1 Project goalIn Lipo-HANABI project we need to develop the following subsystems
i) Sensing system (1st stage): liposome disruption by temperature control.
ii) Amplification system (2nd stage): a chain-reactive disruption of the liposomes activated by the 1st stage.
1-1 1st stage: Sensing systemTo make temperature-sensitive liposomes, we used lipids conjugated with NIPAM polymer.
NIPAM is hydrophilic at room temperature, but switches to hydrophobic over 32 C. When it becomes hydrophobic, it shrinks to avoid water molecules. This structural change of NIPAM induces stress on the surface of liposomes, and consequently disrupts liposomes.
1-2 2nd stage: Amplification systemWe assume that 1st stage liposomes contain key molecules that initiate chain reactive burst of 2nd stage liposomes.
There are two different approaches to realize the 2nd stage.
A) DNA Origami approach
B) Flower DNA approach
1-2-1 DNA origami approachThis approach is inspired by a paper about Membrane-bending proteins In this approach, we name DNA stuck into liposome membrane, Origami-anchor DNA. A lot of DNA origamis are adsorbed on the surface of liposomes by using Origami-anchor DNA. DNA origami is supposed as a stiff, straight board like structure compared with liposome membrane, and as a result, liposome surface gets bending stress. At certain level of the absorbance, liposomes will burst. Also, DNA origamis on the surface of liposome repel each other because of negative charges on DNA backbone. This effect may add more stress on the membrane. We did analysis on this phenomenon (link).
From the reference, we learned that efficient structure design for destabilizing membranes should have the following properties: .
• Having rigid scaffolds
• Having large surface areas to maximize the effect of the scaffold on the membrane
＜Design of DNA origami＞
DNA origami is known as a designable rigid structure made of DNA. We use DNA origami to make rigid scaffolds. In order to meet the requirements, we designed 2D rectanglar DNA origami.
We expect the rectangle DNA origami to work as one scaffold in itself. Following is the design of our rectangular DNA origami.
Fig.4 Rectangular origami
We use caDNAno2 for our DNA origami design. The size of DNA origami is 67.6nm (26 helixes) in width and 127 nm (374 bases) in height. We cut out a smaller rectangle of 10 helixes (161 bases) at one of the corners, so that we could distinguish the two sides with AFM (Atomic Force Microscope) observation. Also, we put 141 staples sticking out from bottom face of the origami. Those staples hybridize with cholesterol-modified Origami-anchor DNA, which has high affinity with lipid membrane.
Fig.6 Unstable liposome
1-2-2 Flower DNA approachThis approach is inspired by a paper about Polymer Flower-micelle.
To adapt Flower-micelle to our project, we should remark the followings.
• Sticking a lot of cholesterol-modified DNA into liposome surface
• Change of DNA’s property by hybridization with Key DNA
In this approach, we call 50nt3’DNA partly hybridized with 10ntDNA, Flower-anchor DNA. 50nt3’DNA is cholesterol-modified and the part of it hybridizes with 10ntDNA. The rest of its 40nt complements Key DNA.
Fig7The hybridization makes Flower-anchor DNA longer. It gives stretching stress to liposome membrane and ends up disrupting liposomes.
We designed the sequences of Flower-anchor DNA and Key DNA by DNA design, software for designing DNA sequences.
Fig.8 Process of flower micelle approach
Fig.9 How to straighten loopこのアプローチは「高分子フラワーミセル」の論文をDNAに応用したものである。 フラワーミセルをリポソームに応用するためには、
このアプローチでは１０ntとそれと一部が相補になっている５０nt3’コレ付きDNAがハイブリしたものをFlower アンカーDNAと呼ぶ。Flower アンカーDNAの一本鎖になっている４０ntの部分に鍵DNAが相補になっており、ハイブリダイゼーションによって持続長が長くなったフラワーDNAはリポソーム膜面に「引っ張り（引き裂き）」ストレスを与え、リポソームを壊すのである。 以下の図のように持続長の変化によって変形するDNAを設計した We designed the sequences of Flower-anchor DNA and Key DNA by DNA design, software for designing DNA sequences.
私たちはこのソフトでFlower DNAとKey DNA のDNAを設計しました。