Synthetic pore-forming toxins stand to expand the scope of biological protein pores. Such artificial systems may be created with DNA. To facilitate their rational design, a fundamental understanding of the interaction between membrane-anchored DNA and lipid bilayers is imperative. Here we explore the interaction of hydrophobically-tagged DNA duplexes with synthetic and cell membranes. The binding of DNA modified with cholesterol or alkyl-phosphorothioate (PPT) to lipid vesicles strongly depends on the position and the chemical nature of the hydrophobic tags and lipid headgroup charge. Lipid tags also influence the dynamic molecular interface to membranes and energetics, as elucidated via MD simulations. For example, longer alkyl-PPT chains provide the most stable anchoring but may disrupt DNA base pairing. In live cells, cholesterol tags lead DNA to homogeneously distribute on the cell surface, while alkyl-PPT DNA clusters on the plasma membrane. The molecular position of DNA at or in the membrane is established by nuclease and sphingomyelinase digestion. Our insight on DNA-bilayer interaction may be applied to rationally design DNA nanostructures for membrane detectors, stimuli-controlled pathways, and synthetic toxins.