The Role of Chemically Innocent Polyanions in Active, Chemically Fueled Complex Coacervate Droplets.

Complex coacervation describes the liquid-liquid phase separation of oppositely charged polymers. Active coacervates are droplets in which one of the electrolyte's affinity is regulated by chemical reactions. These droplets are particularly interesting because they are tightly regulated by reaction kinetics. For example, they serve as a model for membraneless organelles that are also often regulated by biochemical transformations such as post-translational modifications. They are also a great protocell model or could be used to synthesize life-they spontaneously emerge in response to reagents, compete, and decay when all nutrients have been consumed. However, the role of the unreactive building blocks, e.g., the polymeric compounds, is poorly understood. Here, we show the important role of the chemically innocent, unreactive polyanion of our chemically fueled coacervation droplets. We show that the polyanion drastically influences the resulting droplets' life cycle without influencing the chemical reaction cycle-either they are very dynamic or have a delayed dissolution. Additionally, we derive a mechanistic understanding of our observations and show how additives and rational polymer design help to create the desired coacervate emulsion life cycles.

Phase Transitions in Chemically Fueled, Multiphase Complex Coacervate Droplets.

Membraneless organelles are droplets in the cytosol that are regulated by chemical reactions. Increasing studies suggest that they are internally organized. However, how these subcompartments are regulated remains elusive. Herein, we describe a complex coacervate-based model composed of two polyanions and a short peptide. With a chemical reaction cycle, we control the affinity of the peptide for the polyelectrolytes leading to distinct regimes inside the phase diagram. We study the transitions from one regime to another and identify new transitions that can only occur under kinetic control. Finally, we show that the chemical reaction cycle controls the liquidity of the droplets offering insights into how active processes inside cells play an important role in tuning the liquid state of membraneless organelles. Our work demonstrates that not only thermodynamic properties but also kinetics should be considered in the organization of multiple phases in droplets.

Evolution and Single-Droplet Analysis of Fuel-Driven Compartments by Droplet-Based Microfluidics.

Active droplets are a great model for membraneless organelles. However, the analysis of these systems remains challenging and is often limited due to the short timescales of their kinetics. We used droplet-based microfluidics to encapsulate a fuel-driven cycle that drives phase separation into coacervate-based droplets to overcome this challenge. This approach enables the analysis of every coacervate-based droplet in the reaction container throughout its lifetime. We discovered that the fuel concentration dictates the formation of the coacervate-based droplets and their properties. We observed that coacervate-based droplets grow through fusion, decay simultaneously independent of their volume, and shrinkage rate scales with their initial volume. This method helps to further understand the regulation of membraneless organelles, and we believe the analysis of individual coacervate-based droplets enables future selection- or evolution-based studies.

Molecular Design of Chemically Fueled Peptide-Polyelectrolyte Coacervate-Based Assemblies.

Complex coacervated-based assemblies form when two oppositely charged polyelectrolytes combine to phase separate into a supramolecular architecture. These architectures range from complex coacervate droplets, spherical and worm-like micelles, to vesicles. These assemblies are widely applied, for example, in the food industry, and as underwater or medical adhesives, but they can also serve as a great model for biological assemblies. Indeed, biology relies on complex coacervation to form so-called membraneless organelles, dynamic and transient droplets formed by the coacervation of nucleic acids and proteins. To regulate their function, membraneless organelles are dynamically maintained by chemical reaction cycles, including phosphorylation and dephosphorylation, but exact mechanisms remain elusive. Recently, some model systems also regulated by chemical reaction cycles have been introduced, but how to design such systems and how molecular design affects their properties is unclear. In this work, we test a series of cationic peptides for their chemically fueled coacervation, and we test how their design can affect the dynamics of assembly and disassembly of the emerging structures. We combine them with both homo- and block copolymers and study the morphologies of the assemblies, including morphological transitions that are driven by the chemical reaction cycle. We deduce heuristic design rules that can be applied to other chemically regulated systems. These rules will help develop membraneless organelle model systems and lead to exciting new applications of complex coacervate-based examples like temporary adhesives.

Active coacervate droplets as a model for membraneless organelles and protocells.

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 which remains in its active folded state inside the droplets. Finally, we show that in their pathway towards decay, these droplets break apart in multiple droplet fragments. Emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of membrane-less organelles, and we believe that our work could be powerful as a model to study such organelles.