Tailoring Multiphasic Protein Condensates via Liquid‐Liquid Phase Separation

ABSTRACT Many of the membraneless organelles inside cells are multiphasic condensates with complex structural organizations driven by the demixing of phase‐separating proteins. Tailoring the structures of multiphasic condensates by controlling their demixing states is a challenge. Here, we employ two proteins with distinctly different features, including thermal responsiveness, hydrophobicity, and charges: a positively charged RGGRGG protein, which forms phase‐separated condensates below an upper critical solution temperature, and a protein based on an elastin‐like polypeptide, which forms condensates above a lower critical solution temperature. These two proteins demix to form multiphasic condensates with nested and core‐shell structures under variable conditions, which can be tailored by altering the physical and chemical environments. The demixed multiphasic condensates can also be constructed inside Escherichia coli cells, recapitulating the properties of membraneless organelles. We also show that nucleic acids preferentially enrich in the positively charged segment of the multiphasic condensates. Lastly, multiphasic condensates can deliver nucleic acids across the plasma membrane into mammalian cells, enabling cell transfection.