Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers

Abstract

DNA origami nanotechnology is being increasingly used to mimic membrane-associated biophysical phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, we used a set of elongated DNA origami structures with varying placement and number of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single-and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. Our results show that the location and number of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct physical properties of single-and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, we provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.

Modifying Membrane Morphology and Interactions with DNA Origami Clathrin-Mimic Networks

“…GUVs, comprising DOPC with 0.1 mol % fluorescently labeled 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (Atto488-DOPE), were prepared by electroformation in 52 mM sucrose. 16,17 Triskelion dimers with Alexa647 labels (Supporting Figure S1) were assembled in equiosmolar TE-MgCl 2 buffer, incubated for at least 10 min with the GUV suspension diluted 20-fold in the same TE-MgCl 2 buffer (Supporting Figure S14), and observed using confocal microscopy. Dimers were observed to bind to membranes homogeneously and diffuse freely (Figure 2A,B and Supporting Movie M1).…”

Hydrophobic Cholesteryl Moieties Trigger Substrate Cell–Membrane Interaction of Elastin–Mimetic Protein Coatings in Vitro

“…41 The augmented affinity to cell membranes with cholesteryl anchors was further reported with modified hyaluronan, 46 or DNA. 47 The differences in CTA groups on cellular uptake might be explained by the involvement of cells in lipid uptake. Cholesterol can interact with other proteins via both covalent 48 and noncovalent mechanisms, 49,50 thereby regulating protein stability, localization, 51 and activity.…”

Synthetic cell division via membrane-transforming molecular assemblies

“…In this methodology, DNA’s specific base-pairing and self-assembly properties are exploited to use it as a structural material to generate objects of pre-designed shapes [90]. Taking advantage of this programmability, DNA origami has successfully been employed to achieve membrane binding [91, 92] and transformation [93, 94]. For example, we have shown that variably curved DNA origami objects mimicking banana-shaped BAR domains, targeted to membranes via cholesterol anchors, can recognize and deform GUVs [94].…”