Small molecule properties define partitioning into biomolecular condensates

Biomolecular condensates regulate cellular function by compartmentalizing molecules without a surrounding membrane. Condensate function arises from specific exclusion or enrichment of molecules. Thus, understanding condensate composition is critical to characterizing condensate function. While principles defining macromolecular composition have been described, understanding of small molecule composition remains limited. Here we quantified partitioning of ~1700 biologically relevant small molecules into condensates composed of different macromolecules. Partitioning varied nearly a million-fold across compounds but was correlated among condensates, indicating disparate condensates are physically similar. For one system, enriched compounds did not generally bind macromolecules with high affinity under conditions where condensates do not form, suggesting partitioning is not governed by site-specific interactions. Correspondingly, a machine learning model accurately predicts partitioning using only computed physicochemical features of the compounds, chiefly those related to solubility and hydrophobicity. These results suggest that a hydrophobic environment emerges upon condensate formation, driving enrichment and exclusion of small molecules.