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.