Biomod/2013/LMU/origamidesign

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Requirements on Origami design

For the positioning and the connection to the nanodiamonds the DNA-Origami structure has to fulfill some conditions. First of all it 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 see (resolve) the fluorescence nanodiamonds in further experiments separated the structure should also be longer than the resolution limit of our total internal reflection fluorescence microscope (TIRFM). Last but not least high stability is needed to support heavy nanodiamonds.

Structure requirements:

  • Provide biotin groups to link it to the neutravidin
  • It should be stable enough to support the nanodiamonds bound to it
  • Should be sufficiently large to get over the resolution limit of our fluorescence microscope
  • The structure should be easy to synthesize

Plan

Materials included:

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

Biotin

  • functional group which binds to the neutravidin
  • contains amino groups


Short explanation:

To fulfill all this conditions we choose the structure of a simple 6 helix bundle "6HB"(short: 6HB) 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

CadNano interface

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

6HB structure
6HB with free neutravidin

After the ordered staples arrived we put them together with the scaffold in the PCR-Machine (Thermal cycler). The PCR-Machine controls over 12 hours the temperature of the assembling process.


Temperature Time assemble step
80°C 5 min Put every staple and the scaffold open
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. The sample was stored at room temperature (20°C) for a other additional six hours. Since the neutravidin could possibly link toalso connect two 6HBs we add an excessive amount to prohibit clustering. With its four specific binding sites for biotin it acts as an adapter to link the biotin groups of the origami to the nanodiamonds during the final conjugationneutravidin works as a bridge between the biotin of DNA-origami and Biotin-PEG-Mal.

Purification of the structure

Purified 6HB structure modified with attached neutravidin
Gel fluorescence picture

Six hours later we purified the modified structure from the free staples and the free neutravidin. Therefore we used a 0.7 % agarose gel, which ranhas run for 1.5 hours with 60 V. After running the gel we cut the very left and the very right band and stunnedstained both of them with SYBR Gold to know where we had to cut the band. We didn't stain the hole gel to prevent that the origami is going to fluoresce (because of the Sybr Gold) in further experiments with the fluorescence nanodiamonds, otherwise the DNA-origami would have started to fluoresce. Then we cut the unstained band and used the "squeeze method" ???link to protocol to separate the origami structure from the agarose gel. Finally we checked the structure with the TEM and as one can see from the picture the structure looks quite good.

Review

TEM picture of the modified and purified 6HB structure

This means that the 6HB structure fulfills all the conditions set in from the beginning:

  • The modification of staples on both ends of structure with biotin groups allows specific linking to the 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).
  • Finally the 6HB is a commonly used structure which is stable and easy to synthesize.
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