Programmable Zwitterionic Droplets as Biomolecular Sorters and Model of Membraneless Organelles

Abstract

Increasing evidence indicates that cells can regulate biochemical functions in time and space by generating membraneless compartments with well‐defined mesoscopic properties. One important mechanism underlying this control is simple coacervation driven by associative disordered proteins that encode multivalent interactions. Inspired by these observations, programmable droplets based on simple coacervation of responsive synthetic polymers that mimic the “stickers‐and‐spacers” architecture of biological disordered proteins are developed. Zwitterionic polymers that undergo an enthalpy‐driven liquid–liquid phase separation process and form liquid droplets that remarkably exclude most molecules are developed. Starting from this reference material, different functional groups in the zwitterionic polymer are progressively added to encode an increasing number of different intermolecular interactions. This strategy allowed the multiple emerging properties of the droplets to be controlled independently, such as stimulus‐responsiveness, polarity, selective uptake of client molecules, fusion times, and miscibility. By exploiting this high programmability, a model of cellular compartmentalization is reproduced and droplets capable of confining different molecules in space without physical barriers are generated. Moreover, these biomolecular sorters are demonstrated to be able to localize, separate, and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation, and diagnostics.

Complex Coacervate Materials as Artificial Cells

“…Polypeptides have also been extensively studied as complex coacervate-forming macromolecules . Advantages of synthetic polypeptides include the use of natural amino acids as building blocks, variability of side chain functional groups, and possibility for growing, including active droplets. − Synthetic polymers are beginning to be used increasingly for artificial cell assembly . Their use allows for facile variation of charge density and molecular weight, access to alternative charge functional groups, architectures, and incorporation of aspects of natural systems through bioconjugation strategies. − Charge density along the polyelectrolyte backbone has been shown to play a significant role in enzymatic processes inside coacervate droplets.…”

“… 22 − 25 Synthetic polymers are beginning to be used increasingly for artificial cell assembly. 26 Their use allows for facile variation of charge density and molecular weight, access to alternative charge functional groups, architectures, and incorporation of aspects of natural systems through bioconjugation strategies. 27 − 31 Charge density along the polyelectrolyte backbone has been shown to play a significant role in enzymatic processes inside coacervate droplets.…”

“…One elegant example from Capasso Palmiero et al shows how the modularity of synthetic polymers can be used to screen for a wide range of life-like coacervate properties ( Figure 5 ). 26 The authors copolymerized the zwitterionic monomers sulfabetaine methacrylate and sulfobetaine methacrylate, and the resulting polymers self-associatively phase separated into liquid droplets. Incorporation of additional monomers (hydroxyethyl methacrylate, methylacrylamide, benzyl methacrylate, and trimethylammonium ethyl methacrylate), hypothesized to mimic amino acid groups present in specific attractive interactions, e.g., hydrogen bonding, pi–pi aromatic interactions, or electrostatic charge imbalances, led to simple coacervate droplets that could be tuned to uptake certain molecules of interest.…”

“…g) Salt dependence of FITC-BSA uptake and coacervate disassembly. Reproduced with permission from ref ( 26 ). Copyright 2021 The Author(s) distributed under a Creative Commons Attribution License 4.0 (CC BY-NC).…”

Biomolecular condensates: Formation mechanisms, biological functions, and therapeutic targets

“…Biomolecular condensates are membraneless compartments and nonmembrane‐bound bodies, which were first discovered in the 19th century. 1 , 2 They exist throughout eukaryotic cells and play an extremely important role in various biological processes. 3 The formation of these condensates requires their components to undergo a liquid–liquid phase separation or aggregation process, which changes them from a state of dissolution to a state of separation from their surroundings.…”