Highlights d New engineered phase-separating IDPs capable of binding RNA d IDPs co-phase-separate with RNAs into liquid granules in response to heat d IDP-RNA-rich granules regulate translation through mRNA sequestration d Droplet-based protocells temporally inhibit translation in a programmable manner

The phase separation of biomolecules has become the focus of intense research in the past decade, with a growing body of research implicating this phenomenon in essentially all biological functions, including but not limited to homeostasis, stress responses, gene regulation, cell differentiation, and disease. Excellent reviews have been published previously on the underlying physical basis of liquid–liquid phase separation (LLPS) of biological molecules (Nat. Phys. 2015, 11, 899–904) and LLPS as it occurs natively in physiology and disease (Science 2017, 357, eaaf4382; Biochemistry 2018, 57, 2479–2487; Chem. Rev. 2014, 114, 6844–6879). Here, we review how the theoretical physical basis of LLPS has been used to better understand the behavior of biomolecules that undergo LLPS in natural systems and how this understanding has also led to the development of novel synthetic systems that exhibit biomolecular phase separation, and technologies that exploit these phenomena. In part 1 of this Review, we explore the theory behind the phase separation of biomolecules and synthetic macromolecules and introduce a few notable phase-separating biomolecules. In part 2, we cover experimental and computational methods used to study phase-separating proteins and how these techniques have uncovered the mechanisms underlying phase separation in physiology and disease. Finally, in part 3, we cover the development and applications of engineered phase-separating polypeptides, ranging from control of their self-assembly to create defined supramolecular architectures to reprogramming biological processes using engineered IDPs that exhibit LLPS.

Multivalent nanoparticles that target a cell surface receptor that is overexpressed by cancer cells are a promising delivery system for cancer therapy. However, the impact of the receptor density and nanoparticle ligand valency on the cell uptake has not been studied in a system where both variables can be systematically tuned over a wide range. To address this lacuna, we report cell-uptake studies on a genetically engineered breast cancer cell line with tunable ErbB2 expression by a polypeptide micelle with tunable ligand valency. We examined the uptake of ErbB2-targeting micelles at 5 ligand densities and 11 receptor densities. We identified a matching pattern between receptors and ligands in which a receptor-to-ligand density ratio of 0.7–4.5 and a minimum of ∼1.6 bonds are required to initiate receptor-mediated endocytosis. Lower and upper limits of receptor density in the cell-uptake profile suggested a standard by which to categorize breast cancer patients as ErbB2-low, ErbB2-medium, and ErbB2-high, with each group expected to respond differently to multivalent therapeutic nanoparticles. At ErbB2-medium and ErbB2-high levels, increasing the ligand valency to 40-valent ErbB2-targeting peptides for a 20 nm radius nanoparticle accelerated the cell uptake, suggesting that the use of nanoparticles with high ligand valency for drug delivery will greatly benefit patients in these two groups. This study advances our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Complex coacervation of polymers can be a route to the compartmentalization of aqueous solutions. Presented here is a study of the hydrogen-bonded complex coacervation of poly­(acrylic acid) and poly­(ethylene glycol) or Pluronic block copolymers and the ability of these coacervates to encapsulate various ionic and nonionic dyes as well as a pharmaceutical compound within them. The formation of complex coacervate driven by hydrogen bonding is studied as a function of both pH and salt content with turbidimetry and isothermal calorimetry. Small-angle X-ray scattering shows the presence of micelles within Pluronic containing coacervate materials formed with a Pluronic block copolymer concentration higher than its critical micelle concentration. Although dyes generally partition to the coacervate phase, in the absence of salt, dyes that are able to hydrogen bond with the coacervate components are better incorporated into the coacervate. It is observed that the addition of salt to the polymer solutions increases the hydrophobicity of the environment within the coacervate, increasing the ability to sequester dye molecules for which there is no hydrogen bonding with the coacervate components. These materials are characterized with UV–vis spectroscopy, dynamic light scattering, zeta potential measurements, isothermal calorimetry, small-angle X-ray scattering, and fluorescence spectroscopy.

Fullerene molecule covalently functionalized with 12 carboxylic acid groups on its periphery was synthesized, and its solution behavior was explored. The functionalized fullerene molecules behave as hydrophilic macroions in polar solvents by showing strong attractions with each other mediated from their counterions and consequently self-assembling into single-layer, hollow, spherical blackberry-type structures in solvents with moderate polarity. The fullerene molecules are not touching with each other in the assemblies, and the assembly size can be tuned by changing the polarity of the solvents. More importantly, the transition between the self-assembly and the disassembly of the macroions can be easily achieved by changing temperature. The discovery confirms that the semirigid clusters demonstrate the unique solution behavior of macroions and open up a new way to assemble fullerene into functional materials and devices.

The coupling of compartmentalisation with molecular replication is thought to be crucial for the emergence of the first evolvable chemical systems. Minimal artificial replicators have been designed based on molecular recognition, inspired by the template copying of DNA, but none yet have been coupled to compartmentalisation. Here, we present an oil-in-water droplet system comprising an amphiphilic imine dissolved in chloroform that catalyses its own formation by bringing together a hydrophilic and a hydrophobic precursor, which leads to repeated droplet division. We demonstrate that the presence of the amphiphilic replicator, by lowering the interfacial tension between droplets of the reaction mixture and the aqueous phase, causes them to divide. Periodic sampling by a droplet-robot demonstrates that the extent of fission is increased as the reaction progresses, producing more compartments with increased self-replication. This bridges a divide, showing how replication at the molecular level can be used to drive macroscale droplet fission.

The predesigned metal-organic macrocycle ZnQDB(NO) (Zn-QDB) was observed to self-assemble into a hollow, spherical, single-layered "blackberry"-type structure. The self-assembly behaviors of the Zn-QDB are significantly influenced by additional small ions. Specifically, the cations exhibit strong co-ion effects on the interaction between cationic macrocycles which are different from the previously reported co-ion effects of simple anions on anionic polyoxometalates. This unusual phenomenon is due to the unique cation-π interaction between small cations and electron-rich cavity of Zn-QDB, as confirmed by UV-vis, H NMR, and fluorescence spectra. The variation of hydrodynamic radius (R) of assemblies with the changes of solution ionic strength and the type of cations reveals the competition between counterion-mediated attraction and cation-π interaction during the self-assembly process. Furthermore, the cooperativity of cation-π interaction and π-π stacking play a vital role in enhancing the stability of the supramolecular structure.

Two triarmed organic–inorganic hybrid materials based on carboxylic acid-functionalized polyhedral oligomeric silsesquioxane (APOSS) with/without PS linkers are designed and synthesized (tri-PS-APOSS and tri-APOSS). They can both self-assemble into hollow spherical nanostructures in water/organic mixed solvents, as confirmed by light scattering and TEM techniques, yet they possess completely different mechanisms and driving forces. With the PS linkers, the hybrid forms bilayer vesicles similar to surfactants; while without the PS linkers, the hybrid behaves like hydrophilic macroions and assembles into single-layered, vesicle-like “blackberry”-type structure. Consequently, the trend of the assembly size in response to the change of the solvent polarity is different for the two scenarios. This work shows a simple, universal approach of controlling the mechanism and product of the self-assembly process via minor adjustment of the organic–inorganic hybrid structures.