Results and Discussion
All structural designs were done on caDNAno which is an open source plugin developed at Wyss Institute for Autodesk Maya; each design, once completed, was then verified using a software tool called CanDo which is provided by Laboratory for Computational Biology & Biophysics at MIT . CanDo imports the raw data from caDNAno files and outputs a 3D model of the predicted DNA structure. These models show the expected shape, capsule-like figure for the barrels, and the basket weave panel at the end of both barrels. The basket weave end has an intertwined section of scaffold and staples (Figure 2).
Optimizing Folding Conditions
Optimization of the origami structures found that each structure has unique folding salt concentration as well as thermal annealing conditions. The bundle structure folded under 1mM EDTA, 5mM TRIS, 5mM NaCl, and 20mM MgCl2 salt concentrations. Thermal annealing conditions included 12 hour cycle starting at 56 degrees C and ending at 44 degrees C with a rate of -0.2 degrees C /12 minutes. TEM imaging verified the folding of this structure (Figure 3).
The inner barrel was run on a matrix of differing magnesium and calcium concentrations. Optimal conditions included 0.5mM EDTA, 5mM TRIS, 2.5mM CaCl2, and 4.6mM MgCl2. Yield of formation hit its peak at a 12 hour folding reaction starting at 71 degrees C and ending at 44 degrees C with a rate of -0.3 degrees C / 8 minutes. Longer annealing time at this point did not lower the yield, nor did it increase it. Gel results showed a strong dimer band (Figure 4).
The monomer was verified as the properly formed structure (Figure 5). The dimer, however, turned out to be two inner barrel structures non-specifically binding to each other from the panel end, also known as blunt stacking (Figure 6). The basket weaved back panel was also verified to be present in the structure due to the dark negative stain in one end of the capsule, signifying the strong amount of DNA present in that single section.
The outer barrel was initially tried under the same conditions as the inner barrel due to the near identical structural design. However, folding salt concentrations differedslightly consisting of 0.5mM EDTA, 5mM TRIS and 2mM MgCl2. The thermal annealing conditions were the same as the 12 hour annealing optimized for the inner barrel.
Modifications to Assemble Capsule
After verification of the structures forming properly under TEM images, as well as its stability in physiological buffer, modifications were added to the inner barrel help guide the energetically disfavored assembly of these structures. The inner barrel features a series of short 20 bp strands that extend toward the interior of the structure. These staples line up the inner wall of the structure from the lip all the way to the base. Each staple binds to a specific series of regions of the 400 bp loop which extends from one end bundle structure. The staples bind to the loop in successive order to pulling the bundle inside the inner barrel structure. The two structures showed signs of assembly under a post-mix heating of a constant 37 degrees C for one hour. In Figure 3 there is a clear contrast in stain from the body of the inner barrel structure to the basket weave panel; this is due to the difference in the density of DNA in those respective locations. Images of the bundle and barrel solution under with the staple modifications for post-fold assembly show a high negative stain throughout the whole inner barrel structure. (Figure 7). This shows signs of success in the assembly technique by the usage of hybridizing extended staples for 3D origami structures.
Further investigation in the complete assembly should focus first on completing the inner-outer barrel assembly. The 400 bp loop in the outer barrel is tightly held against the lip of the barrel so that the extended staples of the inner barrel can find its hybridizing regions.
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