Recently I have been working on a project related to DNA Origami. The project is titled :
DNA Origami – Creating Complex 2-D Structures by folding of M13mp18 DNA
DNA origami is the nanoscale folding of DNA to create arbitrary two and three dimensional shapes. The specificity of the interactions between complementary base pairs makes DNA a useful construction material through design of its base sequences.
To produce a desired shape, images are drawn with a raster fill of a single long DNA molecule. This design is then fed into a computer program which calculates the placement of individual staple strands. Each staple binds to a specific region of the DNA template, and thus due to Watson-Crick base pairing the necessary sequences of all staple strands are known and displayed.
The DNA is mixed and then heated and cooled. As the DNA cools the various staples pull the long strand into the desired shape. Designs are directly observable via several methods including atomic force microscopy, or fluorescence microscopy when DNA is coupled to fluorescent materials.
- Adobe Photoshop
The majority of our work was done on caDNAno software. CaDNAno is open-source software for design of 3D DNA origami nanostructures. This free open source software is based on Adobe AIR platform. It was written with the goal of providing a fast and intuitive means to create and modify DNA origami designs.
This free software makes heavy use of several fantastic open-source libraries and resources, especially Papervision3D for 3D rendering.
“The goal is to choose a continuous route through the scaffold path and then generate a list of staples that would force the scaffold to adopt that configuration in the test-tube.”
Labeled Snapshot of the Software-
DNA Origami map of India and Gujarat
1. We started out with the basic structure of the map. Layout of the map of Gujarat was taken on to an image editing software. Then we divided the image into smaller grids which can be easy to replicate graphically.
2. Then we moved onto the caDNAno software. Here we firstly emphasized on making the shape of Gujarat almost as it is, using the gird layout map for reference. For this we chose 35 discontinuous single stranded scaffolds which are all in the same plane and anti-parallel. Initially we started working with scaffolds of length 63 bases but later we extended the length of the helices by (5*32) base pairs.
Our goal was to choose a continuous route for the scaffold path and then generate a list of staples that would force the scaffold to adopt that configuration in the test tube.
After this, we used tools provided in the software to cut the helices at specific positions to bring out the desired shape.
3. Once the shape was created and satisfactory we then moved on to the more advanced workings that are making the staple sequences for the map.
4. We made finer adjustments to make the whole map with one strand of DNA by creating crossovers among the scaffolds. The resolution was compromised a bit but the shape did come out well.
5. Then the staple sequence was applied onto the structure by using the Auto-Staple feature in the caDNAno software. It automatically generates staple sequences for the whole map.
6. But then we take care of the imperfections in the scaffolds, many of which have to be checked and taken care of manually with precise care. The scaffold length must be between 16 to 50 bases.
7. After the crossovers and the scaffolds are satisfactory, we move on to coloring the scaffolds with a different color and are ready to extract the staple sequences.
8. After this we added the sequence of m13 derived scaffolds to our design. We chose these scaffolds specifically for our project because they are more than 7000 bases long.
9. We faced many problems initially such as errors in the staple sequences, unstable scaffold structures. But these were taken care-of intelligently by manipulating the structure a bit and then placing the crossovers in strategic positions.
The Staple Sequences are the extracted and copied onto an excel file.