Amphiphilic Lipid–Single-Stranded DNA Conjugate-Mediated Cell Surface Engineering for Programmable Intercellular Tethering and Immune Synapse Formation

Intercellular tethering and interface stability critically influence cellular activation, particularly in solid tumors where physical constraints limit sustained effector-target engagement. In particular, effective immune-synapse formation in natural killer cells requires stable cell-cell contact. However, most existing strategies rely on tumor-antigen-mediated recognition and are therefore vulnerable to antigen heterogeneity and immune escape. Here, we developed an amphiphilic single-stranded DNA (ssDNA)-based surface-engineering strategy that enables controllable and receptor-independent regulation of intercellular interfaces. Lipid-conjugated ssDNA constructs were designed to (a) anchor onto cell membranes, (b) induce sequence-specific association through DNA hybridization, and (c) enable thermally reversible dissociation of tethered cell pairs. This membrane modification was rapidly achieved, and complementary ssDNA pairing markedly increased effector-target tethering, cytotoxic granule and cytokine secretion, and elimination of triple-negative breast cancer cells. Importantly, this platform remained effective in 3-dimensional tumoroid models, where amphiphilic ssDNA enabled robust membrane localization and facilitated natural-killer-cell-mediated tumor disruption. Collectively, these results demonstrate that immune-synapse efficiency could be actively modulated by engineering the physical properties of intercellular interfaces. Moreover, this programmable ssDNA-based platform offers a versatile framework for regulating diverse cell-cell interfaces, with broad applicability across immunotherapy, tissue engineering, and cell-based therapeutic systems.