(316f) Controlled Recruitment of Particles into Biomolecular Condensates

Liquid-liquid phase separation gives rise to distinct cellular compartments called membraneless organelles, essential to cellular processes such as gene expression and nucleocytoplasmic transport. Partitioning of molecules into these condensates is central to regulating the composition and function of condensates. Previous studies have measured the mesh size of condensates to be ~5 nm based on dextran partitioning studies, in which smaller dextrans can partition while larger ones are excluded from condensates. We asked whether larger particles can partition into condensates driven by particle-condensate interactions. We sought to discover the driving forces that determine particle inclusion and exclusion in condensates using polystyrene latex beads with tailored surface chemistries, as models of macromolecular assemblies. Starting with unmodified 500 nm beads with negative surface charge, we found that protein composition alters particle partitioning into condensates. Beads localize at the periphery of condensates of the LAF-1 RGG domain, whereas they partition robustly into condensates formed from SARS-CoV-2 N protein. Next, we found particle PEGylation resulted in exclusion from the condensates. We next leveraged the PEGylated particles as an inert platform upon which we attached specific adhesive moieties. Attachment of biotin to PEGylated particles resulted in bead recruitment into streptavidin-RGG condensates, driven by biotin-streptavidin affinity. By tuning the biotin density around the beads, we demonstrate controlled recruitment of the beads into the condensates, as well as bead size dependence on partitioning. Similarly, polyA20 oligonucleotide conjugation to PEGylated particles results in strong recruitment into N protein and highly polycationic condensates. Polyadenosine (polyA)-coated particles are more strongly recruited inside droplets at low salt conditions, and less so at higher salt, implicating electrostatic interactions. Partitioning is also dependent on oligonucleotide length and density. For example, particles conjugated with polyA5 are excluded from N protein and RGG. We also demonstrate orthogonality by mixing both PS-PEG-biotin and PS-PEG-polyA20 particles with N protein and SA-RGG condensates. Coarse-grained molecular dynamics simulations provide mechanistic insights into our experimental results. Based on our findings, we conclude that large particles can partition into biomolecular condensates through condensate-particle interactions, suggesting that both size and specific binding interactions determine whether macromolecular assemblies can partition into biological condensates.