Project goal

 In Lipo-HANABI project, we need to develop the following two subsystems.

i) Sensing system (First stage): liposome disruption by temperature control.
ii) Amplification system (Second stage): a chain-reactive disruption of the liposomes activated by the First stage.

First stage: Sensing system

 The purpose of First stage is to detect temperature change and release key molecules for the Second stage. This is achieved by temperature-sensitive liposomes containing  the keys. To make the liposome, we used lipids conjugated with NIPAM polymer.
 This structural change of NIPAM induces stress on the surface of the liposome, and consequently disrupts them.
Fig.1 Temperature-sensitive liposome

Second stage: Amplification system

 The purpose of Second stage is to accept the key from the First stage and release a lot of payload molecules in a chain-reaction.
 There are two different approaches to realize the Second stage.
A) DNA Origami approach
B) Flower DNA approach

DNA origami approach

 This approach is inspired by a paper about Membrane-bending proteins (Prinz WA, Hinshaw JE., Crit Rev Biochem Mol Biol., 2009). In this approach, we use “Origami-anchor DNA” which connects DNA Origami with liposome membrane. A lot of DNA origamis are adsorbed on the surface of liposomes by using Origami-anchor DNA. DNA origami is supposed to be a stiff, straight board 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 repel each other because of negative charges on DNA backbones. This effect may add more stress on the membrane.
Fig.2 Stress on liposome membrane

 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

  •  DNA origami is known as a designable rigid structure made of DNA. We use DNA origami to make the rigid scaffolds. In order to meet the requirements, we designed a 2D rectangular DNA origami.
    Fig.3 Rectangular origami

    Fig.4 DNA origami designed by caDNAno
     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 the bottom face of the origami. Those staples hybridize with cholesterol-modified Origami-anchor DNA, which has high affinity with lipid membrane.

    Fig.5 Unstable liposome

    Flower DNA approach

     This approach is inspired by a paper about Polymer Flower-micelle (Yukio Tominaga, Mari Mizuse, Akihito Hashidzume, Yotaro Morishima and Takahiro Sato, J. Phys. Chem. B, 2010). To adapt the Polymer Flower-micelle to our project, the followings are required.

  • Embedding a lot of cholesterol-modified ss DNA on the liposome surface
  • Adding another ssDNA (complementary to the above DNA) which induces a structural change by DNA hybridization
  • The induced structural change on the DNA results in disruption of the liposome

  •  At first, we designed “Flower-anchor DNA”, which is a couple of ss DNAs both having cholesterol modified groups (Fig.6): Flower-anchor1 is 10nt ss DNA and Flower-anchor2 is 50nt ss DNA. Both are cholesterol-modified at their 3’ ends.
     In addition, the 5’ end of the Flower-anchor2 is complementary to Flower-anchor1. When they hybridize, the rest 40nt of Flower-anchor2 remains single-stranded.

    Fig.6 Liposome with Flower-anchor DNA

     The key DNA released from stage 1 liposome is complementary to this single-stranded part. When the key hybridizes on it, a double-stranded section is formed. The length of the section is shorter than its persistence length; therefore it works as a rigid strut. The strut is anchored on the liposome at both ends, thus it extends the membrane. As a consequence, this may lead to drastic conformational change of the liposome, namely, disruption.

    Fig.7 Process of flower DNA approach

    Fig.8 How to disrupt a liposome