Biomod/2014/Kashiwa/Experiment

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  EXPERIMENTS

The Receptor

 Direction

We planned to do experiments in several steps. First, we design the Receptor. Then, the following experiments are conducted in parallel.

• Form the Receptor dimer by using thrombin.
• Emit the Polymerization Initiator by demerization.
• Embedding of Receptor on the liposome.


Forming the Receptor dimerEmiting the InitiatorEmbedding on the liposome

Finally, after success in the experiments, we combine them to attain the dimerization and the Initiator emission on the liposome.


 Experiments

 Folding the Receptor


Folding of the Receptor was corroborated by TEM and 1% agarose gel electrophoresis analysis.
The folded structure of the Receptor was clearly appeared in the TEM image.




 Forming the Receptor dimer

Before the experiment of forming the Receptor dimer with thrombin, we confirmed that thrombin-binding aptamers bind to thrombin. The molar ratio of the aptamers to thrombins was also optimized.

Binding of the aptamers with thrombin was analyzed with Native-PAGE.

The result showed that the aptamers should be added at least 10 times as many as alpha-thrombins for binding.


 Emitting the Initiator



 Embedding on the liposome



In order to certify the penetration of the Receptors to liposomes, a flotation assay was conducted.

In preparation for a floation assay, cholesterol oligomers were hybridized to the Receptors for penetration. The hybridization was confirmed by 1% agarose gel electrophoresis analysis.








In flotation assay, we divided mixture with the Receptors and the liposomes to a few fractions, each of them was analyzed by fluorescence spectrophotometer and 1% agarose gel electrophoresis. This assay showed in which fraction of the mixture includes the liposomes embedded by the Receptors.





The fluorescence intensity of NileRed in each fraction (NileRed dyes the membrane of liposomes.)

The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3.






The ratios of the Receptor in each fraction were analyzed by the density of band.

In sample1 (cholesterol + / liposome +), the ratio of the Receptor in fraction3 was a bit larger than that in fraction2. In contrast, in sample 2, 3 and 4, the ratio of the Receptor in fraction3 was smaller than that in fraction2. As liposomes were mostly seen in fraction3 in both sample 1 and 3, and the ratio of the Receptor in each fraction was different only in sample1, we concluded that this difference was caused by penetration of the Receptor to liposomes.



 Next Steps

In these experiments, we achieved to construct the Receptors and insert them into the liposomes. The very basics of others, formation of the Receptor dimers and emission of the Initiator, were also confirmed. Therefore, we are planning to approach the ones following for next steps.

• Confirm the more advanced mechanism of forming the Receptor dimers and emitting the Initiator.
• Examine conditions for dimerization of the Receptor heterounits on the liposomes, may need changes in design of the Receptor.

 



The Motor Monomer

 Direction


We planned to do experiments in several steps. First, we design the Motor-Monomer and assay the formation of a simple Motor-Polymer. Then, the following experiments are conducted in parallel.

• Control the initiation of the polymerization (forming ring structures and ring-opening polymerization).
• Put the Motor-Monomers into the liposome.


Controlling the polymerizationPutting into the liposome

Finally, after success in the experiments, we combine them to attain the polymerization in the liposome.


 Experiments

 Folding the Motor-Monomer

The assembly condition of the Motor-Monomer was optimezed with concentration of MgCl2, annealing temperature and time. The resulting structure was analyzed by agarose gel electrophoresis.


Multimers (e.g. dimmers) were appeared at lower temperature, so optimum temperature of annealing was 55.0℃.






(Annealing temperature : 55.0℃, M13 : staples = 1 : 2)
The band of monomer at 16mM is bending and its migration distance is different from the one of MoN. It shows that the Monomers were not annealed correctly at 16 mM. There seemed to be little difference at other concentration, so optimum concentration of MgCl2 was the range 10 to 14mM.




(Annealing temperature : 55.0℃, MgCl2 : 10mM)
At the ratio of 6 and 8 a band of a monomer is weak and migrating distance was shorter than the one of monomer. As the graph showed, the ratio of dimer to monomer was low in lane 1 and 2, so optimum ratio of staples to scaffold was 2 or 4.



Folding of the Motor-Monomer was corroborated by PAGE analysis. The folding, however, was not verified by TEM. We considered it was because of hollow structures of the Motor-Monomers.



 Forming the Motor-Polymer

In this experiment, the effect of arm number on polymerization was examined. Two samples, one has three arms and the other has one extruded from the body, was compared. The Monomers comprise two similar structures, the Motor-Monomer A and the Motor-Monomer B. The polymerization was confirmed by mixing the Monomer A and the Monomer B.

The ratio of dimers of three arms monomers roughly increased with time, while the ratio did not change significantly for one arm monomers. It confirmed the polymerization of the Monomers with three arms.



 Next Steps


© 2014 UTokyo Chem & Bio

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