Flat 2D Wireframe DNA Origami

Two-dimensional (2D) DNA origami is widely used for applications ranging from stimulators to single-molecule biophysi.

Traditional single-layer 2D DNA origami exhibits flexibility and bendability in solution;However, this may limit its applicability as a template for 2D structur.

In contrast, 2D wireframe DNA origami is rendered with six helical bundle edges, providing local control over dual orientation, enhancing in-plane rigidi.

Here, we investigate the three-dimensional structure of these assemblies using cryo-electron microscopy (cryo E.

3D reconstruction shows that the solution is highly planar and homogeneous for polygonal objects with or without internal meshes, enabling a resolution of 10° of triangl.

Coarse-grained simulations are consistent with cryo-EM data, providing insights into the molecular structure of this class of 2D DNA origa.

Our findings suggest that these assemblies may be valuable for 2D material applications and geometries that require high structural fidelity as well as local control over dual orientation, rather than parallel dual assemb.

Introduction

DNA origami was invented by.

Rothemund in 2006 (

1) by drawing three-dimensional two-dimensional (2D) geometric figures in DNA, including squares, triangles, stars, and smiley fac.

In the first implementation, straight-parallel DNA duplexes are interconnected with crossed anti-parallel DNA strands that are hybridized into hundreds of shorter synthetic short strands from long scaffold strands from the M13 phage geno.

This straight-line or brick-like fabrication strategy is powerful because it provides direct bracket string routing and binding sequence design manually or using simple computer-aided design tools (2), making the technique widely available to non-exper.

However, since these 2D origami objects are almost entirely visualized on 2D surfaces with atomic force microscopy (AFM) or transmission electron microscopy (TEM), although objects are bent, bent or twisted in 3D solution, they are usually seen is flat (three–5
Although 3D structure prediction tools such as CanDo (6) is used to reduce out-of-plane deformation (7–9), experimental verification of flatness remains elusive, and general design rules for obtaining and maintaining the flatness of arbitrary 2D geometri.

In recent years, the planarity of single-layer parallel-duplex 2D DNA origami has been studied and controlled (10,11), the 3D structure of these components in solution remains elusive, probably because of their flexibility and heterogeneity in solution, as confirmed by solution scattering data (10) and atomic force microscopy (11) An exception is the recent 3D cryo-electron microscopy (Cryo EM) study, which revealed parallel-organized single-layer duplex rectangular origami with remarkable flexibility and curvature, originally implemented by Rothemund (1) and in the same report, the 3D structure of rigid and substantially uniform multilayer brick-like origami was reconstructed to nucleotide resolution (12

Although difficult to achieve and verify experimentally, the planarity of 2D origami is crucial for numerous applications seeking to organize secondary materials with nanoscale precision (

13,14) including basic research on light harvesting and radicals (15–18), single molecule (19–twenty one) and super-resolution imaging (19,twenty two), molecular biophysics (twenty three), photonics (twenty four) Cell Biophysics (25–28) and surface-based patterning and lithography (29,30
Multilayer honeycomb (31) and the square (32) Brick-like origami designs offer an alternative to making single-layer 2D origami, but they achieve planarity while reducing the overall lateral size of objects that can be rendered due to the increased length of the scaffolds required;They may require careful sequence design to reduce or eliminate inherent distortions through structural simulations and iterative feedback from experiments (6,8,9,32), they are largely limited to drawing straight-line geometry consisting of parallel duplexes throughout the object, and (33) or without bending (1,34
Attachment of 2D monolayer origami to surfaces using high-affinity ligand-receptor pairs can be used for partially flat objects, although experimental validation is again challenging due to the perturbed nature of AFM and the low contrast of TEM, and not applicable for many applications for this biochemical fixation strate.

Two-dimensional wireframe DNA origami as an alternative to linear, brick-like origami has recently emerged as an alternative to localize secondary materials in 2D with nanoscale precision and local orientation control over biaxial (

35,36
In contrast to traditional rectilinear brick origami, wireframe geometry can render polyhedral geometry that cannot be accessed by rectilinear duplex components (35,36);They reduce the overall scaffold length required to render a two-dimensional object of a given lateral dimension, because they have an open mesh structure that minimizes the use of DNA, and they also provide bidirectionality that may be required in some applications The local directional control of ,such as organizing chromophores to control molecular excitons (37–39) and photonics (30

The fully automated sequence design tool METIS (Mechanical Augmentation and Three-layer Origami Structures) provides in principle the ability to render flat 2D wireframe objects using multilayer, six-helix bundle (6HB) edges, although so far,

Experimental characterization is limited to 2D imaging, which likewise suffers from potential artifacts from 2D solid supports (35) Here we report the results of 3D cryo-EM to resolve the first example of a planar 2D DNA origami structure using a 6HB wireframe design with lateral dimensions up to 80 .

To test the width and variety of manufacturable 2D objects, we investigated two different types of wireframe objects, polygonal objects with internal structure and polygonal objects without internal structu.

Furthermore, to determine the size of objects that can be rendered in this way, we varied the side lengths from 42 to 210 base pairs (b.

3D reconstructions show that our method can yield homogeneous, planar objects with a resolution of 10 to 18, suggesting minimal structural deviations from flatness, with lateral dimensions up to 80 .

This type of flat DNA origami is made from top to bottom using the fully automatic sequencing algorithm METIS (35) is implemented in the graphical user interface (GUI) ATHENA (40) provides a wide range of applications for the design and patterning of 2D nanomateria.

result

Computer Aided Design of 2D Origami

2D wireframe DNA origami structures based on 6HB edges were drawn using MET.

To study the planarity of origami objects, we chose different target plane geometries, including hexagons, pentagons, and symmetrical and asymmetrical triangl.

