Biomod/2011/LMU/FolD'N'Assemble/Methods
- column-one {display:none; width:1000px;background-color: #0000ff;}
- content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: auto;background-color: #ffffff; }
| HOME |
| THE PROJECT |
| METHODS |
| PROTOCOLS |
| LAB BOOK |
| RESULTS |
| TEAM |
DNA Origami technique
The method we used for this project is called DNA origami technique and was pioneered by Paul Rothemund in 2006. It is a very versatile bottom-up method that allows to design and construct nanoscopic structures with a very high precision.
The main idea behind the method is to force a long scaffold DNA strand to fold into the form of the desired structure. This is accomplished by using short, custom made, DNA strands called staples. These DNA staples have a complimentary sequence to two or more positions on the scaffold strand. When they bind to the scaffold, they connect the two specific points, thereby forcing it to fold. All bases of the scaffold are complemented with DNA staples, with each staple binding at least to two different points.
Paul Rothemundes original article provides a very good basic introduction to the method
http://www.nature.com/nature/journal/v440/n7082/full/nature04586.html
Douglas et al. applied the technique for the first time to 3D structures in 2009
http://www.nature.com/nature/journal/v459/n7245/full/nature08016.html
A very good primer on the technique was also published by Castro et al. in 2009
http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1570.html
Cadnano
The design of the nanopill was made using the CAD software caDNAno. Cadnano allows an easy design of DNA structures, by offering an accessible interface for keeping track of the scaffold path through the 3D structure and assisting the user with the generation and adjustment of complimentary DNA staples.
A very good tutorial with further information and examples is available at
Gel electrophoresis
Gel electrophoresis is an established method to sort molecules by size and charge. The basic principle is to position the samples into wells inside a gel block and to apply voltage to the gel. The charged molecules migrate in the electric field, but due to the matrix structure of the gel, the different sized molecules all travel with varying velocity. After some time, distinct bands of same-velocity molecules form in the gel and can be extracted or analyzed by different methods. Gel electrophoresis can also be used for the purpose of filtration and purification.
For all of the experiments, a 2% agarose gel with TBE (Tris borate EDTA) as running buffer was used. Mg2+ is added in the form of MgCl2 to reduce the repulsion of the closely packed negative DNA backbones and to stabilize the DNA structures. A voltage of 70 V is then applied to the gel block for 3 hours. Afterwards the DNA in the gel is made visible by staining with ethidium bromide. Ethidium bromide (EtBr) intercalates DNA and emits florescence light if excited by UV radiation. DNA that came into contact with EtBr can therefore be visualized by using an UV lamp.
TEM
TEM(Transmission Electron Microscope) is a form of microscopy that relies on electrons, instead of light, to image samples. The electrons interact with the sample and the interaction is imaged by CCD sensors. The advantage compared to light microscopy is that electrons have a relatively low de-broglie wavelength, this makes resolution of objects possible that can not be imaged by light microscopy. The samples are first stained with uranyl acetate, it attaches to the DNA structures and interferes with the electrons. This interaction creates negative images of the structures on the CCD sensor.
Laser Scanner
In order to test loading mechanisms we used Cy5 fluorophores attached to double and single stranded DNA. These fluorophores can be imaged by a laser scanner, even when covered by DNA structures. This allows to detect the attachment and release of strands connected to the structure.
Specifications can be found at the manufacturers homepage
http://www.tecan.com/platform/apps/product/index.asp?MenuID=1406&ID=985&Menu=1&Item=21.10.2
