Biomod/2014/Kashiwa/Motor

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- EXPERIMENTS

 The Moving System: The Motor

This section describes the details of our approach to develop the moving system "Motor" of PoLICe. The approach mainly consists of three steps as follows.


2-1. Production of the Motor-Monomer

Producing the Motor-Monomer is divided into three sections: Folding the Motor-Monomer body, synthesizing divalent streptavidin and equipping the Motor-Monomer body with divalent streptavidin.

2-1(a). Folding of the Motor-Monomer body

Fig.2-1(a)-1. Gel analysis of the Monomers annealed in different time.
Fig.2-1(a)-2. Gel analysis of the Monomers annealed in different temperature.Fig.2-1(a)-3. Gel analysis of the Monomers annealed in different concentration of NaCl.Fig.2-1(a)-4. Gel analysis of the Monomers annealed in different concentration of MgCl2.

In this experiment, the assembly condition of the Motor-Monomer structure was optimized and results were analyzed by agarose gel electrophoresis. The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.

Fig.2-1(a)-5. TEM image for the Motor-Monomer.


The optimum results are as follows.

  • Concentration of MgCl2 : 15 mM
  • Temperature of annealing : 49.1 °C
  • Time for annealing : 2 hours.
  • Concentration of NaCl : 2.5 mM.


The folding is corroborated by the TEM image (in fig.2-1(a)-5.)


2-1(b). Synthesis of divalent streptavidin

Fig.2-1(b)-1. SDS-PAGE showing purified divalent SA.


In this experiment, divalent streptavidin (SA) was synthesized for equipment to the Motor-Monomers.

TaKaRa Competent Cell BL21 was used for protein expression. The purification of divalent SA on the nickel-affinity column was analyzed by SDS-PAGE.


Fig.2-1(b)-2. SDS-PAGE showing the affinity of divalent SA with biotin-modified oligos.


The affinity of divalent SA with biotin-modified oligonucleotides was then analyzed by SDS-PAGE.


2-1(c). Equipment of the Motor-Monomer with SA

We used click reaction, alkyne-azide huisgen cycloaddition, to equip the Motor-Monomer with SA. The click reaction was evaluated by the following steps:

(i) Optimization of condition for click reaction

Fig.2-1(c)-1. Optimum combinations of click reaction.

First, the optimum combination of click reaction between simple alkyne and azide was analyzed. two pairs out of 16 pairs were consequently chosen as the optimum combinations.

Fig.2-1(c)-2. Analysis of click reaction by Native-PAGE (16 pairs.)


Fig.2-1(c)-3. Analysis of click reaction by Native-PAGE (4 pairs.)


Fig.2-1(c)-4. Analysis of click reaction by Native-PAGE (2 pairs.)


Table 2-1(c)-1


Fig.2-1(c)-5.Relation between time and rate of reaction


In this experiment, as the reaction was regarded as pseudo-first-order reaction, trendline was fitted and kon was gotten. There were few difference between 2pairs in reactivity.


(ii) Evaluation of biotin-binding activity of divalent SA

Fig.2-1(c)-6. Analysis of NHS reaction by Native-PAGE (Cy5).


Fig.2-1(c)-7. Analysis of NHS reaction by Native-PAGE (SYBR Gold).


Figure 2-1(c)-1 shows that SA was linked with Cy5-modified oligonucleotide via click reaction. In Fig.2-1(c)-2, because the lane of only Cy5 and NH2-modified oligonucleotide is not stained, the stained bands is from biotin-oligonucleotide. This picture shows modified SA can bind biotin. It may show the activity to bind biotin decreases as the amount of modification of SA increases, but the experiment of modification SA with Cy5-NHS deny. The reason of that may be derived from steric hindrance of Cy5-oligonucleotide which binds to SA.

(iii) Analysis of click chemistry of alkyne and azide-modified Motor-Monomer

Click reaction between azide attached to Motor-Monomer and alkyne was confirmed as a preliminary step of modification of Motor-Monomer with inactivated SA.

Fig.2-1(c)-8. Agarose gel labelled with Cy5.


Fig.2-1(c)-9. Agarose gel stained with EtBr.


The figure shows that the band of Cy5 and Motor-Monomer in lane of 30 h are in the same place. It means that alkyne-Cy5 oligo was attached to Motor-Monomer by click reaction.

(iv) Analysis of click chemistry of azide and alkyne-modified deactivated SA

Fig.2-1(c)-10. Agarose gel labeled with Cy3.


Fig.2-1(c)-11. Agarose gel labeled with Cy5.


Fig.2-1(c)-12. Agarose gel labeled with EtBr.


