Growing functional artificial cytoskeletons in the viscoelastic confinement of DNA synthetic cells

Intracellular structures, like the cytoskeletons, form within a crowded cytoplasm with defined viscoelastic properties. While crowding effects on self-assembly are well studied, the role of viscoelasticity in regulating structure formation remains elusive. Here, we engineer crowded all-DNA synthetic cells (SCs) with tunable viscoelastic interiors to investigate this phenomenon. We introduce a facile approach to integrate multiple DNA barcodes with adjustable concentrations into SCs, enabling selective enrichment of DNA tiles to form artificial cytoskeletons coupled to the viscoelastic SC interior. Distinct mechanistic differences in assembly occur compared to solution or simple crowding. Additionally, we develop light, molecular, and metabolic controls to direct structure formation and create self-sorted SC populations with distinct artificial cytoskeletons. These cytoskeletons enhance the mechanical stability of SCs and support stable contacts with mammalian cells. Our SC system serves as a versatile molecular engineering platform to study self-assembly under viscoelastic constraints and leverage sub-SC structures for challenging applications.