Engineering nanocondensate formation through sequence composition and patterning

Abstract Many biological proteins can assemble into dynamic, non-stoichiometric structures known as biomolecular condensates. Although typically observed at micrometer scales in vitro, recent evidence shows that these condensates often appear as nanoscale assemblies both in vitro and in cells. Moreover, biochemical reactions can be more efficiently promoted in nanoscale condensates than in micron-sized droplets, due to mass-transfer limitations and interfacial effects. Therefore, in analogy with colloids, the function of condensate materials can be engineered by tuning their size distribution. However, controlling the size of condensates remains challenging, as the molecular mechanisms that prevent small condensates from coarsening into larger ones are still poorly understood. Here, we developed and applied a computational pipeline that combines high-throughput molecular simulations, machine learning, and mixed-integer linear programming to design phase-separating peptides that form metastable nanocondensates across a broad range of experimental conditions. In addition to experimentally validating these peptides, we elucidate the underlying molecular mechanisms and derive initial design rules. In particular, we show that scaffold net charge combined with sequence blockiness can lead to high phase separation propensity while simultaneously yielding low interfacial tension, thereby slowing ripening. Moreover, these combined properties induce an electrostatics-driven alignment of molecules at the interface, which generates an additional size-dependent coalescence barrier. We further show that these features are shared by biological proteins, providing a possible mechanistic basis for the widespread occurrence of nanocondensates in biological systems. Altogether, our findings shed light on the molecular mechanisms behind nanocondensate formation, and provide a platform to design nanocondensates for several potential applications in bioengineering and biotechnology. Abstract Figure