Team kansai protocols2

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Top Team Project Results Protocols photos


 Staple DNA strands were purchased from Sigma Genosys (Hokkaido, Japan) and used without further purification.

Self-assembly of DNA origami (Smiley, Cyclops, Bear Trap, Plus shaped DNA origami)

 The formation of DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staple strands (20 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg buffer, 50 µL). This mixture was cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands.

Cyclops to Smiley DNA origami

 Staple strands (20 nM for each strand) in middle of Smiley DNA origami is added to Cyclops DNA origami solution. This mixture was cooled from 50˚C to 25˚C at a rate of -0.1˚C/min to anneal the strands. Cyclops DNA origami connected by stacking was inserted staple strands into the middle and transformed to Smiley DNA origami.

Trapping system

 Constructed Plus shaped DNA origami is added to Bear Trap DNA origami. This mixture was cooled from 50˚C to 25˚C at a rate of
-0.1˚C/min to anneal the strands. Bear Trap DNA origami will catch Plus shaped DNA origami and be transformed rectangular DNA origami.

Physically transforming DNA origami by kinesin

 Microtubules are one of the cell skeletal proteins and consisted with polymerized α and β tubulin dimers. Microtubules are ridgid hollow rods approximately 25 nm in diameter. Along the microtubule axis, tubulin heterodimers are joined end-to-end to form protofilaments. Kinesin, a molecular motor protein can walk along a single protofilament of microtubules toward plus end (β subunit-end) as a good carrier.
 Designed DNA origami is attached to kinesin with interaction between biotin-streptavidin interaction.
・Construction of flow cell
 Flow cells were prepared by placing a cover glass (18 × 18 mm2; MATSUNAMI) on a slide glass (26 × 76 mm2) equipped with a pair of spacers to form a chamber of approximately 4 × 18 × 0.1 mm3 (W × L × H) in dimension. Two cover-slips were attached each other with double-sided sticky tape as spacers.
・Preparation of polymerized Rhodamine-labbeled microtubule(MTs)
 Rhodamine-labeled MTs were obtained by polymerizing rhodamine-tubulin in a polymerization buffer (80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 1 mM guanosine-5′-triphosphate (GTP), 5% DMSO; pH adjusted to 6.8) incubating at 37 0C for 30 min (molar ratio of Tubulin/rhodamine was 1:1/10 The solution containing the MTs was diluted with motility buffer (80 mM PIPES, 1 mM EGTA, 2 mM MgCl2, 0.5 mg mL−1 casein, 1 mM DTT, 4.5 mg mL−1 D-glucose, 50 U mL−1 glucose oxidase, 50 U mL−1 catalase, 10 μM paclitaxel, and ~1% DMSO; pH 6.8).
・Preparation of kinesin-Ni NTA-Biotin
 Same volume of Unlabeled kinesin buffer (200 nM kinesin-1 in 12 mM PIPES, 200 mM NaCl, 2 mM EGTA, 5 mM MgCl2, 5 mM ATP and 10 μM Paclitaxel) and biotinyl Ni-NTA buffer (250 nM biotinyl Ni-NTA, 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2) were mixed and incubated for 30 min at r.t.
・Fluorescence microscopic observation
 Labbeled DNA origami or Rhodamine-labeled MTs in each assays were illuminated with a 100 W mercury lamp and visualized by epifluorescence microscopy by using a Plan Apo 60×/1.40 objective (Olympus). Images were captured using a cooled- CCD camera (Cascade II; Nippon Roper) connected to a PC.

AFM observation

 AFM imaging of DNA origami was performed on a E-sweep system (SII, Japan). The mixture (1 µL) was deposited on freshly cleaved mica, additional 1X TAE/Mg buffer (200 µL) was added, and the imaging was performed in the fluid DFM scanning mode with a BL-AC40TS tip (Olympus, Japan).

Agarose gel electrophoresis

 The assembled products were loaded into agarose gels (1.5% agarose in 1×TAE/Mg2+ aqueous buffer) and subject to gel electrophoresis at 100 V for one hour. The gel was stained with GelStar.