Asymmetric secondary and tertiary streptavidin/DNA complexes selectively formed in a nanometer-scale DNA well

Rectangular DNA motifs, which each have a built-in cavity in the middle, were prepared by bundling nine DNA helices into a U-shaped tile first and then assembling two of them together. The cavity was modified with two or three biotin molecules and was used as a nanometer-sized well to capture a single streptavidin (SA) tetramer size-selectively. Selective, one-step formation of asymmetric secondary or tertiary SA/DNA complexes via bidentate or tridentate capture have been shown by analyses of the complexes with denaturing polyacrylamide gel electrophoresis. When biotinylated DNA was simply added to the solution of SA, only symmetric SA/DNA complexes were formed as a mixture of primary to quaternary complexes, depending on the amount of DNA. However, when the DNA motif was preformed prior to SA addition, the asymmetric secondary complex was preferentially formed. The yield of the asymmetric secondary complex was ca. 50% when biotins were tethered to the two-turn well (6.8 nm wide) via 2.3 nm linkers, whereas the yield of the mixture of symmetric and asymmetric secondary complexes was only 7% for the reaction in bulk solution under the corresponding conditions. The larger the size of the well, the lower the yield became. Selective formation of asymmetric tertiary complex was also successful. Treatment of a SA tetramer captured in a well with a biotinylated, long dsDNA has shown that the captured SA still preserves its binding ability to additional biotinylated target.