The gel images show that the band of Cy3 (inactivated SA) and Cy5 (azide) appeared at the same position . And FRET was observed in the lane of 45 h incubation. These results indicates that Cy5-azide was attached to inactivated SA by click chemistry.

It therefore suggests that alkyne-modified deactivated SA can react with Cy5-azide. And rate of reaction reaches 60 % after 45 hours incubation.

These figures confirm the activity of modified SA to bind biotin-nucleotide.


2-2. Deactivation and activation of the binding activity of streptavidin

Fig.2-2-1. Mechanism of this experiment.


In this experiment, deactivation and reactivation of streptavidin (SA) were confirmed by Native PAGE.

First, two complementary oligonucleotides having biotin or desthiobiotin were annealed. The oligos having biotin is named Linker and the oligos having desthiobiotin is named Blocker. The resulting dsDNA was then mixed with divalent SA for deactivation. After removing excess SA with biotin-beads, ds-DNA bound to divalent SA was cut by HindⅢ for reactivation. Another oligonucleotides having biotin, named Chaser, were then added to replace Blocker.

Blocker was labeled with Cy3 and Chaser was labeled with Cy5 to confirm the replacement by Native-PAGE.


Fig.2-2-2. Cy3 fluorescence image of Native-PAGE. Fig.2-2-3. Cy5 fluorescence image of Native-PAGE.

In fig.2-2-3, multiple bands of SA-dsDNA complexes appears in lane 3 which is caused by non-ideal complexes. It is considered that biotin-beads successfully distinguished the ideal SA-dsDNA complex because the upper band in lane 3 mostly disappeared in lane 4.

In fig.2-2-4, a band at the same position of the ideal SA-dsDNA complex is only shown in lane 7. We therefore confirmed the replacement of Blocker with Chaser after cutting dsDNA by HindⅢ and that the replacement did not happen without HindⅢ.


2-3. Incorporation of the Motor-Monomers into the liposome

In this experiment, Motor-Monomers which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV). Inclusion of Motor-Monomers was observed by confocal microscope.

GUVs were dyed by Nile Red. Yellow green dots indicate the fluorescence of Nile Red and red dots indicate the fluorescence of Q-dots combined with Motor-Monomers.

Fig.2-3-1. Confocal microscope image of GUV including Motor-Monomers.


Fig.2-3-2. Confocal microscope image of GUV not including Motor-Monomers.



Comparing the two pictures, we can see the red dots inside the GUV only in the picture showing the GUVs containing Motor-Monomers in the inner solution. From this fact, we could confirm the inclusion of Motor-Monomers into the GUV. As the excitation and fluorescence wave length of Nile Red and Qdots were close, the membrane of GUV was shown in red even in the picture of GUV not including the Q-dots.


The connection of Qdot to the Motor-Monomer was then confirmed by the agarose gel electrophoresis.

Fig.2-3-3. 1% agarose gel electrophoresis of Motor-Monomers and Q-dots(Cy5)


Fig.2-3-4. 1% agarose gel electrophoresis of Motor-Monomers and Q-dots(EtBr)


Fig.2-3-5. 1% agarose gel electrophoresis of Motor-Monomers and Q-dots(EtBr)



The first picture shows the existence of Qdots and the second picture shows the existence of DNA. Comparing the fourth, fifth and sixth lane from the left in the first picture, we can see the difference in the position of the Qdots. Comparing the fifth and sixth lane from the left in the two pictures, we can see the position of DNA, in this case, Motor-Monomers, and the Q-dots in the same position. This data shows that the Qdots connected to the Motor-Monomers through the connection of biotin and streptavidin.








2-4. Polymerization in solution

Polymerization of the Motor-Monomers to the Motor-Polymer was confirmed as a preliminary step of polymerization in a liposome.

Fig.2-4-1. Agarose gel stained with EtBr shows the dimer band.


Fig.2-4-2. Ratio of the Monomer and the Dimer.


Fig.2-4-3. Relationship between polymerization degree and ratio of Polymer.




Fig.2-4-4. TEM image shows tetramer of the Motor-Monomers.



Fig.2-4-5. TEM image shows trimer of the Motor-Monomers.



Figure 2-4-1 and fig.2-4-2 show that the Motor-Monomer polymerized into the Motor-Polymer when streptavidin were added into Motor-Monomer solution. TEM analysis suggest that the Motor-Monomer without streptavidin were not polymerized in solution, while polymerized upon SA addition, trimer and tetramer were observed in SA(+) sample.

The results suggests that the polymerization were proceeded in solution by adding divalent SA.

© 2014 UTokyo Chem & Bio

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