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The dsDNA-NPs having complementary (i.e., pairing) outermost base pairs spontaneously aggregate under high-salt conditions, but not when their outermost base pairs are mismatched (unpairing). In this study, we used colloidal probe atomic force microscopy to examine how the outermost base pairs affect the interaction between the dsDNA-grafted layers (dsDNA layers). To precisely assess the subtle structural differences in the dsDNA layers, we developed a method for the formation of a homogenous dsDNA layer on gold surfaces using hairpin shaped DNAs. Homogenous dsDNA layers having complementary (G-C) or mismatched (C-C) outermost base pairs were grafted onto the surfaces of colloidal probes and gold substrates. Force distance curves measured in an aqueous medium under high-salt conditions revealed that the surface forces between the dsDNA layers were bilateral in nature and were governed by the outermost base pairs. Between complementary outermost dsDNA layers, the surface force changed from repulsive to attractive with an increase in the NaCl concentration (10-1000 mM). The attraction observed under high-salt conditions was attributed to the site-specific interaction proceeded only between complementary dsDNA terminals, the so-called blunt-end stacking. In fact, between mismatched outermost dsDNA layers, the repulsive force was mostly dominant within the same NaCl concentration range. 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The dsDNA-NPs having complementary (i.e., pairing) outermost base pairs spontaneously aggregate under high-salt conditions, but not when their outermost base pairs are mismatched (unpairing). In this study, we used colloidal probe atomic force microscopy to examine how the outermost base pairs affect the interaction between the dsDNA-grafted layers (dsDNA layers). To precisely assess the subtle structural differences in the dsDNA layers, we developed a method for the formation of a homogenous dsDNA layer on gold surfaces using hairpin shaped DNAs. Homogenous dsDNA layers having complementary (G-C) or mismatched (C-C) outermost base pairs were grafted onto the surfaces of colloidal probes and gold substrates. Force distance curves measured in an aqueous medium under high-salt conditions revealed that the surface forces between the dsDNA layers were bilateral in nature and were governed by the outermost base pairs. 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