blocks. In this context, synthetic cells based on liquid-liquid phase separation, such as complex coacervate droplets, have gained attention because they form spontaneously and readily take up molecules from their environment. More recently, we introduced fuel-dependent synthetic cells—coacervates that require fuel to form and decay unless periodically fueled. These synthetic cells are therefore under selection pressure to survive until the next fueling cycle. However, whereas natural cells rely on internal sub-compartments, complex coacervates typically lack structural heterogeneity. Here, we show that peptide amphiphiles can be activated in situ to generate discrete, phase-separated structures within complex coacervates. A chemically orthogonal stimulus enables spatiotemporal control over peptide assembly without disrupting fuel-driven phase separation. We further identify a reciprocal relationship: peptide assemblies alter the physical properties of the coacervate matrix, while the host compartment directs peptide morphology and assembly kinetics. These hierarchical assemblies function as artificial organelles within fueled synthetic cells. Together, they provide a strategy for integrating orthogonal supramolecular assembly with chemically fueled synthetic cells, enabling internal sub-compartmentalization, enhanced kinetic stability, and programmable morphology.

The bottom-up synthesis of life aims to replicate the characteristics of natural living systems from basic abiotic building blocks. In this context, synthetic cells based on liquid-liquid phase separation, such as complex coacervate droplets, have gained attention because they form spontaneously and readily take up molecules from their environment. More recently, we introduced fuel-dependent synthetic cells—coacervates that require fuel to form and decay unless periodically fueled. These synthetic cells are therefore under selection pressure to survive until the next fueling cycle. However, whereas natural cells rely on internal sub-compartments, complex coacervates typically lack structural heterogeneity. Here, we show that peptide amphiphiles can be activated in situ to generate discrete, phase-separated structures within complex coacervates. A chemically orthogonal stimulus enables spatiotemporal control over peptide assembly without disrupting fuel-driven phase separation. We further identify a reciprocal relationship: peptide assemblies alter the physical properties of the coacervate matrix, while the host compartment directs peptide morphology and assembly kinetics. These hierarchical assemblies function as artificial organelles within fueled synthetic cells. Together, they provide a strategy for integrating orthogonal supramolecular assembly with chemically fueled synthetic cells, enabling internal sub-compartmentalization, enhanced kinetic stability, and programmable morphology.

Synthetic cells are compartments designed to mimic the functions and characteristics of living cells. By constructing synthetic cells from abiotic, basic components (bottom-up), it is possible to investigate the minimal requirements for life and gain insights into fundamental principles of biology. Among the available platforms, complex coacervates are particularly attractive, due to their potential to encapsulate a wide range of biomolecules and other cargo, enabling genotype-phenotype mapping. By coupling coacervate formation to a fueled chemical reaction cycle, the synthetic cells become fuel-dependent, growing in the presence of fuel and decaying in its absence, resembling biological cells. However, constructing cellular substructures for these fuel-dependent synthetic cells, such as cytoskeletons, has remained an unresolved challenge. Here, we show that supramolecular (co)polymers can act as cytoskeletons for the synthetic cells, depending on their condenophilicity (their affinity for the droplet phase). By using two distinct supramolecular building blocks, the condenophilicity can be modulated. Supramolecular copolymers where only some of the monomers bind to the complex coacervate result in the formation of a protruding cytoskeleton in and around the droplet. However, as condenophilicity increases, the polymers partition strongly into the coacervates, leading to the formation of a fully encapsulated cytoskeleton. We found that these internal structures influence the synthetic cell properties, such as morphology and lifespan. Moreover, the synthetic cells can dynamically reconstitute the supramolecular fibers, creating distinct populations within the cells and the surrounding dilute phase. Our results demonstrate that orthogonally assembled structures can serve as cellular substructures for active complex coacervate-based synthetic cells, broadening the existing arsenal of tools to bestow these rudimentary synthetic cells with more life-like properties.

Inspired by nature, chemically fueled reaction cycles provide a general strategy to couple the dissipation of chemical potential to controlled changes in molecular structure, assembly, and material properties. Here, we review how kinetically stable yet thermodynamically activated fuels, catalytic activation–deactivation cycles, and supramolecular transformations integrate into a common design spanning molecular, assembly, and material length scales. We unify terminology across communities using diverse systems into state‐network descriptions that capture reciprocal coupling between chemical and supramolecular states. Central to function is kinetic asymmetry (feedback from the supramolecular state on reaction rates), which biases reaction flux and enables work‐like behavior, including directional motion, tunable lifetimes, self‐healing, oscillations, bistability, and spatiotemporal control of optical, mechanical, and catalytic properties. By contrasting single‐point versus multipoint activation and intramolecular versus intermolecular processes, we extract practical design principles and highlight open challenges, including quantitative kinetic descriptions at the assembly scale, management of waste accumulation under sustained fueling, and scaling chemo‐mechanical coupling toward higher‐order autonomous systems. Overall, this review provides a shared mechanistic language and a set of transferable guidelines for engineering functional, adaptive, chemically powered systems across all length scales.

