Surface Assembly of DNA Origami on a Lipid Bilayer Observed Using High-Speed Atomic Force Microscopy

Abstract

The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on a lipid bilayer surface. During the diffusion of DNA origami on the membrane surface, the formation of lattices and their rearrangement in real-time were observed using high-speed atomic force microscopy (HS-AFM). The formed lattices were used to further assemble DNA origami tiles into their cavities. Various patterns of lattice-tile complexes were created by changing the interactions between the lattice and tiles. For the control of the nanostructure formation, the photo-controlled assembly and disassembly of DNA origami were performed reversibly, and dynamic assembly and disassembly were observed on a lipid bilayer surface using HS-AFM. Using a lipid bilayer for DNA origami assembly, it is possible to perform a hierarchical assembly of multiple DNA origami nanostructures, such as the integration of functional components into a frame architecture.

Keywords: DNA nanotechnology; DNA origami; high-speed AFM; lipid bilayer; surface assembly.

Figure 1

Programmed DNA origami assembly systems. (a) Two-dimensional (2D) assembly systems. Nine different DNA origami assembly system using shape- and sequence-complementarity and AFM image of the 3 × 3 assembly (left). Two-dimensional lattice formation by assembling cross-shaped DNA origami using blunt end stacking interaction. (b) Three-dimensional (3D) assembly systems. Paired blocks can be connected and assembled in a programmed fashion via shape-complementary and blunt end stacking interaction.

Figure 2

Programmed assembly of DNA origami on a supported lipid bilayer. (a) Design of DNA-cholesterol barges. A rectangular DNA origami coated with cholesterol-conjugated anchor strands and Cy3-labeled strands. Cholesterol-conjugated anchors can interact with a lipid bilayer. (b) Time-lapse image of DNA barges in a DOPC/DOPE-mPEG lipid bilayer. (c) Oligomerization of DNA origami tiles induced by the addition of ssDNA linker strands. TIRF images before and after (150 min) addition of the linker strands. Intensity-weighted cumulative histogram of particle intensities 0–150 min after adding the linker strands. (d) Programmed assembly of DNA origami into 1D and 2D assembles by the addition of different connector strands during the DNA origami diffusing on the membrane. (e) AFM image of 2D DNA origami assembly on a DOPC lipid bilayer.

Figure 3

Assembly of DNA origami on a lipid bilayer surface. (a) Cross-shaped origami adsorbed and concentrated on the lipid bilayer surface was attached two-dimensionally (2D) by the stacking interaction between their blunt ends to form 2D lattice structures. (b) AFM images of the lattice assembled on the lipid bilayer. (c) Dynamic process of self-assembly visualized by high-speed AFM (HS-AFM). (d) Two-dimensional close-packed assembles formed with cross-shaped (left), triangle-shaped (middle), and hexagon-shaped (right) DNA origami.

Figure 4

Incorporation of square DNA origami (SQ) tiles into the cavities of a lattice on a lipid bilayer surface. (a) After the formation of a lattice by assembling cross-shaped origami, SQ tiles were introduced to fit into the cavities. (b) HS-AFM images taken after the SQ tiles are further adsorbed on the lipid bilayer surface on the formed 2D lattice. (c) Following the formation of a lattice by assembling cross-shaped origami with T₈-strands, SQ tiles with A₈-strands were introduced to hybridize into the cavities. (d) HS-AFM images after the SQ tiles are further adsorbed on the lipid bilayer surface on the formed 2D lattice. Scanning rate 0.2 frame/s.

Figure 5

Formation of a checkered pattern using AB-lattice and SQ tile. (a) Assembly of AB-lattice and incorporation of SQ tile into the specific cavities. T₈-strands introduced in Cross-A hybridize with A₈-strands introduced to SQ tile. (b) AFM image of AB-lattice. In order to distinguish Cross-A and Cross-B, Cross-A was modified with biotins to be labelled by streptavidin. (c) Incorporation of SQ tiles into the formed AB-lattice. AFM images of SQ-attached AB-lattice after incorporation of SQ tiles.

Figure 6

Formation of 2D assemblies on a phase-separated membrane. (a) Cross-shaped DNA origami was deposited onto a lipid membrane. (b) DOPC forms liquid-disordered (Ld) phase and DPPC forms solid-ordered (So) phases. AFM image of cross-shaped origami (scale bar: 100 nm) and phase-separated membrane on the mica surface. (c) AFM images of cross-shaped DNA origami adsorbed on DOPC/DPPC phase-separated membrane surface at different NaCl concentrations (0–300 mM). (d) Assembly of 3-point star tile (U) and cholesterol-modified tile (CHOL) on a DOPC and DPPC surface. (e) AFM images of assemblies of U tile (top), CHOL tile (middle), and U/CHOL mixture (bottom) on the DOPC/DPPC phase-separated membrane surface.

Figure 7

Direct observation of the assembly and disassembly of photoresponsive hexagon DNA origami. (a) Assembly and disassembly of hexagon origami carrying four photo-switching DNA strands by UV/Vis irradiation. (b) Dimer formation and dissociation of hexagon origami by alternating UV/Vis irradiation, which was quantified by gel electrophoresis. (c) Dynamic dimer formation and dissociation of hexagons in solution by alternating UV/Vis irradiation, which was characterized by fluorescence quenching. (d) Formation of ring-shaped and linear hexagon assemblies and their AFM images. (e) Observation of dynamic assembly and disassembly on a lipid bilayer surface by UV/Vis irradiation. (f,g) Direct HS-AFM observation of disassembly of the dimer on the lipid bilayer surface during UV irradiation (f) and assembly of the monomers during Vis irradiation (g). Arrows indicate the dissociated and associated hexagon origami. Scanning rate 1 frame/s.