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<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a>  <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li>  <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>

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DNA design

To achieve the DNA ciliate and its three modes, we constructed five types of DNA sequences: (1) "deoxyribozyme", attached to the body of the DNA ciliate; (2) "substrate DNA", attached to a glass plate as a DNA track for the track walking mode; (3) "UV-switching DNA", used for the UV-switching device and originally designed by ourselves; (4) "blocking DNA", used for the UV-switching device; (5) "complementary strand for deoxyribozyme", used for constantly-gathering of the DNA ciliate. All these five types of DNA strands worked as we expected (see Experimental Results). Here, we explain the sequence information and the functions of the five types of DNA strands in detail.

1.Deoxyribozyme

5' -(NH₂)-TTATTATTAT CTCTTCTCCGAGCCGGTCGAAATAGTGAAAA-3'
Size: 41 bases
- This DNA was attached to the DNA ciliate body.This DNA has enzymatic cleaving activity for the substrate DNA; i.e., deoxyribozyme. The 31 bases from 3' end of the above strand (shown in red) acts as a deoxyribozyme when it hybridizes with the substrate DNA (2). The 31 bases are same as the sequence of the DNA spider leg (CTCTTCTCCGAGCCGGTCGAAATAGTGAAAA) [スパイダーの論文引用].
- We designed the first 10 bases from 5’ end as a linker between the deoxyribozyme region and the DNA ciliate body (TTATTATTAT). This linker increased the spacing between the DNA ciliate body and the enzymatic activity area of the deoxyribozyme, and thus the deoxyribozyme area easily hybridized with the substrate DNA and exerted its enzymatic activity (see Experimental Results). In addition, we carefully designed the sequence not to cause unexpected intramolecular structures or unexpected hybridization with other DNA in the experimental system.

2.Substrate

5' -(NH₂)-TTTTTTTTTT TTTTCACTAT[rA]GGAAGAG-3'
Size: 28 bases
- The substrate DNA is used for the DNA tracks in the track walking mode. The substrate DNA contains an RNA base at the 21st base from 5' end of the DNA (shown as [rA]). When the deoxyribozyme (1) hybridizes with the substrate DNA, the substrate DNA works as an enzymatic substrete of the deoxyribozyme, resulting in the cleavage of the substrate DNA at the RNA base site. The last 18 bases from 3'end of the substrate DNA are same as the substrate of the DNA spider leg (TTTTCACTAT[rA]GGAAGAG) [スパイダーの論文引用].
- We designed the first 10 bases from 5' end as a linker (TTTTTTTTTT). This DNA was also designed not to make unexpected structures.
The 5' end was modified by an amino group (-NH₂) to be fixed on a glass plate by a silane coupling reaction.

3.UV-switching DNA

5' -(NH₂)-TTTTTT TTTTCACTATTTCGACCGGCTCGGAGAAGAG TTTTT CT X CT X TC-3'(X = azobenzene)
Size: 48 bases + 2 azobenzenes
- The UV-switching DNA was used for an anchoring-DNA spot in the light-irradiated gathering mode. The UV-switching DNA forms a stem-loop structure. The loop consists of five bases (TTTTT), and the stem (CT X CT X TC) has two trans-formed azobenzenes (X ). By UV irradiation, the azobenzenes are isomerized from the trans-form to the cis-form. As a result, the stem with the azobenzenes becomes hard to form the double strand [浅沼先生の論文かWebページ（英語なら）を引用].
  To achieve this switching, it is necessary to design the stem sequence that firmly forms the stem-loop structure at the room temperature but opens the stem-loop structure by isomerization of the two azobenzenes from trans- to cis-form. There are no reports on the opening-and-closing transition of a single molecular by azobenzenes inserted into a stem. Here, we designed the sequences “GAAGAG” and "CT X CT X TC" as the stem and "TTTTT" as the loop by thermodynamic calculations [NUPACKのURLを引用].
- The 7th to 37th bases from 5' end (TTTTCACTATTTCGACCGGCTCGGAGAAGAG) is a complementary sequence for the deoxyribozyme (1). In addition, the 7th to 31th bases from 5’ end (TTTTCACTATTTCGACCGGCTCGGA) are a complementary part for blocking DNA (4) below.
  Before UV irradiation, the stem-loop structure of the UV-switching DNA forms, and the blocking DNA is hybridizing with the UV-switching DNA. Thus, the deoxyribozyme cannot hybridize with the UV-switching DNA. After UV irradiation, the branch migration of the deoxyribozyme for the UV-switching DNA starts from the stem part and the blocking DNA is released. As a result, the deoxyribozyme and the UV-switching DNA form a double strand.
- We designed the first 6 bases from 5' end as a linker (TTTTTT). This is also designed not to make unexpected structures.
- The 5' end is modified by an amino group (-NH₂) to be fixed on a glass plate by a silane coupling reaction.

