This method extends DNA nanotechnology from the source materials of linear oligonucleotide, DAS, into some more circular DAS. The main advantages of these technical are that it is a simple, robust and affordable for most labs. For circular DNA purification, add 20 microliters of formamide to a solution of freshly prepared, intact circular DNA, to a final volume of 140 microliters with mixing.
Load 16 to 20 microliters of circular DNA Solution to each of seven to nine denaturing PAGE gel wells. Mix two microliters of loading dye with eight microliters of the corresponding precursor linear DNA, and inject the mixture into a separate reference well. Then run the gel for about two hours at 10 volts per centimeter, stopping the run when the xylene cyanol FF can be observed about two-thirds down the gel.
Wearing rubber gloves and goggles, transfer the gel onto a fluorescent thin-layer chromatography plate, and use a razor blade to cut off the target gel bands, as shadowed by the UV irradiation. Place the gel bands into a two milliliter microcentrifuge tube for drying, then mash the dried gels into a paste, and add de-ionized water at twice the volume of gel, for overnight shaking at room temperature. The next morning, filter the mixture into a two milliliter microcentrifuge tube, rinsing the sides of the first tube with a small volume of de-ionized water to recover any residual DNA, and filtering again to combine the supernates.
Then purify the circular DNA via ethanol precipitation, and store the DNA at minus 20 degrees Celsius. For one pot annealing, mix two microliters of 10-fold Tris acetate EDTA magnesium buffer, one microliter of each 10 micromolar DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional Tris EDTA buffer in a 200 microliter PCR test tube, to a final volume of 20 microliters. Then anneal the assembly solution in a thermal bottle from 95 to 25 degrees Celsius, over 48 hours.
For two-step annealing of infinite lattices, mix two microliters of 10-fold Tris acetate EDTA magnesium buffer, one microliter of each 10 micromolar DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional Tris EDTA buffer in a 200 microliter PCR test tube, to a final volume of 20 microliters. In the first sub-tile annealing step, anneal each precursor sub-tile solution in a PCR thermocycler, using a fast linear cooling method from 95 to 25 degrees Celsius, over two and a half hours. To prepare the assembly solution, for each of the four infinite lattices, mix 10 microliters of each of the two corresponding sub-tile solutions together, to a final concentration of 0.25 micromolar for each strand.
In the second lattice annealing step, anneal the 20 microliter mixture in a PCR thermocycler, using a slow cooling method. For two step annealing of two finite rectangle lattices, mix one microliter of 10 fold Tris acetate EDTA magnesium buffer, 0.5 microliters of each 10 micromolar DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional Tris EDTA buffer in a 200 microliter PCR test tube, to a final volume of 10 microliters. In the first annealing step, anneal each precursor sub-tile solution in the PCR thermocycler, using the fast linear cooling method, as demonstrated, from 95 to 25 degrees Celsius, over two and a half hours, and mix the annealed 32 sub-tile solutions, each with 2 microliters, together in a 200 microliter PCR test tube, to a final volume of 64 microliters.
Then, in the second annealing step, anneal the 64 microliter mixture in the thermocycler, using the slow cooling method, as demonstrated. For electrophoresis purification of the finite lattices, prepare a native 2%agarose gel and load 20 microliters of the finite lattice solution into each well, along with the addition of 5 microliters of a 100 to 3000 base-pair DNA ladder, in a separate well. After running the gel for two hours at 5 volts per centimeter in an ice water bath, cut the target gel bands at about the level of the 1000 base pair marker under UV light, as demonstrated, and slice the gel bands into fine pieces.
Place the crushed gels into a filter column, and centrifuge the column to extract the lattices. Then, collect the solution for atomic force microscopy imaging. For finite lattice purification by polyethalene glycol, or PEG, mix 50 microliters of the finite lattice solution with an equal volume of 15%PEG 8000 buffer, centrifuge the solution, and re suspend the pellet in 100 microliters of PEG buffer, for an additional centrifugation.
After the second centrifugation, re suspend the pellet in Tris acetate EDTA magnesium buffer, for atomic force microscopy imaging. For infinite 2D lattice atomic force microscope imaging, cleave off a fresh layer of mica, and deposit two microliters of the annealed sample onto the clean surface. Allow the DNA lattices two minutes to absorb to the mica surface, before washing the surface two times, with 100 microliters of de-ionized water per wash, drying the surface with compressed air after the second wash.
To obtain atomic force microscope images of the infinite DNA lattices in air, open the tapping mode in the imaging software, and set the scan size to 0.5 to five micrometers, the scan resolution to 512 lines, and the scan rate between one to 3.5 Hertz. Then, scan the mica surface with triangular atomic force microscope probes. For finite DNA lattices imaging, cleave off a fresh layer of mica, as demonstrated, and deposit 80 microliters of one fold Tris acetate EDTA magnesium buffer onto the clean mica surface.
Next, add five microliters of finite samples into the buffer, and allow the DNA lattices two minutes to absorb to the mica surface, before adding another 50 microliters of Tris acetate EDTA magnesium buffer to the probe. Then, set the scan size to 0.5 to one micrometer, the scan resolution to 256 lines, and the scan rate to 1.5 Hertz, before scanning the surface with the triangular probes, as demonstrated. The circular DNA moves slightly slower than it's precursor linear DNA in the denaturing PAGE, because the pore inside the circular DNA is penetrated and retarded by the gel fibers.
The circular 64 nucleotide assembly families have only one clear and clean band for each assembly, confirming the stability of their monomer motifs. The circular 84 nucleotide assembly families of tiles, however, have smears around their main bands, indicating the presence of minor by-products. Regardless of the minor by products of incorrect associates, excellent lattices with high yields can be assembled from the target monomer motifs.
Each assembly has it's own morphological features in the micrometer scale, such as nanowires, nanotubes, nanospirals, nanoribbons, and nanorectangles. While attempting this procedure, it is important to remember to avoid, particularly, debris contamination during or off the steps. After it's development, this technique paved the way for researches in the field of DNA nanotechnology to explore new family members of some more circular DNA nanotechnology.
