Membraneless organelles, such as stress granules, nucleoli, and P granules, are vital for cellular function. They compartmentalize biochemical reactions through liquid-liquid phase separation (LLPS), a process driven by entropic and ion-pairing effects known as complex coacervation. Recent research highlights the active regulation of some organelles by ATP-dependent cycles, suggesting they exist out of equilibrium as active droplets. However, how cells control the phase separation dynamics of these droplets remains largely unclear. Numerous synthetic complex coacervate droplets serve as models for studying membraneless organelles. While existing models demonstrate reversible droplet formation and dissolution in response to environmental changes (pH, temperature, salt concentration, UV light, or enzymatic reactions), they lack the fuel-driven behavior observed in biological systems. A model incorporating fuel-driven behavior would be crucial for understanding how chemical reactions regulate droplet dynamics and composition. The field of fuel-driven self-assembly focuses on self-assembly regulated by chemical reaction cycles, including a building block activation and deactivation reaction. Irreversible fuel consumption activates the building block, while deactivation reverts the product to the precursor, leading to a transient product and kinetic regulation of the assembly. This concept mirrors biological control mechanisms seen in membraneless organelles. Examples of synthetic dissipative assemblies include fuel-driven fibers and vesicles. This study aims to introduce a model for membraneless organelles based on complex coacervate droplets regulated by a fuel-driven chemical reaction cycle to gain insights into how dynamic assembly and disassembly influence droplet behavior.

The literature extensively describes membraneless organelles and their role in cellular processes. Studies have shown that these organelles rely on liquid-liquid phase separation for compartmentalizing biochemical reactions, often involving proteins and RNA held together through entropic and ion-pairing effects in complex coacervation. The dynamic nature of these organelles, regulated by ATP-dependent reactions, suggests an out-of-equilibrium state. However, understanding the cellular control of their phase separation remains a challenge. Previous research has explored synthetic complex coacervate droplets as models for membraneless organelles, focusing on reversible droplet formation and dissolution in response to various stimuli. These studies have provided insights into droplet formation and dissolution but lacked the dynamic fuel-driven behavior observed in biological systems. The concept of fuel-driven self-assembly has gained traction recently, with studies demonstrating that the self-assembly of molecules can be regulated by chemical reaction cycles, offering valuable insights into the kinetic control of assembly processes.

This study employs a model system for membraneless organelles based on RNA and a peptide whose RNA affinity is controlled by a fuel-driven reaction cycle. The RNA component is homo-polymeric RNA (poly-U), and droplet dynamics are governed by a peptide whose RNA binding is modulated by a chemical reaction cycle. The activation reaction converts a negatively charged aspartate residue on the peptide to an anhydride using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) as fuel, producing EDU (O-acyl urea) as a byproduct. The anhydride spontaneously hydrolyzes back to the aspartate, forming a transient anhydride. The peptide sequence (Ac-FRGRGRGD-OH) was designed such that the anhydride form (+3 charge) interacts with poly-U, inducing complex coacervation, while the precursor form (+1 charge) does not. Droplet formation and dynamics were studied using various techniques. Isothermal titration calorimetry (ITC) measured the interaction strength between the peptide precursor and RNA. Turbidity measurements monitored droplet formation and dissolution using a plate reader. HPLC quantified the fuel, precursor, and product concentrations throughout the reaction cycle. Confocal microscopy visualized droplet morphology, dynamics, and RNA localization. A kinetic model, based on HPLC data, predicted fuel, precursor, and product concentrations over time. The RNA concentration inside and outside droplets was determined using centrifugation and fluorescence spectroscopy. The effects of fuel and RNA concentration on droplet behavior were studied, resulting in a phase diagram. Finally, the behavior of functional RNAs (ligating ribozyme, cleaving ribozyme, and fluorophore-binding aptamer) inside the droplets was investigated through confocal microscopy and fluorescence spectroscopy to assess their activity.

The researchers found that the addition of EDC (fuel) led to the formation of transient RNA-containing coacervate droplets. The droplets' behavior depended on the fuel concentration: low fuel concentrations (7.5-40 mM) resulted in 'dynamic droplets' that quickly formed and dissolved, while higher concentrations (>40 mM) yielded 'metastable droplets' with longer lifetimes. Dynamic droplets showed a strong correlation between product concentration and turbidity, with the turbidity peaking when product concentration was highest and disappearing when the fuel and product were depleted. Metastable droplets, however, persisted even after fuel and product exhaustion, suggesting a kinetically arrested state. Confocal microscopy revealed that dynamic droplets exhibited dynamic behavior, including fusion, vacuole formation, and fragmentation into smaller droplets before dissolution. The metastable droplets showed less fusion and vacuole formation and persisted even after all product had been deactivated, suggesting that the peptide can hold the droplet together. The study further demonstrated that functional RNAs (ribozymes and aptamers) were transiently up-concentrated within the droplets, maintaining their active folded state. This was confirmed by fluorescence spectroscopy, showing that the aptamer-ligand complex localized within the droplets, indicating that functional RNA can remain active within the droplet environment. The critical coacervation concentration was determined to be around 0.9 mM of the anhydride product. The ratio of product to RNA significantly differed between dynamic and metastable droplets, with metastable droplets showing a higher ratio, potentially contributing to the longer lifetimes. In summary, the fuel-driven system allowed for the observation of several hallmarks of membraneless organelles: emergence, decay, rapid exchange of building blocks, and functionality.

This study successfully developed a fuel-driven model of membraneless organelles using RNA-containing coacervate droplets. The findings demonstrate how chemical reaction kinetics regulate droplet behavior, including formation, dissolution, and RNA partitioning. The observation of dynamic processes such as fusion, vacuole formation, and fragmentation provides valuable insights into the complex dynamics of biological membraneless organelles. The ability of the droplets to transiently up-concentrate functional RNA highlights their potential as a protocell model. The results significantly advance our understanding of how chemical reactions influence LLPS, a key mechanism in cellular organization. This model offers a powerful tool to study the effects of various factors on droplet behavior, such as protein modifications, post-translational modifications, and the role of RNA. The fuel-driven nature of the system allows for a closer approximation to the dynamic and out-of-equilibrium conditions present in living cells.

This research presents a novel fuel-driven model for membraneless organelles based on complex coacervate droplets, showcasing dynamic behavior reminiscent of biological counterparts. The system successfully demonstrates transient up-concentration of functional RNA and exhibits processes like fusion, vacuole formation, and fragmentation, providing a valuable platform for further exploration of the intricate mechanisms governing membraneless organelles. Future studies could focus on incorporating self-replicating RNA sequences and exploring the potential of these droplets as minimal protocells.

The model system, while a significant advancement, may not fully capture the complexity of biological membraneless organelles. The peptide used is a simplified representation of the more complex proteins involved in biological phase separation. Furthermore, the in vitro environment might differ from the intracellular environment, potentially influencing droplet behavior. The study focuses on a specific set of conditions and further investigation is needed to assess the model's robustness and applicability across different contexts. The methods used, while effective, might have limitations in precisely quantifying the concentrations of some components in the dynamic system.