To evaluate the effect of internal meshes on flatness, we plotted hexagons and pentagons with and without internal wireframe support (Table S
Finally, as before, generate pentagons with different side lengths (40) explores the effect of lateral dimension on the obtained flatne.

Flatness of 2D Wireframe Origami

The METIS algorithm exploits the 6HB edge motif to obtain the structural rigidity of the edge and the maximum number of vertex crossings between each duplex in adjacent edges, giving the entire object overall stiffness and ideal flatness (

figure 1
While this multilayer design with multidirectional vertex connections has previously been shown to enhance in-plane structural integrity compared to DX (double-crossover) based objects consisting of only two duplexes per edge,In this work, AFM and TEM characterization could not determine the out-of-plane deformation and flatness of the assembled structure in solution (35

Using different target boundary geometries, including hexagons and pentagons, we first use Cryo-EM to evaluate the flatness of these objects with and without internal meshes (

figure 2) in order to improve the in-plane structural fidelity of the target 2D wireframe structure, it was verified by AFM and TEM (35) cryo-EM imaging and 3D reconstruction confirmed that, regardless of the presence or absence of an internal grid, the object not only has precise interior angles, but also remains flat in the range of ~6 nm in its lateral 80 nm dimension (figure 2) hexagons and pentagons have an internal mesh structure with a minimum side length of 84 bp, while the corresponding hollow structures specify a minimum side length of 106 bp for hexagons and 122 bp for pentagons, to obtain the same 80 nm diamet.

as shownFigure 2ADuring the imaging process, the hexagonal internal mesh structure directly opposite our design is consistent with our design, which is consistent with its AFM and TEM images (35), however, these structures are now frozen in solution without any surface confineme.

Individual DNA structures in different orientations are also identifiable in vitreous ice, the planarity of which is evident in different orientations of 2D objects, most notably on vertically oriented 2D objects (F.

S

Similar observations were made for pentagonal structures with internal meshes (F.

S3), as well as hexagonal and pentagonal structures without internal mesh.

As sho.

For S4 and S5, both hollow hexagons and pentagons are planar in solution and appear as a straight line when oriented vertically in cryo-EM imagi.

While most objects are planar at the single-particle level, occasionally, structures also show some curvature (Figs S2 to S
However, the single-particle inhomogeneity is low enough that it is easy to generate 2D class averag.

In addition to enabling the planarity of complex wireframe origami objects, these 3D reconstructions provide the first reported examples of 2D cryo-EM-DNA origami structures that are 80 nm in size to our knowled.

In another study it was observed that objects rotated along one axis showed not only flatness but also lack of overall distortion (12
While most of the 6HB edges in our structure show no twist, the inner mesh of the pentagon object does show a left-handed twist, which is evident on the inner spoke edg.

This is consistent with the correlation coefficient of the pseudoatomic model with the density map: the correlation coefficient of the pentagon structure of the inner mesh is 75, which is lower than that of the hexagonal structure of the inner mesh (8
For hollow hexagons and pentagons, the correlation coefficients were 84 and 83, respectively (Table S
The resolutions of these four objects are also comparable, the inner meshes of the hexagon and pentagon are reconstructed to 18 and 17 respectively? The resolution of the two hollow structures is reconstructed as 16? resoluti.

Influence of edge length on flatness

For the flatness obtained by wireframe origami without an internal mesh, we choose a pentagon as the model geometry and investigate whether changing the edge length affects the flatne.

Comparing the minimum edge length pentagon structures of 84 and 122 bp shows that flatness remains unchanged despite changes in edge length, based on Fourier-Shell Correlation (FSC) curves with a resolution of 16(Figure 3Afi.

S5 and S
For both structures, the generated pseudo-atom model predictions (Protein Database or PDB files) agree well with the density maps, with a correlation coefficient of 83, and no significant edge distortions regardless of edge leng.

Because the difference in edge length between the two structures is not an integer multiple of the number of DNA helix turns, the pattern of pin crossings at the vertices of the two pentagons is different, although the vertex angles of the two objects are the same (F.

S
The fact that the resolution, model fit, and quality of the two cryo-EM structures are identical suggests that our vertex design parameters are generally val.

A discrete advantage of using 6HB edges compared to single DNA duplexes or DX-based edges is that their relatively large persistence length of 1 to 2 μm correlates with their rigidity (41,42), which is critical for the structural integrity of wireframe DNA origami, especially the hollow structures fabricated he.

In order to further study the change of edge length, five pentagon structures with side lengths ranging from 42 to 210 bp (14 to 71 nm) were characterized by cryo-electron microscopy (Figure 3B) of smaller size pentagons with side lengths of 42–126 bp can be easily adapted to different orientations in vitreous ice, and their planarity can be observed in cryo-EM imagi.

In contrast, pentagons with an edge length of 168 bp are essentially regular, although they adopt fewer apparent orientations during imaging due to their larger size (~100 nm in diamete.

When the edge length reaches 210 bp, kinks and bends are observed at the edges and vertices of the pentagon structu.

According to the cryo-EM imaging results, the upper limit of the outer dimension of METIS is ~100 nm to achieve planar targets with precise inner target angles, The edge length is ~60nm (Figure 3B

Rendering symmetrical and asymmetrical objects

To test whether asymmetric objects can maintain flatness, we examined asymmetric and symmetric triangles with a maximum side length of 84bp, the overall resolution of the reconstruction was 11 and 13°, and the flatness was again maintained with the target interior angle

Figure 5) The oxDNA2 coarse-grained model is used because it accurately represents the thermodynamic and mechanical properties of DNA, and at the same time it enables long time simulations with lower computational cost compared to the classical all-atom model (44,45

twenty three), the activator (15–18), photonics (twenty four), and plasmonics (14) in other respec.