Biomod/2014/ASU/Discussion

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Biomod/2014/ASU

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Discussion


Goal 1: Creation of a DNA antibody capable for binding a target protein.

Outcome: We designed a DNA antibody with the dimensions 28nm x 9nm, incorporating four oligonucleotide sequences at positions corresponding to the size of the target protein, alpha-thrombin. The DNA antibodies were imaged by atomic force microscopy, which showed small rectangular structures measuring the correct dimensions (see Figure R1).


Goal 2: Demonstration of DNA antibody binding to protein using positive control DNA strands.

Outcome: Positive control DNA antibodies were made by incorporating two aptamers with sequences known to bind alpha-thrombin into different arrangements on our DNA antibody (see Figure D9). Electrophoretic mobility shift assay showed band shifts for each tile incubated with protein relative to just the tile (see Figure R3) suggesting that the positive control DNA antibodies did bind alpha-thrombin. This shows that the DNA antibodies work as a system to bind a target protein with respect to the selection process.


Goal 3: Demonstration of DNA antibody binding to protein using randomized DNA oligonucleotides.

Outcome: DNA antibodies were created using randomized oligonucleotides. Two methods were used to characterize binding between the DNA antibody and alpha-thrombin: EMSA (electrophoretic mobility shift assay) and column chromatography.

The EMSA proved inconclusive because shifted bands representing DNA antibodies that bound protein were too faint to detect (see Figure R4). It is suggested that this is due to only a very small fraction of the pool of random DNA antibodies would be able to bind the protein.

The column chromatography method used NHS ester beads which we functionalized to bind alpha-thrombin. We used a method of column chromatography in which only DNA that bound to the protein would remain in the column whereas that which did not would run through. The problem we encountered was that the DNA also bound nonspecifically to the beads. Thus, this method would select for some DNA which did not actually bind the protein (see Figure R7). Because of the sensitivity of PCR, these strands were also very amplified and the process was not very selective. In the future, other types of columns will be investigated such as those using magnetic beads, in the hopes that they will not bind DNA antibodies nonspecifically.

Overall, it is very possible that DNA antibodies were generated from the random pool which were able to selectively bind alpha-thrombin However, these DNA antibodies might be very few in number and the methods used so far were unable to detect the tiles adequately.


Goal 4: Demonstration of the recovery of the DNA antibody after binding to the protein and the amplification of the randomized oligonucleotide strands for confirmation by sequencing.

Outcome: In order to generate more copies of DNA antibodies which bind the protein, PCR was performed on recovered, selected tiles using primers for the oligonucleotide strands. This amplifies multiple copies of the selected oligonucleotide strands which can be used to further enrich the oligonucleotide pool incorporated into DNA antibodies for another round of selection. Figure R9 demonstrates that the primers were able to amplify randomized oligonucleotides through PCR, which suggests that the recovery and amplification of selected DNA antibodies is possible.


Goal 5: Creation of molecular dynamics simulation as a visualization tool for the project.

Outcome:A pdb file of the DNA antibody was generated and used for visualization. This file was also prepared for molecular dynamic simulations, but proper resources to run the simulations were not available. Future simulations are planned in order to have a better visual representation of the interaction between the DNA antibody and alpha-thrombin.



The unique programmability of DNA nanotechnology opens up a host of possibilities for a DNA structure with the selectivity and function of an antibody. By incorporating highly specific and selective binding functionality into DNA tiles and other nanostructures, DNA antibodies open the possibility for further, highly diverse interactions of DNA nanotechnology with proteins. Applications of this research include, but are not limited to, targeted drug delivery systems, increasing efficiency in enzymatic pathways, and a new economical way to isolate proteins.


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