Biomod/2012/TeamSendaiA/Project

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Team Sendai Top

Project plan

Our project is divided large three parts, Selector, Gate and Liposome.
So we'll do each experiments abreast and finally we'll mix.
我々のプロジェクトは大きく三つに分けることができる。セレクター、チャネル、リポソームである。そこで我々はこの三つの実験を並行して行う。それぞれの実験がうまくいったところで組み合わせる。(D-Hertを分解した画像をこの下に欲しい)

Selector

Working Selector


Inside the gate, a cascade of three single stranded DNAs is planted. We named the DNAs Selector1, Selector2, and Selector3 from the outside of the gate. In addition, another Selector, which is called Selector4, is in the vesicle.
[In the gate] Selector1and 2 have complementary sequences to a target oligonucleotide here and there and consecutive adenine sequences in other portion. We made an attempt to capture a target distant from the gate with high specificity. So we lay out a long Selector1. By catching a target and making loops, it can shrink and go in the gate.
Selector3 is complementary to the target, but it is shorter than the target. When it binds to the target, the upper end of the target makes a toehold.
We designed the inner Selector has higher bonding energy. So once the outer-most ssDNA binds to a target, the target is passed to the inner-ones one by one.
[In liposome] Selector4 is perfectly complementary to the target. After the target reaches Selector3, Selector4 conveys the target into liposome.

NameSequence(5' to 3')Tm(°C)
target* - ACTAGTGAGTGCAGCAGTCGTACCA
Selector1AAAAAAAAAAAAAAAAATGGTACAAAAAAAAGACTGAAAAAAAACTGCA30.6
Selector2AAAAAAAAAAATGGTACAAAAGCTGCA36.5
Selector3TGGTACGACTGCTGCA62.3
Selector4TGGTACGACTGCTGCACTCA68.0

*Red-orange and blue-green regions are complementary DNA sequences.
*Black region is added to differ the molecular weight of each sample(for distinguishing them during electrophoresis).

Experiment page

Gate

Strategy

Gate should be able to transport the target with selector inside gate and go through cell membrane.
To transport the target with selector, we decided to make hexagonal tube as gate.
The reasons we adopted hexagonal tube as gate are that surfaces of hexagonal tube are suitable for being attached selector,
high strength of honeycomb structure are easy to be observed. To go through cell membrane, we placed the staple attached lipid on center of gate.
We expect gate is introduced liposome simultaneously with creation of liposome.
In addition, we attached edge of the gate to adenine staple like a "mustache" to make easy watching by AFM and interrupt other DNA approaching.
We think because of our selector 1 is enough long, only target is transported into the gate.

Structure image

This is the hexagonal tube design by caDNAno using honeycomb structure.



Experiment page


Membrane

We use liposomes as a model of cell membrane. To insert cell-gate into the
liposome, we stretched out ssDNA of 10 nt from the side of the hexagonal tube. Then,
we extend the complementary ssDNA, and modified the cholesterol at the end of them.
We choose the cholesterol because cholesterol is strongly hydrophobic. We expect
that cholesterol penetrate into the hydrophobic portion of the liposome. We confirmed
the tube modifying the cholesterol by electrophoresis. We use fluorescein to confirm
that the tube insert into the liposome correctly.
(細胞膜のモデルとして、リポソームを使用する。リポソームにセルゲートを刺さるように
するために、六角形筒の横から10塩基のシングルストランドDNAを伸ばした。さらに、
相補的なシングルストランドDNAを伸ばして、それらの末端にコレステロール修飾を行
った。コレステロールを修飾したのは、コレステロールが強い疎水性であるからである。
コレステロールがリポソームの疎水性部分に入り込むことで、筒がリポソームに刺さりや
すくなると考えた。コレステロール修飾は電気泳動で確かめた。筒がリポソームに刺さ
っているかを確かめる方法として、蛍光分子を利用する。)
Experiment page

Future

Single stranded DNA can bind not only to DNA, also to various biomolecules like RNA and proteins. By changing Selector DNA sequences in the gate, we’ll capture biomolecules just as we want to.
In the future, we expect to make the molecular robot which can transport the object to inside the cell and collect the object from the cell using Cell-gate.
And now we use liposome as a model of the cell.
We think that by using liposome not only as a model of the cell but a bag to put something into,
we can make organelles to collect unnecessary object and release medicine.
将来的にはこのセルゲートを使うことで細胞内への物質の運搬、細胞内からの物質の回収ができるロボットの作製を目指している。
また、実験を行うにあたって細胞のモデルとしてリポソームを現在用いているが、単にモデルとしてリポソームを用いるだけでなくこのリポソームでできた袋を蓄積場として、
不要な物質の回収や、優良物質の散布ができる小器官のようなものを作製し細胞内で働かせるといった応用も考えられる。

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