NruI restriction enzyme

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Intact λDNA (0.3 μg mL−1, 3010, Takara, Shiga, Japan) shown in step 1 of Fig. 2 was first digested by a restriction enzyme, NruI (1168A, Takara), at 37 °C for one hour, providing six fragments (Fig. 2, step 2).	Fragments of 4.6 kbp and 6.7 kbp were purified for labeling with biotin and digoxin, respectively, using NucleoSpin Extract II (740609, Macherey-Nagel, Düren, Germany) after electrophoresis in 0.8% agarose gel. Each fragment was labeled at 37 °C for one hour in separate Eppendorf tubes following the user instructions for the nucleic acid labeling kits used, Label IT® Digoxin Labeling Kit (MIR3325, Mirus, Madison, WI, USA) and Label IT® Biotin Labeling Kit (MIR3425, Mirus), as shown in step 3 of Fig. 2. Fragments were purified from free digoxin or biotin by G50 microspin purification columns (Mirus).

Protocol tips
Intact λDNA (0.3 μg mL−1, 3010, Takara, Shiga, Japan) shown in step 1 of Fig. 2 was first digested by a restriction enzyme, NruI (1168A, Takara), at 37 °C for one hour, providing six fragments (Fig. 2, step 2).
Downstream tips
Fragments of 4.6 kbp and 6.7 kbp were purified for labeling with biotin and digoxin, respectively, using NucleoSpin Extract II (740609, Macherey-Nagel, Düren, Germany) after electrophoresis in 0.8% agarose gel. Each fragment was labeled at 37 °C for one hour in separate Eppendorf tubes following the user instructions for the nucleic acid labeling kits used, Label IT® Digoxin Labeling Kit (MIR3325, Mirus, Madison, WI, USA) and Label IT® Biotin Labeling Kit (MIR3425, Mirus), as shown in step 3 of Fig. 2. Fragments were purified from free digoxin or biotin by G50 microspin purification columns (Mirus).

Publication protocol

"For the self-assembled molecular configuration, two specific binding mechanisms are adopted: avidin–biotin and antigen–antibody bindings. The target λDNA molecule was modified as shown in Fig. 2. Intact λDNA (0.3 μg mL−1, 3010, Takara, Shiga, Japan) shown in step 1 of Fig. 2 was first digested by a restriction enzyme, NruI (1168A, Takara), at 37 °C for one hour, providing six fragments (Fig. 2, step 2).
Fragments of 4.6 kbp and 6.7 kbp were purified for labeling with biotin and digoxin, respectively, using NucleoSpin Extract II (740609, Macherey-Nagel, Düren, Germany) after electrophoresis in 0.8% agarose gel. Each fragment was labeled at 37 °C for one hour in separate Eppendorf tubes following the user instructions for the nucleic acid labeling kits used, Label IT® Digoxin Labeling Kit (MIR3325, Mirus, Madison, WI, USA) and Label IT® Biotin Labeling Kit (MIR3425, Mirus), as shown in step 3 of Fig. 2. Fragments were purified from free digoxin or biotin by G50 microspin purification columns (Mirus). Biotinylated fragment was first ligated to intact λDNA using T4 ligase (2011A, Takara), and this was followed by the ligation of the digoxin-labeled end. Each ligation process was performed overnight at 15 °C. The resulting DNA has a
length of 59.8 kbp (Fig. 2, step 4), and was labeled with 1 μM YOYO-1 (Y3601, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for fluorescent observations. To evaluate the DNA functionalization process, agarose
gel electrophoresis and membrane blotting were employed after each ligation process. Fully functionalized DNA was electrophoresed and transferred onto a membrane (Hybond N, GE Healthcare, Little Chalfont, UK) for biotin and digoxin detection by blue color development of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) to alkaline phosphatase. A DIG nucleic acid detection kit
(1175041, Roche, Basel, Switzerland) was used for digoxin detection, and streptavidin-alkaline phosphatase (21324, Pierce, Rockford, IL, USA) was used together with the kit for biotin detection by following the user instructions with some modifications. Tubulin (7 mg mL−1) was purified from four porcine brains. Part of the tubulin was labeled with tetramethylrhodamine (C1711, Molecular Probes, Invitrogen) and
Biotin XX, SE (B1606, Molecular Probes, Invitrogen) according to standard methods [27, 28]. Then the unlabeled tubulin was mixed with rhodamine-labeled tubulin and biotin-labeled tubulin in the ratio 9:3:4 and polymerized for 30 min at 37 °C to give partially biotinylated and fluorescent microtubules. Diluted microtubules (70 μg mL−1) were stabilized by paclitaxel (20 μM, T1912, Sigma, St. Louis, MO, USA) in BRB80 (80 mM PIPES, 2 mM MgCl2, 1mM EGTA, pH 6.8). His6-tagged kinesin was purified by Ni−NTA affinity (30210, Qiagen, Hilden, Germany). The kinesin sequence from Homo sapiens (amino sequence 1-573) was ligated
into the pET30b (Novagen, Madison, WI, USA) plasmid to give the His6-tag for the subsequent purification, and transformed into an Escherichia coli Rosetta(DE3)pLysS (Novagen). This construct was a gift from Prof. Y. Hiratsuka of the School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST). Protein concentrations were quantified using a Coomassie (Bradford) Protein Assay kit (23200, Pierce). Their activities were examined by in vitro gliding assay. "

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