Covalent Positioning of Single DNA Molecules for Nanopatterning

Abstract

Bottom-up micropatterning or nanopatterning can be viewed as the localization of target molecules to the desired area of a surface. A majority of these processes rely on the physical adsorption of ink-like molecules to the paper-like surface, resulting in unstable immobilization of the target molecules owing to their noncovalent linkage to the surface. Herein, successive single nick-sealing facilitated the covalent immobilization of individual DNA molecules at defined positions on a dendron-coated silicon surface using atomic force microscopy. The covalently-patterned ssDNA was visualized when the streptavidin-coated gold nanoparticles bound to the biotinylated DNA. The successive covalent positioning of the target DNA under ambient conditions may facilitate the bottom-up construction of DNA-based durable nanostructures, nanorobots, or memory system.

Keywords: DNA ligation; DNA-based nanomachine; atomic force microscopy; covalent bond; dendron-coated surfaces; molecular writing.

Figure 1

Successive DNA ligation for covalently positioning single DNA molecules. (A) Scheme of four-step process: (Step 1) A free 47mer DNA (in blue) modified with a biotin residue (represented by the black triangle) linked at its 5′ end is hybridized to the complementary 71mer (c71mer) DNA (in gray) immobilized on the tip of the AFM cantilever in the DNA ligation solution; (Step 2) The AFM tip is made to approach a 24mer DNA immobilized on the dendron-coated surface to introduce a single nick between the 5′ end (that is, a phosphate group) of the 24mer DNA and the 3′ end (that is, a hydroxyl group) of the 47mer DNA. The nick can be sealed by the DNA ligase, which forms a new phosphodiester bond in the nick; (Step 3) After a pause of 30 s, the AFM tip is pulled upward to allow the hybridization of the c71mer DNA with a new free 47mer DNA. The direct immobilization of a single 47mer DNA is performed repeatedly at different points for DNA patterning; (Step 4) Prior to visualizing the nanoscale DNA patterns, the streptavidin-coated gold nanoparticles are employed to label the ligated DNA, taking advantage of the high affinity between streptavidin and biotin. The wide triangle (in green) on the bottom surface represents the 27-acid dendron molecules coated on the silicon substrate. (B) Representative three single-peaked force-distance curves for the unbinding events between the 24mer DNA on the 9-acid dendron-coated bottom surface and hybridized c71mer/47mer DNA on the 27-acid dendron-coated AFM tip (left) and those between the ligated 71mer DNA and the c71mer DNA (right).

Figure 2

Visualization of the positions of the 71mer DNA. (A) DNA positioning map: successive single DNA nick-sealing was performed at different positions in the 1.5 µm × 1.1 µm area to create an N-shaped pattern. The center of each square of the positioning map was made to coincide with the point at which the single DNA nick-sealing was validated. (B) Scheme for AFM force mapping: an AFM tip with the c71mer DNA scanned the surface of a square depicted in (A) to confirm the presence of the ligated 71mer DNA with the 5′-biotin. (C) Representative force map: the unbinding force (from 0 to 80 pN) recorded in each section of a square was indicated by the color scale. NS: non-specific unbinding force. (D) Force map obtained from the 2nd round of force mapping of the region dashed in red in (C). (E) SEM image of the streptavidin-coated AuNPs on the silicon substrate. (F) Overlap of the SEM image with the DNA positioning map. Scale bars: 500 nm in (E,F).

Figure 3

Evaluation of the DNA-AuNP complexes. (A) A single streptavidin-coated AuNP was imaged in the area surrounding a point where a single 71mer DNA with a terminal biotin moiety was covalently immobilized. The position of the DNA-AuNP complexes is indicated by the blue-lined square. (B) No AuNP was detected at a point where a single 71mer DNA was positioned. The position of a single DNA without AuNP is indicated by the blue-dotted square. (C) A AuNP was detected outside the DNA positioning map. The position of the AuNPs non-specifically bound to the silicon substrate is indicated by the gray-dotted square.