4.Blocking DNA

5' -TCCGAGCCGGTCGAAATAGTGAAAA-3'
Size: 25 bases
- Blocking DNA is the DNA which hybridizes with UV-switching DNA and prevent hybridization between deoxyrobozyme and UV-switching DNA.
- Blocking DNA is complimentary to UV-switching DNA segment which is the 7th to 31th bases from 5' end (TTTTCACTATTTCGACCGGCTCGGA).
- Blocking DNA’s sequences is equal to a part of deoxyribozyme. The part is 25 bases from deoxyribozyme’s 3’ end. However, the blocking DNA doesn’t have deoxyribozyme activity because this DNA doesn’t have 6 bases which need for deoxyribozyme activity. Accordingly, in the future, if both substrate and UV-switching DNA are attached, released blocking DNA doesn’t cleave substrate.
This DNA was used for UV-switching DNA devices.

5.Complementary DNA for deoxyribozyme

5' -(NH₂)-TTTTTT TTTTCACTATTTCGACCGGCTCGGAGAAGAG-3'
Size: 48 bases
- The last 31 bases from 3' end (TTTTCACTATTTCGACCGGCTCGGAGAAGAG) are a complete complementaty part for deoxyribozyme, and also, these bases are equal to the UV-switching DNA's 37 bases from 5' end
- We designed the first 6 bases from 5' end as a linker (TTTTTT). This is also designed not to make unexpected structures. As a result, we decide using this linker.
- The 5' end is aminated to be fixed on a glass plate.
This DNA was used for construction of DNA tracks.

