Protein Phase Separation Arising from Intrinsic Disorder: First-Principles to Bespoke Applications.

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.

Quantitative Study of the Interaction of Multivalent Ligand-Modified Nanoparticles with Breast Cancer Cells with Tunable Receptor Density.

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.

Engineered Ribonucleoprotein Granules Inhibit Translation in Protocells.

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.

Hierarchical self-assembly of zwitterionic dendrimer-anionic surfactant complexes into multiple stimuli-responsive dynamic nanotubes.

Zwitterionic materials attract a wide range of attention due to their unique molecular structures and properties, which make them an interesting candidate to solve multiple problems e.g. in biological and industrial applications. Here, we show that the incorporation of zwitterions into supramolecular assemblies of ionic building blocks can be an effective way to design responsive nanostructures with well-defined morphologies. We report the hierarchical assembly of stimuli-responsive nanotubes with tunable diameters in aqueous solutions via the selective attachment of anionic surfactants to dendrimers with uniquely engineered zwitterionic peripheries. We found that the packing number of the dendrimer-surfactant hybrids can be reversibly controlled, which will trigger their assembly into tubular-like structures. These tubes can grow up to the micro-scale, their diameter is responsive to the ionic strength of the solution, and they can reversibly assemble/disassemble with a change in pH. To the best of our knowledge, this is the first example of dynamic nanotubes formed through controlled ionic interactions involving zwitterionic dendrimers in solution. This not only provides a bottom-up method to make stimuli responsive and dynamic tubes but also introduce a pathway to design complicated nanostructures by controlling the electrostatic interactions of building blocks using zwitterionic functionalities.

Strong Co-Ion Effect via Cation-π Interaction on the Self-Assembly of Metal-Organic Cationic Macrocycles.

The predesigned metal-organic macrocycle Zn3QDB3(NO3)4 (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, 1H NMR, and fluorescence spectra. The variation of hydrodynamic radius (Rh) 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.

Supramolecular Assembly of Poly(propyleneimine) Dendrimers Driven By Simple Monovalent Counterions.

The self-assembly of semiglobular, positively charged poly(propyleneimine) (PPI) dendrimers with small monovalent counterions (e.g., Cl(-)) in water/acetone mixtures was investigated. We showed that PPI dendrimers can assemble into hollow, spherical, single-layered blackberry-type structures mediated by the presence of monovalent counterions. The effects on the assembly of changing the solvent polarity and adjusting the pH were further investigated to confirm the presence of electrostatic interactions and hydrogen bonding as the driving forces. Results showed that PPI dendrimers form stable, hollow spheres in 5-20% v/v acetone/water and that the size of the spheres decreases monotonically as the solvent polarity and/or the charge on the dendrimers (i.e., lower solution pH) increases. This is the first example to show that small monovalent counterions can trigger attraction among PPI dendrimers (or broadly defined polyelectrolytes) that is strong enough to bring them together to form large, stable supramolecular assemblies, which indicates that these organic macroions have similar solution behavior to more-well-defined inorganic molecular macroions.