The study explores the design and application of peptide–DNA nanotechnology to engineer synthetic cytoskeletons for the bottom-up construction of artificial cells. Inspired by actin-binding proteins, the researchers developed a library of peptide–DNA crosslinkers with varying lengths, valencies, and geometries. These crosslinkers enable the formation of tactoid-shaped bundles with tunable aspect ratios and mechanics when peptide filaments hybridize through DNA. When confined in cell-sized water-in-oil droplets, these bundles can be localized at the cortex or within the lumen, regulating the passive diffusion of payloads and allowing for reversible recruitment and release of signals. The spatial arrangement of the peptide–DNA cytoskeletons can be controlled by the degree of bundling, and the mechanical properties of the bundles can be tuned by the length of the DNA crosslinkers. The study demonstrates that the peptide–DNA nanotechnology platform can mimic the functions of natural cytoskeletons, including cargo diffusion, signal scaffolding, and shape deformation, making it a powerful tool for constructing functional synthetic cells.The study explores the design and application of peptide–DNA nanotechnology to engineer synthetic cytoskeletons for the bottom-up construction of artificial cells. Inspired by actin-binding proteins, the researchers developed a library of peptide–DNA crosslinkers with varying lengths, valencies, and geometries. These crosslinkers enable the formation of tactoid-shaped bundles with tunable aspect ratios and mechanics when peptide filaments hybridize through DNA. When confined in cell-sized water-in-oil droplets, these bundles can be localized at the cortex or within the lumen, regulating the passive diffusion of payloads and allowing for reversible recruitment and release of signals. The spatial arrangement of the peptide–DNA cytoskeletons can be controlled by the degree of bundling, and the mechanical properties of the bundles can be tuned by the length of the DNA crosslinkers. The study demonstrates that the peptide–DNA nanotechnology platform can mimic the functions of natural cytoskeletons, including cargo diffusion, signal scaffolding, and shape deformation, making it a powerful tool for constructing functional synthetic cells.