Regulating nucleic acid catalysis using active droplets.

Cells use transient membraneless organelles to regulate biological reaction networks. For example, stress granules selectively store mRNA to downregulate protein expression in response to heat or oxidative stress. Models mimicking this active behavior should be established to better understand in vivo regulation involving compartmentalization. Here we use active, complex coacervate droplets as a model for membraneless organelles to spatiotemporally control the activity of a catalytic DNA (DNAzyme). Upon partitioning into these peptide-RNA droplets, the DNAzyme unfolds and loses its ability to catalyze the cleavage of a nucleic acid strand. We can transiently pause the DNAzyme activity upon inducing droplet formation with fuel. After fuel consumption, the DNAzyme activity autonomously restarts. We envision this system could be used to up and downregulate multiple reactions in a network, helping understand the complexity of a cell's pathways. By creating a network where the DNAzyme could reciprocally regulate the droplet properties, we would have a powerful tool for engineering synthetic cells.

Liquid spherical shells are a non-equilibrium steady state of active droplets.

Liquid-liquid phase separation yields spherical droplets that eventually coarsen to one large, stable droplet governed by the principle of minimal free energy. In chemically fueled phase separation, the formation of phase-separating molecules is coupled to a fuel-driven, non-equilibrium reaction cycle. It thus yields dissipative structures sustained by a continuous fuel conversion. Such dissipative structures are ubiquitous in biology but are poorly understood as they are governed by non-equilibrium thermodynamics. Here, we bridge the gap between passive, close-to-equilibrium, and active, dissipative structures with chemically fueled phase separation. We observe that spherical, active droplets can undergo a morphological transition into a liquid, spherical shell. We demonstrate that the mechanism is related to gradients of short-lived droplet material. We characterize how far out of equilibrium the spherical shell state is and the chemical power necessary to sustain it. Our work suggests alternative avenues for assembling complex stable morphologies, which might already be exploited to form membraneless organelles by cells.

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.

Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer-Dependency of Potent Antimitotic Bioactivity.

Druglike small molecules with photoswitchable bioactivity-photopharmaceuticals-allow biologists to perform studies with exquisitely precise and reversible, spatial and temporal control over critical biological systems inaccessible to genetic manipulation. The photoresponsive pharmacophores disclosed have been almost exclusively azobenzenes, which has limited the structural and substituent scope of photopharmacology. More detrimentally, for azobenzene reagents, it is not researchers' needs for adapted experimental tools, but rather protein binding site sterics, that typically force whether the trans (dark) or cis (lit) isomer is the more bioactive. We now present the rational design of HOTubs, the first hemithioindigo-based pharmacophores enabling photoswitchable control over endogenous biological activity in cellulo. HOTubs optically control microtubule depolymerisation and cell death in unmodified mammalian cells. Notably, we show how the asymmetry of hemithioindigos allows a priori design of either Z- or E- (dark- or lit)-toxic antimitotics, whereas the corresponding azobenzenes are exclusively lit-toxic. We thus demonstrate that hemithioindigos enable an important expansion of the substituent and design scope of photopharmacological interventions for biological systems.

Substituent Effects in Chain-Breaking Aryltellurophenol Antioxidants.

2-Aryltellurophenols substituted in the aryltelluro or phenolic parts of the molecule were prepared by lithiation of the corresponding tetrahydropyran-protected 2-bromophenol, followed by reaction with a suitable diaryl ditelluride then deprotection. In a two-phase system containing N-acetylcysteine as a co-antioxidant in the aqueous phase, all of the compounds quenched lipid peroxyl radicals more efficiently than α-tocopherol, with three to five-fold longer inhibition times. Thus, these compounds offer better and longer-lasting antioxidant protection than recently prepared alkyltellurophenols. Compounds with electron-donating para substituents in the aryltelluro or phenolic part of the molecule showed the best results. The mechanism for quenching peroxyl radicals was considered and discussed with respect to the calculated O-H bond-dissociation energies, deuterium-labelling experiments and studies of thiol consumption in the aqueous phase.