Biomod/2012/UTokyo/UT-Hongo/Function

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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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Structure

In this section we will describe the methods for designing our DNA Shell.

Outline

We designed the structure shell-shaped so that it can efficiently capture target molecule with a dramatic change in the fluorescence for easy detection. We actually created three shell-shaped structures.

  • Plane shell
  • Shell with florescent molecules and quenching molecules attached
  • Shell with florescent molecules, quenching molecules, and biotin attached




Structure of the body

DNA origami is comprised of two elements, the scaffold and the staples. Scaffold is like the main body of the whole origami, and we can shape the structure as we like by attaching staples. Following is the description of how scaffold and staples were designed.

  • Scaffold：We used a typical DNA material called M13. It has approximately 7250 base pairs and it is an ideal number for our shell. A part of this DNA self-hybridizes, so we had to cut that part off for further design.
  • Staple：The characteristic of hybridization is greatly affected by the number of base pairs. In order to make it go efficiently, we standardized the number of base pairs to 32bp. We had difficulty in deciding the position of staples because it must be strongly attached to the body. Also, we wanted to control the flexibility of the shells in response to the purposed functions. To realize this demand, staples at the junctions of the shell was designed so as not to affect other parts. By doing so, the junctions of the shell became more flexible while the whole body became stronger at the same time.

Adding functionality

In order to add functionality to the body, we extended some of the staples so they could bond with DNA fragments that are modified. Therefore, we added 13 base pairs to the staples which originally had 32 base pairs. We needed to attach 12 single-stranded DNAs with fluorescent molecules (named as fluorescent DNAs) and two functional single-stranded DNAs to one side, and 12 single-stranded DNAs with quenching molecules (named as quenching DNAs) and two functional single-stranded DNAs to the other side. We had to think up of 4 different sequences that would bind to either 1.fluorescent DNAs, 2.quenching DNAs, 3.functional DNAs to the fluorescent side, and 4.functional DNAs to the quenching side. We designed the structure so that the functional part is 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 the fluorescent molecules and the quenching molecules face each other.)

Generally, in designing moderate arrangement of staples it is required to make sure that the staples are hybridized only at the points we want hybridization to occur. That is, we have to keep the energy for hybridization high in inappropriate areas and keep it low in the correct areas. In order to achieve this requirement, we focused on these three points.

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

the energy of hybridization would become the lowest in the appropriate area, which means that the possibility of achieving the correct hybridization will increase.

  • To keep the melting temperature of correct hybridization low, and to keep the melting temperature of incorrect hybridization high
We achieved this requirement through trial and error.
  • To avoid steric hindrance
If we hybridized the arrangements extended from staples and functionalized parts simultaniously, there would be some steric hindrance. To weaken this effect, we set a realm where the staples will not be hybridized. Concretely, we embedded two T (thymine) bases in the staples.

Simulation Techniques

To simulate the energy of hybridization and melting temperature, we used the software named DINAMelt and NUPAC supported on online. Since there is no absolute solutions for design, we needed to get over the difficulties by 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.

The image shown below is one example of simulation　using　DINAMelt (http://mfold.rna.albany.edu). This is a simulation of the melting temperature and the possibility of correct hybridization.　 This is simulated under a situation of

  • From 0°C by 1°C to 100°C as DNA
  • [A0] = 1.6e-07 M, [B0] = 1.6e-07 M  ;[A0] is concentration of extended staple and [B0] is concentration of modified functional part
  • [Na+] = 0.2 M, [Mg++] = 0.0125 M

As seen below, a hybridization of A and B (appropriate hybridization) is dominant at low temperature(from 0°C to 30°C).

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