Membrane Activity of a DNA-Based Ion Channel Depends on the Stability of Its Double-Stranded Structure

Diana Morzy, Himanshu Joshi, Sarah E. Sandler, Aleksei Aksimentiev, and Ulrich F. Keyser
Nano Letters 21(22) 9789-9796 (2021)
DOI:10.1021/acs.nanolett.1c03791  BibTex

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Structural DNA nanotechnology has emerged as a promising method for designing spontaneously inserting and fully-controllable synthetic ion channels. Earlier, we have shown that such membrane-spanning DNA-based channels can scramble lipids 10,000 faster than any known biological enzyme. However, both insertion efficiency and stability of existing DNA-based ion channels leave much room for improvement. Here, combining our MD simulations with the experiments performed by Keyser (Cambridge U., UK) lab, we demonstrated an approach to overcoming the unfavorable DNA-lipid interactions that hinder the formation of a stable transmembrane pore. In all-atom MD simulations, we observed that the DNA insertion-driving cholesterol modifications, when introduced at an end of a DNA strand, cause fraying of the terminal base pairs as the DNA nanostructure adopts its energy-minimum configuration in the membrane. The fraying of base pairs distorts nicked DNA constructs when embedded in a lipid bilayer. To avoid the undesirable distortion in the structure of DNA, we introduced a DNA nanostructures that do not have discontinuities (nicks) in their DNA backbones. This non-nicked DNA nanostructure leads to considerably more stable DNA-induced conductive pores and inserts into lipid membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows to design and maintain membrane-spanning helices in a tilted orientation within the lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

Abstract

DNA nanotechnology has emerged as a promising method for designing spontaneously inserting and fully controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based membrane channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA–lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications can cause fraying of terminal base pairs of nicked DNA constructs, distorting them when embedded in a lipid bilayer. Importantly, we show that DNA nanostructures with no backbone discontinuities form more stable conductive pores and insert into membranes with higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows the design and maintenance of membrane-spanning helices in a tilted orientation within the lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

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1 μs long equilibrium MD simulation of 4nm-2x structure. The complementary DNA strands are shown using turquoise and orange spheres, and the backbone of DNA is shown in tubular representation. The lipid bilayer is shown using turquoise lines and a representative carbon atom (C22) of the lipid headgroup is shown as turquoise and yellow spheres to distinguish the upper and lower leaflets, respectively. The cholesterol molecules are shown in green; water and ions are not shown for the sake of clarity.

1 μs long equilibrium MD simulation of 8nm-2x structure. The complementary DNA strands are shown using turquoise and orange spheres, and the backbone of DNA is shown in tubular representation. The lipid bilayer is shown using turquoise lines and a representative carbon atom (C22) of the lipid headgroup is shown as turquoise and yellow spheres to distinguish the upper and lower leaflets, respectively. The cholesterol molecules are shown in green; water and ions are not shown for the sake of clarity.

1 μs long equilibrium MD simulation of 8nm-0x structure, having a continuous DNA backbone.  The complementary DNA strands are shown using turquoise and orange spheres, and the backbone of DNA is shown in tubular representation. The lipid bilayer is shown using turquoise lines and a representative carbon atom (C22) of the lipid headgroup is shown as turquoise and yellow spheres to distinguish the upper and lower leaflets, respectively. The cholesterol molecules are shown in green; water and ions are not shown for the sake of clarity.