Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

The biological stabilization of DNA nanostructures is essential for future in-vivo applications, such as targeted drug delivery. However, DNA gets degraded in the body by various enzymes. Here we present a coating technique that protects DNA nanostructures from enzymatic degradation.

The main advantages are that our coating comprises non-toxic compounds that are already used in living organisms and that the functionalization of the surface remains possible. This functionalization is important to incorporate molecular sensors for target recognition and switches for drug cargo release. Polycation coatings significantly increases the cellular uptake of encapsulated DNA origami structures, so this method can open the door for applications of DNA origamis in diagnostics and therapeutics.

The cellular uptake is also required for potential gene delivery applications of the DNA nanotechnology. I consider that the method is quite straightforward and can be easily mastered. To begin, measure the concentration of purified DNA origami.

Use two microliters of TB buffer as the blank to calibrate ultraviolet spectrophotometer. And then measure DNA origami concentration at 260 nanometers. Then calculate the amounts of phosphates in the purified DNA origami solution, using formula two, as described in manuscript.

Prepare 15 microliters of the DNA origami structure by including one nanomole of phosphate, using TB buffer for dilution. Add 13.2 microliters to TB to 1.8 microliters of chitosan. And add 15 microliters of the chitosan solution to 15 microliters of the DNA origami to develop a DNA origami chitosan polyplex with an N/P ratio of eight.

Continue preparing polyplexes of different N/P ratios, using calculations as described in the manuscript. Prepare 80 millimeters of a tube for 2%agarose gel. Add 352 microliters of 2.5 molar magnesium chloride and then pre-stain it with 10 microliters of DNA gel stain.

Pour the gel solution into a dry gel box. Insert a gel electrophoresis comb and let it solidify at room temperature for 15 to 30 minutes. Meanwhile supplement the running buffer with 11 millimolar magnesium chloride.

Mix 10 to 20 microliters of each sample with 20%of 6X loading buffer. Load the samples into the agarose gel wells, including a naked DNA origami as control. Run the gel at 70 volts at room temperature for two and a half to three hours.

Then use a UV scanner to visualize the gel. To perform a DNase I protection assay, add four microliters of each polyplex compared at different N/P ratios, 1.5 microliters of 10X DNase I buffer, 8 microliters of TB, and 1.5 microliters of DNase I to a separate 2 milliliter PCR tube. Then incubate the same at 37 degrees celsius in a thermocycler for 24 hours.

To digest DNase I, add two microliters of 20 milligrams per milliliters proteinase K and incubate for 30 minutes at 37 degrees celsius in a thermocycler. Then, to unravel the core DNA nanostructures add 3.6 microliters of 50 milligrams per milliliter 40 kilodaltons dextran sulfate. Then use these samples and fresh DNA origami as control to perform agarose gel electrophoresis as previously described.

Excise the band corresponding to loaded samples from the gel. To extract the DNA origami, use a squeeze freeze extraction column and follow the manufacturer's instructions and continue with negative stain transmission electron microscopy imaging as described in the manuscript. Mix 6.5 microliters of purified 36 nanomolar HRP-NR with 6.5 microliters of polycations of varying concentrations.

To prepare a polyplex at N/P ratios of one, two, four, 10, and 20, prepare a blank group by mixing 6.5 microliters of distilled water with 6.5 microliters of polycations of various concentrations corresponding to the N/P ratios of one, two, four, 10, and 20. Then add 12.5 microliters of each of these prepared solutions to a separate well of a 96 well plate. Then add 72.5 microliters of HEPES buffer to each well and mix.

Add 10 microliters of 15 millimolar ABTS and then 5 microliters of a 12 millimolar hydrogen peroxide to each well and mix by pipetting up and down. Finally, use a multimode plate reader to measure absorbance at 421 nanometers over four hours. After analyzing polyplexes using a gel retardation assay, there was a shift in the naked nanostructure towards the cathode, probably due to counterbalancing of the negative charge of phosphate groups upon binding to the polycations, and the size increase of the complex.

When an extra amount of polyanions, such as dextran sulfate, was added, polyplex formation was reversed. Dextran sulfate of higher molecular weight showed to be more efficient compared to lower molecular weight. After analyzing the polyplexes using negative stain transmission electron microscopy, micrographs showed naked DNA origami nanostructures, linear polyethylenimine polyplexes after decapsulation, chitosan polyplexes, images of naked and protected DNA origami subjected to magnesium depletion, enzymatic degradation, and serum digestion.

In the presence of DNase I, after two hours, naked nanostructures were completely digested, whereas the linear polyethylenimine polyplexes of chitosan encapsulated DNA origami stayed intact in the presence of 10 units per milliliter DNase I.Linear polyethylenimine polyplexes protect the DNA nanostructures more efficiently compared to chitosan. When the N/P ratio of the polyplex is increased, DNA digestion was lower. After coating enzyme, or after more functionalized DNA origami, with linear polyethylenimine polyplexes and chitosan at variant N/P ratios, no remarkable interference with enzymatic activity was observed.

On the other hand, the kinetic of HRP enzyme dramatically changed after binding to the DNA origami. It is important to start with well purified, DNA origami structures. There exists a stabler strand that can also bind to polycations.

They are difficult to be separated from the DNA origami polyplexes. Our coating molecules are also used in gene therapy applications. This means the DNA nanostructures can be used in future to alter the genome of living organisms.

For agarose gel staining, usually molecules are used that intercalate the DNA and are potentially mutagenic. Please follow the safety instructions of your laboratory when staining DNA.