Team kansai result

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Top Team Project Results Protocols photos

Contents

Stage

Fig. 5. AFM images of  (a) Stage,  (b) Convex DNA origami and (c) Stage + Convex DNA origami. (High resolution data of Stage + Convex DNA origami.)
Fig. 5. AFM images of (a) Stage, (b) Convex DNA origami and (c) Stage + Convex DNA origami. (High resolution data of Stage + Convex DNA origami.)

 First, We constructed the stage and the result is shown in Fig. 5 (a). As you can see, the stage was successfully constructed. However, most stages make complexes by blunt-end π–π stacking interactions. To suppress making complexes, thymine spacers were inserted in staple DNAs which is located at side of the stage.

Convex DNA origami

 Next, we tried to construct the convex DNA origami and the result is shown in Fig. 5 (b). Convex DNA origami was successfully constructed.

Stage + Convex DNA origami

 It was confirmed that Stage and Convex DNA origami were constructed. Next, we tried to combine them. The result is shown in Fig. 5 (c) and confirmed that Stage was combined with Convex DNA origami which width was observed ~17 nm. At first time, the yield was ~20% and a reason of the low yield is that the mixture was only incubated at room temperature without slowly annealing. After this, Stage + Convex DNA origami was constructed with slowly annealing from 40℃ to room temperature and the yield rose up to ~60%.

Agarose gel electrophoresis

Fig. 6. The result of electrophoresis. Lane 1, M13mp18 ssDNA. Lane 2, Stage. Lane 3, Convex DNA origami. Lane 4, Stage + Convex DNA origami. (Raw data)
Fig. 6. The result of electrophoresis. Lane 1, M13mp18 ssDNA. Lane 2, Stage. Lane 3, Convex DNA origami. Lane 4, Stage + Convex DNA origami. (Raw data)

 Formation of Stage + Convex DNA origami was confirmed by electrophoresis and the result is shown in Fig. 6. It was confirmed to construct Stage and Convex DNA origami because of the bands of Lane 2 (in red parentheses) and Lane 3 (in orange parentheses) have different electrophoretic mobility compared with Lane 1. A band having low mobility was observed in Lane 4 (in blue parentheses). It shows that Stage + Convex DNA origami was constructed.

Formation of a molecular spider in a ditch

Fig. 7. AFM image of formation of molecular spider in a ditch
Fig. 7. AFM image of formation of molecular spider in a ditch

 Then, we tried to form a molecular spider on the first scaffold.  Fig. 7 is the result of it. There was few molecular spider in a ditch. We thought that streptavidin might be spoiled.

Confirmation of forming a molecular spider

 We confirmed that streptavidin can bind with biotin by electrophoresis (Fig. 8). Lane 1 is the ratio of Streptavidin to biotinylated DNA equals the ratio of 1 to 1, Lane 2 is the ratio of 1 to 2, Lane 3 is the ratio of 1 to 3, Lane 4 is the ratio of 1 to 4. The higher the ratio of biotinylated DNA, the lower electrophoretic mobility the band became. This shows that streptavidin combines with biotinylated DNA regularly.

Fig. 8. PAGE analysis of streptavidin-biotinylated DNA conjugate.
Fig. 8. PAGE analysis of streptavidin-biotinylated DNA conjugate.












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