Vertical DNA Nanostructure Arrays: Facilitating Functionalization on Macro-Scale Surfaces

Abstract

The capability for varied functionalization and precise control at the nanoscale are significant advantages of DNA nanostructures. In the assembly of DNA nanostructure, the surface-assisted growth method utilizing double-crossover (DX) tile structures facilitates nucleation at relatively low concentrations on the surface based on electrostatic interactions, thereby enabling crystal growth over large areas. However, in surface-assisted growth, the geometrical hindrance of vertical structures on the DX tile structure surface makes it challenging to conjugate DNA nanostructures into fabricated surfaces. Here, the surface-assisted growth method was employed to extend the DX tile growth for forming vertical structure arrays on the substrate, providing attachment sites for functionalization on uniformly covered substrates at the macroscopic scale. Additionally, the spacing of the vertical structure arrays was demonstrated to be controllable through the strategic design of the repeating unit tiles that construct the DX crystals.

Keywords: AFM; DNA nanotechnology; aptamer; double-crossover tile; macro-scale; supporting substrate.

1

Schematics and AFM images of vertical structure array surfaces. (a) Schematic illustration of the DX tiles used in the experiments. The chains of different colors represent distinct nucleic acid strands. These strands form DX tiles, represented as hexagonal prisms. A DX tile without a vertical structure on the tile is depicted as a brown hexagonal prism, while a DX tile containing a vertical structure on the tile with an attachment site (8-nucleotide, shown in a red circle) is illustrated as an ivory hexagonal prism with a yellow cylinder (right). (b–d) The DX tile forms a higher-order crystal structure by the repetitive combination of unit-tiles according to the design. Red outlines show the unit-tiles of each higher-order crystal structure: (b) Two unit-tiles (2-tile): One brown and one ivory hexagonal prism; (c) Four unit-tiles (4-tile): Three brown and one ivory hexagonal prisms; (d) Six unit-tiles (6-tile): Five brown and one ivory hexagonal prisms. (e–g) AFM images of the crystal structure correspond to the schematic illustrations above. The black inset boxes illustrate the DX crystal structure according to the unit-tiles, while the yellow inset box displays a magnified AFM image to observe the DX crystal structures at the tile level (white scale bars: 200 nm; yellow scale bars: 30 nm).

2

Three different surface-assisted growth methods for vertical structure array surfaces. (a) Method I: Thermal annealing from 95 to 25 °C with DNA strands (colored chains) forming the DX crystal structure and the substrate (ochre-colored square). (b) Method II: Thermal annealing from 40 to 25 °C with separately prepared individual tiles (brown and ivory hexagonal prisms) assembling the DX crystal structure and the substrate. (c) Method III: Thermal annealing from 40 to 25 °C with prepared crystal structures (crystal consists of brown and ivory hexagonal prisms) and the substrate. (d–f) AFM images of the crystal structure correspond to the methods above. Inset bar graphs show the number of receptor attachment sites for each AFM image (scale bar: 100 nm).

3

Characterization of TBA15 functionalized vertical structure arrays on the surfaces. (a) Schematic image of the thermal annealing process of the TBA15 array. AFM images of the TBA15 arrays on the (b) 2-tile, (c) 4-tile, and (d) 6-tile surfaces, respectively. The black inset boxes illustrate the DX surfaces and the dots on an ivory hexagonal prism represent the attached TBA15 on the DX tile. (e) Bar graphs of the average heights of vertical structures, each with an attachment site and TBA15s attached vertical structures. (f) The average of 50 measured distances between nearest-neighbor TBA15s in each direction, like those indicated in the yellow inset box in (b). (g) The average number of TBA15s in 0.25 μm2 areas. (white scale bars: 200 nm; yellow scale bars: 50 nm).

4

Characterization of the fluorescent signal from TBA15-functionalized surfaces. (a) Schematic depiction of TBA15, including unpaired nucleotides for hybridization with an attachment site and modifications for selective fluorescence emission. The red-colored sequences represent the hybridization domain (8 nt) complementary to the sequences of an attachment site of vertical structure arrays. The green-colored sequences represent the TBA15 sequences (15 nt), with a fluorophore (TAMRA) modification. The gray-colored sequences represent the detachment strand (6 nt), with a quencher modification at the 3′-end. Once thrombin binds to TBA15, it releases out the detachment strand with the quencher (red dashed box). (b) Fluorescence intensity of TBA15-functionalized surfaces for each unit-tile and method, with thrombin at a concentration of 200 nM. Each orange-colored bar graph indicates the quenched fluorescence intensity under the given conditions. (c) Each bar graph corresponds to the fluorescence intensity of a single functionalized surface following a unit-tile design. The coefficient of variation (CV) is shown for each unit-tile surface.