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To simulate the energy of hybridization and melting temperature, we used the software named DINAMelt and NUPAC supported on online.
To simulate the energy of hybridization and melting temperature, we used the software named DINAMelt and NUPAC supported on online.
There are not tangible design solutions, therefore it needs us to get over the difficulties of trial and error  
 
これらのエネルギーに関する計算は、CADnanoでは出来ず、明確な設計解がないため、試行錯誤的な操作が必要となり、本当に大変な作業でした。エネルギーと融解http://openwetware.org/wiki/OpenWetWare:Copyrights温度の計算には、nupac及びdinameltという、ウエブ上の計算ソフトを用いました。
There are not tangible design solutions, therefore it needs us to get over the difficulties of trial and error; It was serious and troublesome work.
この計算には多大な人的労力を要しました。なぜなら、複数のstapleに同じ配列を延長する必要があるため、同じ配列を延長させたそれらのstapleが、すべて安定にハイブリする必要があり、そのような配列を探し出すのは、キャンパス内におとされた5セントコインを探し出すようなものでした。
In designing the arrangement extended from staples, we need to attach the same arrangement to the staples attached with functional parts.
This work of discovering moderate arrangement cost us immense labor, because it is required to make these staples added only ONE pattern
of arrangement hybridized stably. The work of discovering a arrangement compatible to several staples was like finding one penny lost somewhere in campus.

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<li class="toppage"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo">Top</a></li> <li class="motives"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro">Motives</a></li> <!-- <li class="design"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Function">Design</a></li> --> <li class="result"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly">Design & Results</a> <ul class="submenu"> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Assembly_of_the_DNA_Shell">Assembly of the DNA Shell</a></li> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Capturing_ability">Capturing Ability</a></li> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Immobilizing_on_microfluidic_device">Immobilizing on microfluidic device</a></li> <li><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Supporting_Enzyme">Supporting Enzyme</a></li> </ul> </li> <li class="method"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Method">Method</a></li> <li class="futurework"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork">Progress & Beyond</a></li> <li class="team"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Team">Team</a></li> <li class="acknowledgement"><a href="/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement">Acknowledgement</a></li> 

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Base sequence design

Outline

We designed the structure shell-shaped because it can efficiently capture target molecules and fluorescence change can be detected with high sensitivity. We actually created three shell-shaped structures. ・Plane shell ・Shell with fluorescent molecules and quenching molecules attached ・Shell with fluorescent molecules, quenching molecules, and biotin attached

We used single-stranded M13 mp3 for the body of our DNAorigami. The structures are shown below. 画像

Structure of the body

DNAorigami comprises of two elements, scaffold and staple. Scaffold is the base of the origami, and we can design the shape we like by attaching staples. We contrived in two ways. 　scaffold：We used a typical DNA material called M13. It has approximately 7250 base pairs and it is an ideal number to observe. In addition, a part of this DNA self-hybridize, so we had to cut that part off to design.

　staple：The characteristic of hybrid is greatly affected by the number of base pairs. In order to make efficient, we standardized the by to 32. We had difficulty in deciding the position of staples because it should be strongly attached to the body. Plus, we wanted to control the movability of the shells in response to the purposed functions. To realize this demand, staples at the junctions of the shell was designed not to affect other parts. By doing so, the junctions of the shell became more flexible and strong at the same time.

Adding functionality

So as to add functionality to the body, we extended some of the staples so they bond with DNA 　fragments with functions. Concretely, we added 13 bases to the staples which originally had 32 base pairs. We attached 12 fluorescent molecules and two functional parts to one side, and 12 quenching molecules and two functional parts to the other side. The sequences that we designed are sequence to attach fluorescent molecules, a sequence to attach quenching molecules, a sequences to attach functional parts to the fluorescent side, and a sequence to attach functional parts to the quenching sie. We attached the functional part to the center of the side, and surrounded by fluorescent/quenching molecules. (The yellow sequences in the image are the staples to attach fluorescent/quenching molecules, the red sequences are the staples to attach functional parts. For quenching molecules to function, we had to design the positions so that fluorescent molecules and quenching molecules to face each other.

Generally, in designing moderate arrangement of staples it is required to make sure that the staples are hybridized at the point we are driving at. That is, we have to keep the energy to hybridize high in inappropriate area and keep it low in appropriate area. In order to achieve this requirement, we contrived in these three points.

  • To make sure that the complementary arrangements of the designed arrangements should not appear inappropriate area
The　combination of Watson-Crick base pair is stable, therefore if there are no other possibility of hybridization of staple and scaffold in inappropriate area,

the energy of hybridization becomes lowest in appropriate area, this means that the possibility we achieve the correct hybridization increases.

  • To keep the melting temperature of correct hybridization low, and keep the melting temperature of incorrect hybridization high
We achieved this requirement by a trial-and-error method.

（ただし、完全にランダムに設計したわけではありません。経験的にstapleを構成する塩基のうちGCの含量が50%にすると、融解温度がいい感じになるそうです。いる?）

  • To avoid steric hindrance
If we hybridized the arrangements extended from staples and functional parts entirely, there would appear a steric hindrance. To weaken this effect,

we set a realm in where the staples are not hybridized. Concretely, we embedded two T (thymine) in the staples.

To simulate the energy of hybridization and melting temperature, we used the software named DINAMelt and NUPAC supported on online.

There are not tangible design solutions, therefore it needs us to get over the difficulties of trial and error; It was serious and troublesome work. In designing the arrangement extended from staples, we need to attach the same arrangement to the staples attached with functional parts. 

This work of discovering moderate arrangement cost us immense labor, because it is required to make these staples added only ONE pattern

of arrangement hybridized stably. The work of discovering a arrangement compatible to several staples was like finding one penny lost somewhere in campus.

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