Stability of DNA-tethered lipid membranes with mobile tethers

Abstract

We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M.; Lowe, R. D.; Chan, Y-H. M.; Ganesan, P. V.; Boxer, S. G. J. Struct. Biol.2009, 168, 190-9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility, and patch membrane edge were also investigated. On the basis of these results, a model for the mechanism of patch destruction is developed.

Figure 1

Schematic diagram of DNA-tethered lipid bilayer patches formed by rupture of giant unilamellar vesicles on two different substrates [1]. In (A), the tethering DNA is covalently attached to the substrate and so the DNA tethers are not mobile (the lateral density can be controlled; the remainder of the surface is passivated with phosphate groups [1]). In (B), the tethering DNA is displayed on the surface of a supported lipid bilayer so the DNA tethers are laterally mobile. This design brings two fluid bilayers into close proximity defined by the length of the DNA tether (~ 8 nm for a 24mer hybrid; ~ 16 nm for a 48mer hybrid). While tethered lipid bilayer patches formed using immobile tethers are stable irrespective of their composition, those formed from lipids such as Egg PC using mobile tethers are not. See supplementary Figures S1 and S2 for mechanism of GUV tethering and patch formation.

Figure 2

Schematic diagram of the architecture used to create a 2nd tethered lipid membrane patch by GUV rupture on top of a first bilayer patch which is tethered using fixed DNA on the surface and whose composition can be varied at will (c.f. Figure 1A). The GUVs used to form the first story tethered patch display both the antisense sequence to bind the GUV and patch to the fixed DNAs on the surface (black DNA) and a second DNA sequence, orthogonal to the first, which is used to tether the second story GUV and patch (red DNA). In the example described in the text, the lower tethered bilayer is a 60:40 mixture of DPPC and cholesterol, while the upper bilayer is Egg PC. This strategy is used to test the dependence of the second story patch stability on DNA hybrid diffusion, but also is the basis for much more complex designs.

Figure 3

An example of the tethered lipid bilayer patch disintegration process monitored by fluorescence microscopy. A tethered lipid bilayer patch composed of Egg PC lipids and containing a small amount of Texas Red labeled lipid is formed by GUV rupture and spreading on a mobile supported bilayer as illustrated in Figure 1B. The time of formation is defined as t = 0 (see Figure S2). The initial area is outlined, and it is observed that the patch shrinks from this boundary over time. The bright spots outside of the patch are bound mid-size GUVs.

Figure 4

Comparison of a disintegrating patch formed with egg-PC and a patch formed with DPPC and cholesterol (30min after formed). While both of patches are formed on mobile DNA tethers, a DPPC/cholesterol patch is stable. The disintegrating EggPC bilayer patch of figure 3 (15min) is shown again for side-by-side comparison. Disintegrated parts of patch bilayer are illustrated as faded color.

Figure 5

The dissipation of DNA tethers when a tethered patch disintegrates. (A) The DNA is labeled by Cy5 so that the trajectory of DNA hybrids can be traced. EggPC GUVs containing Cy5 single strand DNA (see Materials and Methods) was used to form tethered bilayer patches on a supported lipid bilayer displaying complementary unlabeled DNA. The patch is visualized by the Cy5 fluorescence and is relatively homogeneous over the patch area. As the patch disintegrates, a halo of Cy5 intensity builds up around the patch, suggesting that the patch destruction is accompanied with lateral dissipation of DNA tethers. A scale bar corresponds to 10 μm. (B) Schematic illustration of the patch destruction process consistent with what is observed in (A) and the stability data summarized in Table 1. A part of the tethered lipid bilayer migrates away from the patch with DNA hybrid tethers