@@ Line 21: / Line 21: @@
 *5' -(NH<sub>2</sub>)-<font color="#696969">TTATTATTAT</font> <font color="#dc143c">CTCTTCTCCGAGCCGGTCGAAATAGTGAAAA</font>-3'
 *Size: 41 bases
 **This DNA was attached to the DNA ciliate body.This DNA has enzymatic cleaving activity for the substrate DNA; i.e., deoxyribozyme. The 31 bases from 3' end of the above strand (shown in <font color="#dc143c">red</font>) acts as a deoxyribozyme when it hybridizes with the substrate DNA (2). The 31 bases are same as the sequence of the DNA spider leg (<font color="#dc143c">CTCTTCTCCGAGCCGGTCGAAATAGTGAAAA</font>) [スパイダーの論文引用].
 **We designed the first 10 bases from 5’ end as a linker between the deoxyribozyme region and the DNA ciliate body (<font color="#696969">TTATTATTAT</font>). This linker increased the spacing between the DNA ciliate body and the enzymatic activity area of the deoxyribozyme, and thus the deoxyribozyme area easily hybridized with the substrate DNA and exerted its enzymatic activity (see Experimental Results). In addition, we carefully designed the sequence not to cause unexpected intramolecular structures or unexpected hybridization with other DNA in the experimental system.
@@ Line 27: / Line 26: @@
    </tr>
 </table>
 ==2.Substrate==
 <table width="100%">
@@ Line 39: / Line 34: @@
 *5' -(NH<sub>2</sub>)-<font color="#696969">TTTTTTTTTT</font> <font color="#0000ff">TTTTCACTAT</font>[<font color="#ff8c00">rA</font>]<font color="#0000ff">GGAAGAG</font>-3'
 *Size: 28 bases
 **The substrate DNA is used for the DNA tracks in the track walking mode. The substrate DNA contains an RNA base at the 21st base from 5' end of the DNA (shown as [rA]). When the deoxyribozyme (1) hybridizes with the substrate DNA, the substrate DNA works as an enzymatic substrete of the deoxyribozyme, resulting in the cleavage of the substrate DNA at the RNA base site. The last 18 bases from 3'end of the substrate DNA are same as the substrate of the DNA spider leg (<font color="#0000ff">TTTTCACTAT</font>[<font color="#ff8c00">rA</font>]<font color="#0000ff">GGAAGAG</font>) [スパイダーの論文引用].
 **We designed the first 10 bases from 5' end as a linker (<font color="#696969">TTTTTTTTTT</font>). This DNA was also designed not to make unexpected structures.
 *The 5' end was modified by an amino group (-NH<sub>2</sub>) to be fixed on a glass plate by a silane coupling reaction.
 </td>
    </tr>
 </table>
 ==3.UV-switching DNA==
 <table width="100%">
@@ Line 67: / Line 51: @@
   </tr>
 </table>
-<!--
-*5' -(NH<sub>2</sub>)-<font color="#696969">TTTTTT</font> <font color="#0000ff">TTTTCACTATTTCGACCGGCTCGGAGAAGAG</font> <font color="#ff8c00">TTTTT CT </font><font color="#8b008b">X </font><font color="#ff8c00">CT </font><font color="#8b008b">X</font> <font color="#ff8c00">TC</font>-3' (<font color="#8b008b">X </font> means azobenzene. )
-*Size: 48 bases + 2 azobenzenes
-**UV-switching DNA is used for the scaffold in light-irradiated gathering mode. We designed this DNA by ourselves. UV-switching DNA has a five bases’ loop (<font color="#ff8c00">TTTTT</font>) and there are two azobenzenes (<font color="#8b008b">X </font>) in the one side of the stem (<font color="#ff8c00">CT</font><font color="#8b008b">X</font><font color="#ff8c00">CT</font><font color="#8b008b">X</font> <font color="#ff8c00">TC</font>).
-**By spotting UV, azobenzenes are isomerized (trans to cis), so the part which contains azobenzenes becomes hard to form double strand. It is known that UV-switching can be realized by using this principle. (ref)
-**To achieve this switching, it is necessary to design the stem which forms the loop firmly in the room temperature and opens the loop by isomerizing of two azobenzenes. We didn’t find the precedent which succeeded in opening and closing at a single molecular by azobenzenes which are inserted into a stem, so the designing is very difficult. After trial and error, we designed to use “GAAGAG” and "<font color="#ff8c00">CT</font><font color="#8b008b">X</font><font color="#ff8c00">CT</font><font color="#8b008b">X</font><font color="#ff8c00">TC</font>" as the stem and "<font color="#ff8c00">TTTTT</font>" as the loop.
-**The 7th to 37th bases from 5' end (<font color="#0000ff">TTTTCACTATTTCGACCGGCTCGGAGAAGAG</font>) is a complete complementary part for the deoxyribozyme. In addition, the 7th to 31th bases from 5’ end (<font color="#0000ff">TTTTCACTATTTCGACCGGCTCGGA</font>) are a complementary part for blocking DNA. Consequently, the 32th to 37th bases from 5’ end (<font color="#0000ff">GAAGAG</font>) are a complementary part for deoxyriboyme and not a complementary part for blocking DNA, so branch migration is started from this part and blocking DNA is released. Moreover, these 6 bases are a part which makes stem, so this part is blocked when the loop is closed. By this structure, branch migration doesn’t happen when the loop is formed.
-**We designed the first 6 bases from 5' end as a linker (<font color="#696969">TTTTTT</font>). This is also designed not to make unexpected structures. As a result, we decide using this linker.
-**The 5' end is aminated to be fixed on a glass plate.
-*This DNA was used for [[Biomod/2011/TeamJapan/Tokyo/Project/Results#UV-switching system|UV-switching DNA devices]].
--->
 *5' -(NH<sub>2</sub>)-<font color="#696969">TTTTTT</font> <font color="#0000ff">TTTTCACTATTTCGACCGGCTCGGAGAAGAG</font> <font color="#ff8c00">TTTTT CT </font><font color="#8b008b">X </font><font color="#ff8c00">CT </font><font color="#8b008b">X</font> <font color="#ff8c00">TC</font>-3'(<font color="#8b008b">X </font> = azobenzene)
 *Size: 48 bases + 2 azobenzenes

Biomod/2011/TeamJapan/Tokyo/Achievements/DNA Devices: Difference between revisions

Revision as of 10:42, 2 November 2011

DNA design

1.Deoxyribozyme

2.Substrate

3.UV-switching DNA

4.Blocking DNA

5.Complementary DNA for deoxyribozyme

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