Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases—homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

Significance: Vesicular assemblies play crucial roles in the subcellular organization as well as in biotechnological applications. Classically, the ability to form such assemblies was primarily assigned to lipids and lipid-like amphiphilic molecules. Here, we show that disordered RNA−protein complexes can form vesicle-like ordered assemblies at disproportionate mixture compositions. We also show that the ability to form vesicular assemblies is generic to multicomponent systems where phase separation is driven by heterotypic interactions. We speculate that such vesicular assemblies play crucial roles in the formation of dynamic multilayered subcellular membraneless organelles and can be utilized to fabricate novel stimuli-responsive microscale systems.

The form and function of biomolecular condensates are intimately linked to their material properties. Here, we integrate microrheology with molecular simulations to dissect the physical determinants of condensate fluid phase dynamics. By quantifying the timescales and energetics of network relaxation in a series of heterotypic viscoelastic condensates, we uncover distinctive roles of sticker motifs, binding energy, and chain length in dictating condensate dynamical properties. We find that the mechanical relaxation times of condensate-spanning networks are determined by both intermolecular interactions and chain length. We demonstrate, however, that the energy barrier for network reconfiguration, termed flow activation energy, is independent of chain length and only varies with the strengths of intermolecular interactions. Biomolecular diffusion in the dense phase depends on a complex interplay between viscoelasticity and flow activation energy. Our results illuminate distinctive roles of chain length and sequence-specific multivalent interactions underlying the complex material and transport properties of biomolecular condensates.

Proteomic studies have shown that cellular condensates are frequently enriched in diverse RNA molecules, which is suggestive of mechanistic links between phase separation and transcriptional activities. Here, we report a systematic experimental and computational study of thermodynamic landscapes and interfacial properties of protein-RNA condensates. We have studied the affinity of protein-RNA condensation as a function of variable RNA sequence length and RNA-protein stoichiometry under different ionic environments and external crowding. We have chosen the PolyU sequences for RNA and arginine/glycine-rich intrinsically disordered peptide (RGG) for proteins as a model system of RNA-protein condensates, which we then investigate through in vitro microscopy measurements and coarse-grained molecular dynamics simulations. We find that crowding and RNA chain length can have a major stabilizing effect on the condensation. We also find that the RNA-protein charge ratio is a crucial variable controlling stability, interfacial properties, and the reentrant phase behavior of RGG-RNA mixtures.

Biomolecular condensates are dynamic intracellular entities defined by their sequence- and composition-encoded material properties. During aging, these properties can change dramatically, potentially leading to pathological solidlike states, the mechanisms of which remain poorly understood. Recent experiments reveal that the aging of condensates involves a complex interplay of solvent depletion, strengthening of sticker links, and the formation of rigid structural segments such as beta fibrils. In this study, we use various coarse-grained models to investigate how solvent expulsion, biopolymer chain rigidity, and the lifetimes of sticker contacts influence the viscoelastic properties and aging dynamics of condensates. We find that the rigidity of the biopolymer backbone is essential for replicating the predominant elastic behavior observed in experiments. In contrast, models using fully flexible chains—an assumption common in simulations of intrinsically disordered proteins—fail to exhibit a dominant elastic regime. We also demonstrate that altering the solvent content within condensates affects the crossover between storage and loss moduli. This suggests that desolvation plays a significant role in condensate aging by promoting the transition from a viscous to an elastic state. Furthermore, the lifetime of sticker pairs profoundly influences the mature state of the condensates; short-lived stickers lead to a Maxwell fluid behavior, while longer-lived, irreversibly cross-linked stickers result in solidlike properties, consistent with the Kelvin-Voigt model. Finally, by incorporating the chain rigidification, desolvation, and sticker pair formation into a nonequilibrium dynamic aging simulation, we show the molecular mechanism of forming solid shells around the condensate surfaces observed in a recent experimental report. Published by the American Physical Society 2024