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<div id="header"><h1><p style="background:#CCFFFF; color:purple;"><font face=cursive size="6"><B>  Team FIT </font> </B></p></a></h1></div>
         


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<td cellspacing="10" cellpadding="10" width="1055" width="180" height="60" bgcolor="#BAD3FF"><a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka"><B><font face=cursive color="#003366" size="3">Top</font></B></a></td>
<td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Introduction.html"><B><font face=cursive color="#003366" size="3">Introduction</font></B></a></td>
<td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Approach and Goals.html"><B><font face=cursive  color="#FF6633" size="3" >Approach and Goals</font></B></a></td>
<td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Method.html"><B><font face=cursive color="#003366" size="3">Method</font></B></a></td>
<td cellspacing="10" cellpadding="10" width="1300" width="180" bgcolor="#BAD3FF"><a href="fit_Results and Discussion.html"><B><font face=cursive color="#003366" size="3">Results and Discussion</font></B></a></td>
<td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Member.html"><B><font face=cursive color="#003366" size="3">Member</font></B></a></td>
<td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Sponsor.html"><B><font face=cursive color="#003366" size="3">Sponsor</font></B></a></td>    </tr></table>


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<h2 p style="background:#FFFFFF; color:#003366;"><font face=cursive size="5"><B> Approach and Goals </font></B></p></h2>


<a name="header"></a>
<img src="/images/4/46/Fitaologo.PNG" alt" alt="" width="422" height="98" hspace="0" align="left">


<div style="padding: 35px;">
<hr>
<br>




<h3><p>&nbsp;We paid attention to complementary base pair of DNA Single strand DNA of two can be connected even if I make a mistake, and base pair matches it to some extent. The fewer and fewer mismatched base pair, Cohesion is high. We tried to construct the Pop-up Pirate using DNA and porous silica particle.  <br>
<ul id="dropmenu">


<p>
  <li><a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka#home">Home</a>
<table align="left">
<td><div style="width: 330px;"><img alt="" src="/images/c/c7/Dnakurohige.gif" width="320" height="240" border="0" / ><p>(*^_^*)</p><div></td><tr>
<td><div style="width: 330px;"><img alt="" src="/images/b/b7/Fitfiani.gif" width="320" height="240" border="0" / ><p>(*^_^*)</p><div></td></table>


&nbsp;Pop-up Pirate is one of the famous Japanese toy .Players bury a sward into the barrel. When the player bury into the specific position, the doll will be fly away. The barrel is constructed with the single strand DNA binding porous silica particle. Complemental DNA is a roll of the doll. But there are same mismatch between barrel and doll DNAs. Fully complement single strand DNA in a sword. Firstly, doll DNA is mixed with the barrel. Then doll DNA is bound to the barrel with complementally base pairing. Next, sward DNA is added to the solution. The doll DNA would be released firm the barrel because affinity of the sward DNA and the barrel .DNA is strange than that of the doll and the barrel DNAs. At looks like the doll fly away from the barrel.  <br>
    <ul>
&nbsp;In order to detect the different state of DNA binding, FRET system is used. The barrel DNA is labeled with fluorescent bye TAMRA, and the barrel DNAs are bound, TAMURA was located near the FITC.  <br></p>


      <li><a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka#abs">Abstract</a></li>
      <li><a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka#vid">Video</a></li>
 
    </ul>
  </li>
  <li><a href="fit_Introduction.html#pro">Projects</a>
    <ul>
      <li><a href="fit_Introduction.html#back">Background & Motivation</a></li>
      <li><a href="fit_Introduction.html#goal">Project Goals</a></li>


&nbsp;  Therefore, FITC is excited by the 488nm laser light and TAMRA is excited by the fluorescent of FITC. <br>
    </ul>
  </li>
  <li><a href="fit_Approach and Goals.html#des">Design</a>
    <ul>
      <li><a href="#ear">Early Design</a></li>
      <li><a href="#fin">Final Design</a></li>
     
    </ul>
  </li>
  <li><a href="fit_Method.html#met">Method</a>
    <ul>
      <li><a href="fit_Method.html#a">Preliminary Experiment</a></li>
      <li><a href="fit_Method.html#b">Synthesis of the “Barrel” particles and the “Doll” particles</a></li>
      <li><a href="fit_Method.html#c">Combining the Doll particles with the Barrels particles</a></li>
　　　<li><a href="fit_Method.html#d">Pop-up of the doll particle</a></li>
　　　<li><a href="fit_Method.html#e">Materials</a></li>
  </ul>
  </li>
  <li><a href="fit_Results and Discussion.html#">Result and Discassions</a>
    <ul>
      <li><a href="fit_Results and Discussion.html#a">Preliminary Experiment</a></li>
      <li><a href="fit_Results and Discussion.html#b">Synthesis of the “Barrel” particles and the “Doll” particles</a></li>
      <li><a href="fit_Results and Discussion.html#c">Combining the Doll particles with the Barrels particles</a></li>
      <li><a href="fit_Results and Discussion.html#d">Conclusions</a></li>
　　  </ul>
  </li>
  <li><a href="fit_Member.html#team">Team</a>
    <ul>
      <li><a href="fit_Member.html#men">Menber</a></li>
      <li><a href="fit_Member.html#spo">Sponsor</a></li>
     
    </ul>
  </li>
</ul>




&nbsp;Then we observed the red fluorescent around the silica particles under the confocal fluorescent microscope. When we added the sward DNA and FITC labeled doll DNA is released from the TAMRA labeled barrel DNA .We could not observed any fluorescent around  <br clear="all"><br>


</p></h3>
<a name="ear"></a>
<div align="lest" style="margin: 1px 60px;">
<center><font size="6" color="#000022" face="Arial"><b>Early Design</b></font></center>


<font size="4" color="#000022" face="Arial">


<p>
<table align="left">
<td><div style="width: 330px;"><img alt="" src="/images/e/e2/Hy3.png" width="300" height="230" border="0" / ><p> Fig. 4 Early design of nano-pop-up pirate with DNA-origami (a) before and (b) after addition of the 'Sword' DNA. </p><div></td><tr>
<td><div style="width: 330px;"><img alt="" src="/images/9/97/Hy4-3.png" width="300" height="200" border="0" / ><p>Fig.5　 Process of the final design </p><div></td><tr>
<p>
&nbsp;
We firstly planned to fabricate the barrel, the doll, and the sword of the pop-up pirate on the nanoscale with DNA-Origami. DNA-origami doll is combined with the DNA-origami barrel through the hybridization. The doll DNA and the barrel DNA are designed as mostly complementary but partially mismatched (Fig 4a). If we add a sword DNA which is perfectly complementary with the barrel DNA, the DNA-origami doll will be replaced with the sword DNA and will be released（Fig 4b）.<br>
It is easy to design the shape of the barrel and the doll only with DNA origami.
However, in order to directly observe how the DNA-origami doll jump out from the DNA-origami barrel, it is necessary to perform real-time observation of DNA origami with high-speed atomic force microscopy (AFM) in liquid. This observation is supposed to be technically very difficult. In addition, there is a problem of the instability, difficulty in large scale synthesis, and high cost of the DNA-origami when we consider the development of the present study to applications.
<br>


</p>


<hr>


</div>


</div>
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<a name="fin"></a>
<center><font size="6" color="#000022" face="Arial"><b>Final Design</b></font></center>




<table align="left">


<p>
&nbsp; 
<center>
<b>ssDNA-modified silica particles as the 'Doll' and the 'Barrel'</b></center>
<br>
&nbsp;
To solve the problems in the early designs, we fabricate the nanopop-up pirate using ssDNA-modified colloidal silica particles as the doll and the barrel. For the colloid system, the pop-up event can be observed on real-time with an optical microscopy with no technical problem; the observation is much easier than the DNA-origami system which require the high-speed AFM. The use of silica particles is also advantageous because we can synthesize the large amount of the samples at low cost.(Fig 6) We can also control the particle size and shape, and even porosity in view of future applications. In addition, in the case of the mesoporous silica, the form resembles to that the barrel with many holes! The modification of DNA is possible because the silica can be chemically modifiable Si-OH groups on the surface.<br>
First we prepare 'barrel particle'. We synthesize the silica particle and modify its surface with the barrel ssDNA. We then prepare the 'doll particle' in the similar way to the 'barrel particle'. We modify the silica particle with the doll DNA, which is mostly complementary but partially mismatched with the barrel DNA. Then, the doll particle and the barrel particle are combined by DNA hybridization. The base sequences of the ssDNAs of the 'barrel DNA', and the 'doll DNA' are shown in Fig 5. The bases marked by blue color are mismatched so that the Doll DNA will be displaced with a more complementary sword-DNA. <br>
<br><br>
<br><br><br>
<center>
<b> 2. Fully complementary or partly mismatched DNA as the 'Sword' </b> </center>
<br>
&nbsp;
To the doll-barrel pair particle, we add the DNA, which is more complementary with the barrel DNA, as the 'Sword'. Then, the doll-DNA is displaced with the sword DNA and the doll particles will fly away by Brownian motion.<br>
The base sequences of the Sword-DNAs are shown in Table 2. The Sword-DNA-1 is fully complementary with the Barrel-DNA so that the Sword-DNA1 will easily hybridize with the Barrel-DNA. The base sequence of Sword-DNA-2 and Sword-DNA-3 have the one and two mismatches, respectively. We will check the specificity for the pop-up event (displacement of the doll-DNA) among these sword-DNA-1, -2, and -3.<br>
<br>
<center>
<b> 3. Visualization of the pop-up using fluorescence molecules and FRET </b> </center>
<br>
&nbsp;
The observation of the pop-up event may be difficult only with the DNA-modified silica system. This problem is solved using the fluorescent molecules. There are three purposes to use the fluorescent molecules:<br>
1. To easily characterize the modification of DNA to the particles.<br>
2. To clearly observe the colloidal particle with the fluorescence microscopy.<br>
3. To easily follow the situation of the double chain formation of the barrel-DNA and sword DNA by detecting fluorescence resonance energy transfer (FRET).<br>
Here we use FITC and TAMRA as the fluorescence probes. The chemical structure of FITC and TAMRA are  Fig. 4 and the excitation wavelength and the fluorescent wavelength are summarized in Table 2. TAMRA is attached to the 5'-ends of the Sword-DNAs, while FITC is attached to the 3'-end of the barrel DNA. DNA for the doll particle is not attached with the fluorescent molecule to distinguish from the barrel particle. We check the hybridization of barrel DNA with the sword DNA by observing the change of fluorescent color. With the excitation by Ar-laser (488 nm), the FITC at the barrel emits green fluorescence, while, after hybridization, the green fluorescence will be quenched and the red fluorescence from TAMRA will be observed due to FRET.<br>
<br>
<table align="left">
<td><div style="width: 330px;"><img alt="" src="/images/f/fb/Table2.jpg" width="420" height="240" border="0" / ><p>  </p><div></td><tr></table>






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Latest revision as of 21:00, 25 October 2014

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<a name="header"></a> <img src="/images/4/46/Fitaologo.PNG" alt" alt="" width="422" height="98" hspace="0" align="left">



  • <a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka#home">Home</a>
  • <a href="fit_Introduction.html#pro">Projects</a>
    • <a href="fit_Introduction.html#back">Background & Motivation</a>
    • <a href="fit_Introduction.html#goal">Project Goals</a>
  • <a href="fit_Approach and Goals.html#des">Design</a>
    • <a href="#ear">Early Design</a>
    • <a href="#fin">Final Design</a>
  • <a href="fit_Method.html#met">Method</a>
    • <a href="fit_Method.html#a">Preliminary Experiment</a>
    • <a href="fit_Method.html#b">Synthesis of the “Barrel” particles and the “Doll” particles</a>
    • <a href="fit_Method.html#c">Combining the Doll particles with the Barrels particles</a>
      • 　　　
    • <a href="fit_Method.html#d">Pop-up of the doll particle</a>
      • 　　　
    • <a href="fit_Method.html#e">Materials</a>
  • <a href="fit_Results and Discussion.html#">Result and Discassions</a>
    • <a href="fit_Results and Discussion.html#a">Preliminary Experiment</a>
    • <a href="fit_Results and Discussion.html#b">Synthesis of the “Barrel” particles and the “Doll” particles</a>
    • <a href="fit_Results and Discussion.html#c">Combining the Doll particles with the Barrels particles</a>
    • <a href="fit_Results and Discussion.html#d">Conclusions</a>
      • 　　
  • <a href="fit_Member.html#team">Team</a>
    • <a href="fit_Member.html#men">Menber</a>
    • <a href="fit_Member.html#spo">Sponsor</a>


<a name="ear"></a>

Early Design

  We firstly planned to fabricate the barrel, the doll, and the sword of the pop-up pirate on the nanoscale with DNA-Origami. DNA-origami doll is combined with the DNA-origami barrel through the hybridization. The doll DNA and the barrel DNA are designed as mostly complementary but partially mismatched (Fig 4a). If we add a sword DNA which is perfectly complementary with the barrel DNA, the DNA-origami doll will be replaced with the sword DNA and will be released（Fig 4b）.
It is easy to design the shape of the barrel and the doll only with DNA origami. However, in order to directly observe how the DNA-origami doll jump out from the DNA-origami barrel, it is necessary to perform real-time observation of DNA origami with high-speed atomic force microscopy (AFM) in liquid. This observation is supposed to be technically very difficult. In addition, there is a problem of the instability, difficulty in large scale synthesis, and high cost of the DNA-origami when we consider the development of the present study to applications.


<a name="fin"></a>

Final Design


<img alt="" src="/images/e/e2/Hy3.png" width="300" height="230" border="0" / >

Fig. 4 Early design of nano-pop-up pirate with DNA-origami (a) before and (b) after addition of the 'Sword' DNA.

<img alt="" src="/images/9/97/Hy4-3.png" width="300" height="200" border="0" / >

Fig.5　 Process of the final design

 

ssDNA-modified silica particles as the 'Doll' and the 'Barrel'


  To solve the problems in the early designs, we fabricate the nanopop-up pirate using ssDNA-modified colloidal silica particles as the doll and the barrel. For the colloid system, the pop-up event can be observed on real-time with an optical microscopy with no technical problem; the observation is much easier than the DNA-origami system which require the high-speed AFM. The use of silica particles is also advantageous because we can synthesize the large amount of the samples at low cost.(Fig 6) We can also control the particle size and shape, and even porosity in view of future applications. In addition, in the case of the mesoporous silica, the form resembles to that the barrel with many holes! The modification of DNA is possible because the silica can be chemically modifiable Si-OH groups on the surface.
First we prepare 'barrel particle'. We synthesize the silica particle and modify its surface with the barrel ssDNA. We then prepare the 'doll particle' in the similar way to the 'barrel particle'. We modify the silica particle with the doll DNA, which is mostly complementary but partially mismatched with the barrel DNA. Then, the doll particle and the barrel particle are combined by DNA hybridization. The base sequences of the ssDNAs of the 'barrel DNA', and the 'doll DNA' are shown in Fig 5. The bases marked by blue color are mismatched so that the Doll DNA will be displaced with a more complementary sword-DNA.





2. Fully complementary or partly mismatched DNA as the 'Sword'


  To the doll-barrel pair particle, we add the DNA, which is more complementary with the barrel DNA, as the 'Sword'. Then, the doll-DNA is displaced with the sword DNA and the doll particles will fly away by Brownian motion.
The base sequences of the Sword-DNAs are shown in Table 2. The Sword-DNA-1 is fully complementary with the Barrel-DNA so that the Sword-DNA1 will easily hybridize with the Barrel-DNA. The base sequence of Sword-DNA-2 and Sword-DNA-3 have the one and two mismatches, respectively. We will check the specificity for the pop-up event (displacement of the doll-DNA) among these sword-DNA-1, -2, and -3.

3. Visualization of the pop-up using fluorescence molecules and FRET


  The observation of the pop-up event may be difficult only with the DNA-modified silica system. This problem is solved using the fluorescent molecules. There are three purposes to use the fluorescent molecules:
1. To easily characterize the modification of DNA to the particles.
2. To clearly observe the colloidal particle with the fluorescence microscopy.
3. To easily follow the situation of the double chain formation of the barrel-DNA and sword DNA by detecting fluorescence resonance energy transfer (FRET).
Here we use FITC and TAMRA as the fluorescence probes. The chemical structure of FITC and TAMRA are Fig. 4 and the excitation wavelength and the fluorescent wavelength are summarized in Table 2. TAMRA is attached to the 5'-ends of the Sword-DNAs, while FITC is attached to the 3'-end of the barrel DNA. DNA for the doll particle is not attached with the fluorescent molecule to distinguish from the barrel particle. We check the hybridization of barrel DNA with the sword DNA by observing the change of fluorescent color. With the excitation by Ar-laser (488 nm), the FITC at the barrel emits green fluorescence, while, after hybridization, the green fluorescence will be quenched and the red fluorescence from TAMRA will be observed due to FRET.

<img alt="" src="/images/f/fb/Table2.jpg" width="420" height="240" border="0" / >




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