Biomod/2013/LMU/assembly
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Nanodiamond DNA-Origami conjugation
Finally all the necessary steps for the assembly of the structure are 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 of the nanodiamonds with the 6HB. Due to the high specificity of the biotinylation the nanodiamonds are placed exactly on the designed spots, what 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. Beside the modification of the chemical process of the conjugation the simplest method to increase the yield is an appropriate purification process. Regarding this we tried different methods.
We first tried to purificate the sample by gel. In this method molecules with a different mass to charge ratio can be seperated by applying high voltage to the sample. In doing this 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, with particles of same mass but of bigger charge moving faster than less charged particles. By carefully adjusting the time and applied voltage, the molecules can be seperated. For more information on the exact protocol see: Gel purification.
Due to the mass of the nanodiamonds we thought we could seperate empty and occupied 6HB well. But it turned out that this method which is routinely used for other DNA-Origami structures does not work in our case. We either got results where different speeds could not be observed or the fluorescence signal was too weak in comparison with the initial concentration. Since our gel apparatus only can seperate 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 used in gel purification destroys the coating of the nanodiamond which is not based on chemical linking but dipole force. The mechanical destruction of the assembly while moving through the gel could also be an explanation. Unfortunately a final reason why a gel purification does not work in our case could not be verified.
To seperate our samples efficiently we currently use a centrifuge. Here only the mass of the particles is important which benefits us because of the big mass of the nanodiamonds. The heavy successfully synthesized arrangements will sink to the ground and the unoccupied 6HB can 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 the conjugation and purification of the samples we checked if the assembly worked properly. Therefore we imaged the solution with the TEM, using the sample preperation 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 comparing it to the total number of 6HB in the pictures. Because the final yield of our samples is still low we are currently putting some efforts 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 attachement of nanodiamonds to DNA-Origami for the very first time.
