Biomod/2013/LMU/assembly

From OpenWetWare

Jump to: navigation, search


Nanodiamond DNA origami conjugation

Conjugation process via neutravidin
Conjugation process via neutravidin

Finally all the necessary steps for the assembly of the structure have been taken. The nanodiamonds are coated, thus well dispersed and functionalized with biotin (see: Coating). Also the 6HB is synthesized and is connected to neutravidin on both ends (see: Origami-Design). Since the 6HB solution is purified, it contains almost no free neutravidin, which would otherwise connect to the coated nanodiamonds, preventing proper assembly of the final structure.

The neutravidin is biotinylated by the PEG anchors of the nanodiamond finalizing the conjugation with the 6HB. Due to the high specificity of the biotinylation the nanodiamonds are placed exactly on the designed spots, which can be verified by TEM images. Since the yield of this process is low, the sample has to be purified afterwards.

Purification

For further use of the sample in optical or medical applications a sufficient yield of the final structure in the solution is necessary. Apart from the modification of the chemical process of the conjugation the simplest method to increase the yield is an appropriate purification process. Regarding this we tested different methods.

We first tried to purifiy the sample by gel. In this method molecules with a different mass to charge ratio can be separated by applying high voltage to the sample. As a result, the charged molecules of the solution start to move. The direction of the movement is determined by the sign of the particles charge. The speed of the movement is mostly determined by the charge to mass ratio: Particles of the same mass but of bigger charge are moving faster than particles with a lower charge. By carefully adjusting the time and applied voltage, the molecules can be separated. For more information on the exact protocol see: Gel purification.
Due to the mass of the nanodiamonds we thought we could separate empty and occupied 6HB well. But this method which, which is routinely used for other DNA origami structures, turned out to be not usable in our case. We either got results where different speeds could not be observed or the fluorescence signal was too weak in comparison to the initial concentration. Since our gel apparatus only can separate particles moving in the same direction a reason for that could be that the overall charge of the final arrangements is opposite to the charge of the pure 6HBs. Another way to explain this is that the strong electric field that is used during the gel purification process, destroys the coating of the nanodiamonds, which is based on dipole force. The mechanical destruction of the assembly caused by moving through the gel could also be an explanation. Unfortunately, we could not identify the definite reason why the gel purification did not work in our case.

To separate our samples efficiently we currently use a centrifuge. Here only the mass of the particles is important. This benefits us because of the great mass of the nanodiamonds. The successfully synthesized arrangements are heavier and will therefore sink to the ground. The unoccupied 6HB can now be removed with a pipette. By adjusting the time of the sample in the centrifuge we could improve the final yield of our samples.

Results

After conjugation and purification of the samples, we checked if the assembly worked properly. Therefore we imaged the solution with the TEM, using the sample preparation method described in the protocols section. Since the structure was designed to bind specifically to a certain spot on the 6HB, the success of the conjugation can be checked easily. In addition to that, the yield of the method can be determined by counting the properly conjugated arrangements by comparing it to the total number of 6HB in the images. At the moment we are able to obtain a yield of roughly 10%, which is still low. Therefore we are currently putting some effort into both improving the used purification method and finding the optimum chemical conditions for the conjugation. Some TEM images of the synthesized structures are shown in the figures below. Therefore we can report specific attachment of nanodiamonds to DNA origami for the very first time.

Final nanodiamond 6HB arrangement: Model and TEM images (Scaling: 100nm)
Final nanodiamond 6HB arrangement: Model and TEM images (Scaling: 100nm)
Personal tools