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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.