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The orientation of surface-grafted dsDNA assemblies relative to the surface depends strongly on the nature of the employed thiol anchor. This was shown by ssDNA capture probe strands of 20 bases grafted to a gold surface by three dithiane rings or a single mercaptohexyl group. The capture probe strands were hybridized to one end of complementary ssDNA strands (target) comprising 40, 60, or 80 bases (T , T , and T ). At the other end of the targets a fluorophore-labeled reporter probe ssDNA strand of 20 bases was hybridized. To stiffen the DNA assemblies, the targets T and T were further hybridized to ssDNA patches of 20 or 40 bases. Whether the fluorescence intensity, and thus the distance between surface and fluorophore, increases or decreases with increasing target length depends on the thiol anchor. Attempts were made to heal the nicks that are present in the formed dsDNA assemblies by ligation. For enzymatic ligation, the presence of a phosphate at the 5'-end of the reporter probe and a patch is required, which may also influence the fluorescence intensity.
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