Biomod/2011/DAIICT/DANanoTrons:Project

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Abstract

The research on the constructions of 3D origami is attracting many researchers to explore the possibility for making useful tools as well as novel structures. In particular, in April 2011 Hao Yan group has demonstrated curved surfaces using 3D DNA origami. Motivated by this in this project we aim to develop highly complex non-trivial 3D structure of Taj Mahal. This will involve new algorithms and software development. We have identified the basic building blocks of Taj Mahal namely Decagonal Dome (29nm radius, 10 crossovers, 8 rings), Cylindrical Tubes (10nm radius tubular), Pyramidal Tops (13nm radius, 5 crossovers, 5 rings), Cubical Body (85nm width) and square base. Combining these basic shapes is still a challenge and experimental validation for the same is needed. Prior to this work, we have also created the 2D structures of the map of India and Gujarat state. Currently, we utilize tools like caDNAno, Nanorex and Autodesk Maya.

DNA Origami - An introduction

Origami is the traditional Japanese folk art of paper folding. The goal of this art is to transform a flat sheet of material into a finished sculpture through folding and sculpting techniques. The technique of DNA origami (as well as many other techniques of DNA nanotechnology) will be used in future to build smaller, faster computers and many other devices. Developed by Paul Rothemund at the California Institute of Technology, the process involves the folding of a long single strand of viral DNA aided by multiple smaller "staple" strands. These shorter strands bind the longer in various places, resulting in various shapes including a smiley face and a coarse map of the Americas, along with many three dimensional molecules such as cubes DNA origami is the nanoscale folding of DNA to create arbitrary two and three dimensional shapes at the nanoscale. The specificity of the interactions between complementary base pairs makes DNA a useful construction material through design of its base sequences.

One of the practical limitations of DNA origami has been the length of the scaffold strand that is used. The reason for this is because it is difficult to get long single-stranded scaffolds longer than around 7000-8000 nucleotides, inexpensively and in high yield.

We will be using the software caDNAno initially for demonstration of the origami.

Basic Concept

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. The DNA created would be able to devour certain pollutants in the air, or be able to determine which cells in a person's body are cancerous and destroy them before the cancer spreads.

caDNAno

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.

Features

• Very easy – No programming required
• Effectively Integrated 2D and 3D interfaces
• Visual cues to aid design process
• Export formats: SVG, X3D, JSON
• Platform independent
• Open source License

Our Goal for the first task

“Our 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.”

Procedure Adopted for 2D DNA Origami

Map of Gujarat and Map of India -

1. We started out with the basic structure of the map. A Layout of the map of Gujarat (shown adjacent) was taken on to an image editing software. Then we divided the image into smaller grids which is 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 to (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 made installed scaffold crossovers among the 35 helices. The resolution was compromised a bit but the shape did come out well.

5. Then the staples were 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 the software doesn’t generate perfect staple sequences. Thus we then take care of the imperfections in the staples, many of which have to be checked and taken care of manually with precise care. The staples’ length must be between 16 to 50 bases. Moreover care needs to be taken that the staple crossovers and the scaffold crossovers are more than 5 bases apart.

7. After the designed staples, the scaffolds and the crossovers are satisfactorily stable, we move on to coloring the scaffolds with a different color and are ready to get the staple sequences.

8. Next we added the sequence of M13mp18 derived scaffolds to our design because they are more than 7000 bases long and readily available for use.

9. We faced many problems initially such as errors in the staple sequences due to unstable scaffold structures. But these were taken care-of intelligently by manipulating the structure a bit and then placing the crossovers in strategic positions.

10. The Staple Sequences are the extracted and copied onto an excel file. See results section for the sequence.

Analyzing caDNAno generated files

Initially we used to manually study the caDNAno files that were generated and it used to be a lot of work. So we created a few tools in Java for analyzing caDNAno generated files which made our analysis work a lot easier. The results that we generated can be seen in the results section of the Wiki.

3D DNA Origami

We decided to move our research up one level and focus on 3d origami structures. We needed to have a target structure for working on 3D-DNA origami so we decided to try to model the world-famous Taj-Mahal.

We decided to look up some CAD softwares which could help us achieve our Goal. The most interesting ones were :

• caDNAno - We were familiar with caDNAno since we had already used it while creating 2D structures. However there was a problem with 3d modelling - we found it too complex to use caDNAno for making large 3d structures.
• CANDO - could not get the software due since we figured that it is not open for free use to general public.
• Nanorex - got the software along with source code - would be using it

We read Hao Yan's paper {paper title} - {give a short summary of Hao Yan's paper and what we learnt and how we used it}

We decided to get clear with basics of developing the structure for this we need to start by creating basic 3d structures using Hao Yan's modelling concepts and CAD technology available to us

1. First we created a sphere and wrapped it with material assumed to be DNA, according to principles stated by hao yan, just for the purpose of understanding through modelling. We came up with some models and decided to host a couple of them online : [| BIOMOD Experiments]

2. Next we started learning Nanorex and after having gone through all the tutorials, we came up with some simple 3D structures which we can use in the Taj Mahal structure.

3. Next we brainstormed over how to join the individual pieces of 3D structures together. This was the most tricky part and the key to our success.

First guess was using tile staplers. However, when we researched we found out that it is only used for scaling up 2D-DNA origami structures. Thus it was not of much use to us for our Goal.