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We discuss preparation of experimental models for multi-compartment membraneless organelles in which distinct compositions are maintained indefinitely for macromolecule-rich phases in contact with each other. These model systems are based on the physical chemistry phenomenon of complex coacervation. In complex coacervation, liquid-liquid phase separation occurs due to ion pairing interactions between oppositely charged polyelectrolytes. This mechanism can drive the associative phase separation of proteins and nucleic acids, the major macromolecular components of membraneless organelles. Here we provide examples, advice and practical considerations for the design, generation, and analysis of multi-compartment complex coacervates. These structures are of interest to compartmentalize the interior of artificial cells and as models for the intracellular membraneless organelles of biological cells. 

We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid–liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature ( T C ). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles.