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<a name="header"></a>  
<div id="header"><h1><p style="background:#CCFFFF; color:purple;"><font face=cursive size="6"><B>  Team FIT </font> </B></p></a></h1></div>
<img src="/images/4/46/Fitaologo.PNG" alt" alt="" width="422" height="98" hspace="0" align="left">
         


<hr>
<br>
<table> <tr align="center">
<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>


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


<ul id="dropmenu">


<div style="padding: 35px;">
  <li><a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka#home">Home</a>


    <ul>


<h3><p>&nbsp; DNAの相補的塩基対に着目した。その二本の一本鎖DNAはある程度塩基対がミスマッチしていても結合することができる。そのミスマッチの塩基対が少ないほど結合力が高い。この優劣を用いて黒髭危機一髪の作製を試みた。<br>
      <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>
<p>
 
<table align="left">
    </ul>
<td><img alt="" src="/images/c/c7/Dnakurohige.gif" width="400" height="300" border="0" / ></td><tr>
  </li>
<td><img alt="" src="/images/b/b7/Fitfiani.gif" width="400" height="300" border="0" / ></td></table>
  <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; 黒髭危機一髪とは日本発祥の玩具である。土台の樽と飛び出す黒髭人形から成り立ち、土台に剣を刺し、特定の箇所に刺すことで黒髭人形が飛び出す仕組みである。樽に相当するDNA(樽DNA)と人形に相当するDNA（人形DNA）をミスマッチさせた相補鎖の状態で結合させておく。その後、樽DNAとマッチするDNA（剣DNA）を混合させる。そうすること、人形DNAと剣DNAが入れ替わり、人形DNAが飛んでいくように見える。このような、機構で目視検出可能である選択的に物資を放出するデバイスの作製を試みた。（図：DNA origamiのみで作ろうとしたものをアニメーションで表現）<br>
    </ul>
&nbsp; しかしながら、全ての部品をDNA origamiで作製することは観察面、コスト面を考慮すると現実的ではない。そこで、シリカ粒子にDNAを結合させることにより、コストを削減することができ、また観察を容易にすることができる。また不完全な塩基対から完全な塩基対に置き換わったか顕微鏡で確認することは難しいので、2種類の蛍光分子を用いて確認できるようにした。<br></p>
  </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; 蛍光分子は蛍光顕微鏡の単一レーザーの波長が488nmであるため、励起波長が488nmであるFITC（ハイパーリンク）とFITCの蛍光波長で励起するTAMRA（ハイパーリンク）を用いた。<br>


<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>


&nbsp; 樽DNAとミスマッチな塩基対を有する人形DNAの末端に緑色の蛍光分子を修飾し、樽DNAの末端に緑色の波長を吸収して赤く蛍光する分子を修飾する。樽DNAに剣DNAを混合する。人形DNAの時は二つの蛍光分子の相互作用により赤く蛍光し、人形ＤＮＡが先ほどの剣DNAと置き換わることで消光する。検出される光強度が変化することで数値として明確に放出されたか表すことができる<br clear="all"><br>
<font size="4" color="#000022" face="Arial">
(図：完成形をアニメーションで表現)
</p></h3>


<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>
</body>
</html>


<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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