DNA nanotechnology makes use of hydrophobically-modified constructs to create synthetic membrane protein mimics. However, nucleic acid structures exhibit poor insertion efficiency, leading to a low activity of membrane-spanning DNA protein mimics. It is suggested that non-ionic surfactants improve insertion efficiency, partly by disrupting hydrophobicity-mediated clusters. Here, we employed confocal microscopy and single-molecule transmembrane current measurements to assess the effects of the non-ionic surfactant octylpolyoxyethylene (oPOE) on the clustering behaviour, as well as membrane activity of cholesterol-modified DNA nanostruc-tures. Our findings uncover the role of aggregation in preventing bilayer interactions of hydro-phobically-decorated constructs, and we highlight that premixing DNA structures with the sur-factant does not disrupt the cholesterol-mediated aggregates. Yet, we observe the surfactant’s strong insertion-facilitating effect, particularly when introduced to the sample separately from DNA. Critically, we report a highly efficient membrane-spanning DNA construct from combining a non-aggregating design with the addition of the oPOE surfactant.DM acknowledges funding from the Winton Programme for the Physics of Sustainability and the Engineering and Physical Sciences Research Council (EPSRC, project ref. 1948702). MS acknowledges funding from the Friedrich Naumann Foundation, the Jane Bourque-Driscoll Fund and the Cambridge Philosophical Society. UFK acknowledges the ERC Consolidator Grant (De-signer-Pores 647144)
