Biomod/2014/Kashiwa/Motors

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EXPERIMENTS

<a name="background"> The Moving System: The Motor</a>

<img src="http://openwetware.org/images/5/57/Monomerexperiments.png" width="300px" height="170px" align="right" style="margin:10px;">

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.

  • <a href="#2-1">2-1. Production of the Motor-Monomer </a>
    • <a href="#2-1-1"> 2-1(a). Folding of the Motor-Monomer body </a>
    • <a href="#2-1-2"> 2-1(b). Synthesis of divalent SA </a>
    • <a href="#2-1-3"> 2-1(c). Equipment of the Motor-Monomer with divalent SA </a>
  • <a name="#2-1"></a>
  • <a href="#2-2">2-2. Deactivation and reactivation of the binding activity of streptavidin</a>
  • <a name="2-1-1"></a>
  • <a href="#2-3">2-3. Incorporation of the Motor-Monomers into the liposome </a>


2-1. Production of the Motor-Monomer

<a name="2-1-1"></a>

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

<img src="" width="200px" height="120px>

Fig.2-1(a)-1. Gel analysis of the Monomers annealed in different time.

<img src="" width="200px" height="120px>

Fig.2-1(a)-2. Gel analysis of the Monomers annealed in different temperature.

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.


<img src="" width="200px" height="120px>

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

<img src="" width="200px" height="120px>

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

The optimum results are as follows.

  • Concentration of MgCl2 : 15 mM
  • Temperature of annealing : 45.3 °C
  • Time for annealing : 5 hours.
  • Concentration of NaCl : 2.5 mM.


<img src="">

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

The folding is corroborated by the TEM image.<a name="2-1-2"></a>

</div>


2-1(b). Synthesis of divalent streptavidin

<img src="">

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.


<img src="">

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.<a name="2-1-3"></a>


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: <p class="menu">(i) Optimizing condition for click reaction

</p>

(i) Optimization of condition for click reaction

<img src="http://openwetware.org/images/e/e4/Korekashiwa.png"><img src="http://openwetware.org/images/5/53/Kore2Tetra.png">


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


The reaction time was then analyzed with the combination.


Fig.2-1(c)1. Reaction time

</div>

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. </p>

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

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.

<img src="http://openwetware.org/images/3/33/Gonzaclick2.png">

Fig.2-1(c)-4. Agarose gel labelled with Cy5.

<img src="http://openwetware.org/images/9/90/Gonzaclick.png">

Fig.2-1(c)-5. 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

<img src="http://openwetware.org/images/4/48/Inactivated-SA-cy3.png">

Fig.2-1(c)-6. Native-PAGE of oligos-SA labelled with Cy3.

<img src="http://openwetware.org/images/e/e1/Inactivated-SA-cy5.png">

Fig.2-1(c)-7. Native-PAGE of oligos-SA labelled with Cy5.


The photos of gel shows that the band of Cy3 (inactivated SA) and Cy5 (azide) appeared at the same palace. And FRET was observed in the lane of 45 h incubation. These results implies that Cy5-azide was attached to inactivated SA by click chemistry. →Alkyne modified inactivated SA can react with Cy5-azide. And rate of reaction reaches 60 % after 45 hours incubation.

<a name="2-2"></a>


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


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

<img src="">

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.


<img src="">

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

<img src="">

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.

<a name="2-3"></a>

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.

<img src="">

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

<img src="">

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.

<img src="">

Fig.2-1-3. Agarose gel electrophoresis showing existence of Qdots.

<img src="">

Fig.2-1-3. Agarose gel electrophoresis showing existence of DNA.


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.




<a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Receptor"><img src="http://openwetware.org/images/0/0c/ReceptorswitchKashiwa.png" onmouseover="this.src='http://openwetware.org/images/a/a3/Receptorswitch2Kashiwa.png'" onmouseout="this.src='http://openwetware.org/images/0/0c/ReceptorswitchKashiwa.png'" height="160px" width="240px" style="padding:5px 60px;"></a>


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