Requirements on Origami Design
For the positioning and the connection to the nanodiamonds the DNA origami structure has to fulfill certain conditions. First of all the design should contain biotin anchors to enable a specific and definite linking to the neutravidin, which is conjugated to the coated diamond. It should also be simple so that it is easy to synthesize. In order to clearly resolve different fluorescence nanodiamonds in further experiments the structure should also be larger than the resolution limit of our total internal reflection fluorescence microscope (TIRFM). Last but not least high stability is needed to support heavy nanodiamonds.
- It should provide biotin groups to link it to the neutravidin
- It should be stable enough to support the nanodiamonds bound to it
- It should be sufficiently large to get over the resolution limit of our fluorescence microscope
- It structure should be easy to synthesize
DNA single strand of the M13 bacteriaphage
- 7560 bases long
- DNA bases sequence is known
- used as scaffold for the origami structure
DNA single strand staples
- Designed with cadnano to form the structure together with the scaffold
- Two staples are modified with biotin on one end
- Functional group which binds to the neutravidin
- Contains amino groups
To fulfill all this conditions we choose the structure of a simple 6 helix bundle with biotin anchors on both sides. It was first designed with cadnano and than synthesized in the lab with a PCR-machine. After finishing the synthesis an excessive amount of neutroavidin was added. Finally the free neutroavidin and staples were removed by gel purification.
Design: Step by step
Design of the structure with cadnano
For the scaffold we used a 7560 bases DNA strand of the M13 bacteria where the sequence is known. Based on the known sequence by use of cadnano the staples can be designed to form a 6HB together with the scaffold. One of the staples placed on each end of the 6HB was modified with a biotin group. Finally special designed staples were ordered from "MWG biotech". For more information on cadnano visit: cadnano.org
Synthesize the structure in the lab
After the ordered staples arrived, we put them together with the scaffold in the PCR-Machine (Thermal cycler). The PCR-Machine controls the temperature of the assembling process for a period of 12 hours.
|80°C||5 min||Opens every staple and scaffold|
|79°C - 60°C||- 1°C every 4 min (altogether 80 min)||Pre assembly: staples beginning to order|
|60°C - 25°C||-1°C every 15 min (altogether 525 min = 8,75 h)||Assembly every staple finds it right place|
|4°C||go directly to 4°C and stay there till the end of the program||Store the assembled structure|
After 12 hours in the PCR-Machine we added neutravidin to the solution and then stored the sample at room temperature (20°C) for six hours. Since the neutravidin could also connect to two different 6HBs, we added an excessive amount of neutravidin to prevent clustering. With its four specific binding sites for biotin neutravidin acts as a link between the biotin of DNA origami and Biotin-PEG-Mal.
Purification of the structure
Six hours later we purified the modified structure in order to remove the free staples and the free neutravidin. Therefore we used a 0.7 % agarose gel, which has run for 1.5 hours at 60 V. After running the gel we cut the very left and right band and stained both with SYBR Gold to know where we had to cut the band. We didn't stain the whole gel, otherwise the DNA origami would have started to fluoresce. Then we cut the unstained band and used the "squeeze method" to separate the origami structure from the agarose gel. Finally we confirmed the profile of our structure via TEM measurements.
This shows that the 6HB structure fulfills all the conditions above:
- Modification of staples on both ends of structure with biotin groups allows specific linking to neutravidn.
- With a length of around 400 nm the distance between the diamonds should be in the resolution range of our total internal reflection fluorescence microscope (TIRF).
- 6HB is a commonly used structure which is stable and easy to synthesize.