Biomolecular condensates formed through liquid-liquid phase separation play central roles in intracellular organization and regulation. Replicating such dynamic compartmentalization using minimal synthetic components remains exceptionally challenging inside living cells. Here, we report short cationic peptides that undergo directed complex coacervation in living cells through preferential interactions with endogenous polyanionic biomolecules. Although the peptides were designed to contain a mitochondrial targeting motif, the Arg residues and cellular RNA guide the supramolecular interactions toward selective enrichment of liquid-like coacervates in nucleolar regions. In vitro studies reveal that polymeric RNA mimics promote coacervation far more efficiently than ATP, establishing RNA-peptide interactions as the principal driving force. In cells, nucleolar complex coacervates form rapidly and exhibit liquid-like behavior with fast molecular exchange. Importantly, the assemblies are transient and reversible: sustained peptide supply maintains the condensed state, whereas substrate depletion triggers droplet dissolution and recovery of cellular function. These findings demonstrate that endogenous biopolymer distributions can guide and participate in the formation of synthetic coacervates with minimalistic peptides, achieving reversible reorganization of intracellular components. More broadly, this work provides a framework for engineering synthetic coacervates with nonequilibrium, life-like features that operate in direct exchange with living cellular environments.

In the field of biomolecular condensates and synthetic systems, it is an open question whether liquid droplets can undergo self-sustained oscillations of formation and dissolution. To unravel the minimal physicochemical prerequisite for such droplet oscillations, we present a simple model composed of only two independent chemical components with their diffusive and chemical fluxes governed by nonequilibrium thermodynamics. There is turnover of fuel that maintains a chemical reaction away from equilibrium, leading to active droplets. We find that a single active droplet undergoes a saddle-node bifurcation in the droplet volume upon increasing the fueling strength. Strikingly, the active droplet becomes excitable upon adding a further chemical reaction. For sufficient fueling, the system undergoes self-sustained oscillations.

Phosphorylation is among the most ubiquitous and essential posttranslational modifications in biological systems. It is regulated by highly complex enzymatic networks. Here, we explore enzyme-free phosphorylation in a designed peptide system. Specifically, we use phosphorylation to modulate coiled-coil (CC) assembly and dynamics. This exploits a nonbiological reaction cycle to phosphorylate and dephosphorylate a histidine residue in an α-helix, enabling or disabling CC formation with a second helix. Dephosphorylation is kinetically gated─it is 25× faster in the CC compared to the nonassembled state. As a result, the cycle of phosphorylation, CC formation, dephosphorylation, and CC disassembly is ratcheted. The minimal synthetic phosphorylation cycle provides design principles and fulfills requirements to perform work mimicking the function of molecular motors, walkers, and pumps.

Synthetic cells are compartments designed to mimic the functions and characteristics of living cells. By constructing synthetic cells from abiotic, basic components (bottom-up), it is possible to investigate the minimal requirements for life and gain insights into fundamental principles of biology. Among the available platforms, complex coacervates are particularly attractive, due to their potential to encapsulate a wide range of biomolecules and other cargo, enabling genotype-phenotype mapping. By coupling coacervate formation to a fueled chemical reaction cycle, the synthetic cells become fuel-dependent, growing in the presence of fuel and decaying in its absence, resembling biological cells. However, constructing organelles for these transient synthetic cells, such as membranes and cytoskeletons, has remained an unresolved challenge. Here, we show that supramolecular (co-)polymers can act as organelles for the synthetic cells, depending on their condenophilicity (their affinity for the droplet phase). By using two distinct supramolecular building blocks, the condenophilicity can be modulated. Supramolecular copolymers where only some of the monomers bind to the complex coacervate result in the formation of a membrane structure around the droplet. However, as condenophilicity increases, the polymers partition strongly into the coacervates, leading to the formation of a cytoskeleton. Moreover, we found that these organelles influence the synthetic cell properties, such as morphology and their lifespan. Our results demonstrate that orthogonally assembled structures can serve as organelles for active complex coacervate-based synthetic cells, broadening the existing arsenal of tools to bestow these rudimentary synthetic cells with more life-like properties.

Synthetic cells are compartments designed to mimic the functions and characteristics of living cells. By constructing synthetic cells from abiotic, basic components (bottom-up), it is possible to investigate the minimal requirements for life and gain insights into fundamental principles of biology. Among the available platforms, complex coacervates are particularly attractive, due to their potential to encapsulate a wide range of biomolecules and other cargo, enabling genotype-phenotype mapping. By coupling coacervate formation to a fueled chemical reaction cycle, the synthetic cells become fuel-dependent, growing in the presence of fuel and decaying in its absence, resembling biological cells. However, constructing organelles for these transient synthetic cells, such as membranes and cytoskeletons, has remained an unresolved challenge. Here, we show that supramolecular (co-)polymers can act as organelles for the synthetic cells, depending on their condenophilicity (their affinity for the droplet phase). By using two distinct supramolecular building blocks, the condenophilicity can be modulated. Supramolecular copolymers where only some of the monomers bind to the complex coacervate result in the formation of a membrane structure around the droplet. However, as condenophilicity increases, the polymers partition strongly into the coacervates, leading to the formation of a cytoskeleton. Moreover, we found that these organelles influence the synthetic cell properties, such as morphology and their lifespan. Our results demonstrate that orthogonally assembled structures can serve as organelles for active complex coacervate-based synthetic cells, broadening the existing arsenal of tools to bestow these rudimentary synthetic cells with more life-like properties.