Defined Bilayer Interactions of DNA Nanopores Revealed with a Nuclease-Based Nanoprobe Strategy.

DNA nanopores are a recent class of bilayer-puncturing nanodevices that can help advance biosensing, synthetic biology, and nanofluidics. Here, we create archetypal lipid-anchored DNA nanopores and characterize them with a nanoprobe-based approach to gain essential information about their interactions with bilayers. The strategy determines the molecular accessibility of DNA pores with a nuclease and can thus distinguish between the nanopores' membrane-adhering and membrane-spanning states. The analysis reveals, for example, that pores interact with bilayers in two steps whereby fast initial membrane tethering is followed by slower reorientation to the puncturing state. Tethering occurs for pores with one anchor, while puncturing requires multiple anchors. Both low and high-curvature membranes are good substrates for tethering, but efficient insertion proceeds only for high-curvature bilayers of the examined lipid composition. This is likely due to the localized lipid misalignments and the associated lower energetic barrier for pore permeation. Our study advances the fields of DNA nanotechnology and nanopores by overcoming the considerable experimental hurdle of efficient membrane insertion. It also provides mechanistic insights to aid the design of advanced nanopores, and offers a useful route to probe bilayer orientation of DNA nanostructures.