Elena Georgiou: DNA Origami – Lipid Membrane Interactions Controlled by Nanoscale Sterics

 While DNA nanotechnology excels at precisely tuning the shape and dimensions of 2D and 3D nanostructures spanning from the nanoscale[1-6] to the macroscale,[1, 7, 8] semifluid lipid bilayers stand out by compartmentalizing hydrophobic environments,[9] setting up concentration gradients for energy conversion,[10] and providing lateral diffusive platforms for enhanced molecular assembly.[11] Combining DNA nanotechnology with lipid membranes can unlock considerable synergy as illustrated by a range of DNA nanostructures that can bind to membranes,[12-15] mimic cellular cytoskeletons,[16] shape membranes into biologically unprecedented forms,[2-5] define lipid domains,[17] selectively label leaflets,[18, 19] measure membrane curvature,[20] and sort liposomes by size.

[21] The hybrid approach also helps study lipid exchange between membrane bilayers,[22] spatially activate membrane proteins,[23, 24] tune endosomal uptake,[25] sense intracellular interactions,[26] facilitate macrostructure assembly,[27, 28] and even help produce synthetic protocells and proto-tissues.[29-32] In complementary approaches, DNA nanostructures can be inserted into lipid membranes to emulate the function of membrane proteins, including receptors,[33] nanopores,[34, 35] gated channels,[36] membrane force sensors,[37] and lipid flippases.[38] Designing bilayer-interacting DNA nanostructures hinges on attached hydrophobic anchors that insert into the lipid bilayer.[39, 40] Cholesterol is the most prominent anchor, yet tocopherol,[41] porphyrins,[42] alkyl chains,[40] and polypropylene oxide have also been successfully used.[43]

Understanding DNA-membrane interaction is of fundamental scientific relevance but also helps guide rational engineering of DNA nanostructures. Several studies have investigated how cholesterol-mediated anchoring depends on temperature, lipid composition, the number of cholesterol anchors, and buffer conditions.[44-49] However, very few studies[44, 49] have explored the impact of nanoscale 3D steric and geometric effects even though these factors principally contribute to ligand-binding specificity in both chemistry and biology[50-52] and are likely key for controlling DNA nanostructure binding to the lipid bilayer. Unresolved questions are how the DNA nanostructure-membrane interactions depend on the nanoscale steric accessibility of the available anchors in a 3D DNA nanostructure.

A related question is how the interaction is influenced by the nanoscale membrane morphology in terms of global vesicle curvature but also the irregular local curvatures which can result from non-homogenous lipid composition.[53] Ideally, these questions should be addressed with ensemble techniques for efficient throughput but also complemented with single-molecule analysis to obtain further insight into the binding mechanism. A comprehensive understanding of the influence of nanoscale steric factors would be of scientific value and help identify design rules for efficient membrane interaction and nanostructure engineering, such as for size-specific vesicle discrimination to identify diagnostically relevant exosomes.[54, 55]

Here, we examine how nanoscale factors influence DNA nanostructure-membrane interactions. We devise a T-shaped DNA nanoprobe (DNP) structure with prominent nanoscale geometry featuring a baseplate and a tip (Figure 1A). The 3D geometry of the DNP offers different steric environments, from flat to highly recessed. Reflecting its modular design, DNP is also a highly addressable 3D breadboard to place the cholesterol membrane anchors into the various steric environments, which is ideal for probing the influence of nanoscale geometry on membrane binding. To cover the range of accessibilities, we use 20 different DNP variants, which are organized into groups 1–4, each comprising up to five cholesterol tags (Figure 1B, DNP-1 to DNP-4, top row; Figure S1, Supporting Information).

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