Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

The overall goal of the following experiment is to form complex DNA nanostructures with single stranded tiles. This is achieved by mixing carefully designed synthetic DNA strands in desired buffer solution to form the desired DNA nanostructure as a second step. The sample is a kneeled during which structure self-assembly takes place.

Next, native agros gel electrophoresis and atomic force microscope imaging are performed in order to verify the successful formation of the nanostructures. The results show DNA nanostructures self-assembled based on native agros, gel electrophoresis and atomic force microscope imaging. Generally individuals new to this method will struggle because sample preparation of such a self-assembly seemed to be complicated Prior to starting this procedure obtained DNA component strands of specified sequences dissolved in RNAs free water from an oligonucleotide manufacturer in Vbo 96 well plates pipette one microliter from each of the 362 wells for the 24 helices by 28 turns.

Rectangle add 100 micromolar per strand into a two milliliter test tube. Then add 138 microliters of distilled deionized water to the test tube to make a stock solution. At a concentration of 200 nano molar per strand.

Add 50 microliters of the 200 ano molar stock solution. 10 microliters of 10 x annealing buffer A and 40 microliters of distilled deionized water to a 0.2 milliliter PCR tube. Following this ane a sample for 17 hours in a thermal cycler cooling from 90 to 25 degrees Celsius.

After preparing a native 2%agros gel prestained with SYBR safe, run it for two hours at 100 volts in an ice water bath. When finished image the gel using a gel scanner with an appropriate filter for the SYBR safe stain on a blue light Transluminator excise the dominant band with similar mobility to the band of 1, 500 base pairs of the one Kilobase DNA ladder using a sharp clean razor blade. Place the desired gel pieces in a spin column and then crush the gel into fine pieces using a micro tube Pele then centrifuge at 438 G for three minutes at four degrees Celsius.

Following centrifugation. Measure the concentration of the DNA using an ultraviolet spectrophotometer at 260 nanometers. At this point, peel off MICA attached to a specimen metallic disc using scotch tape to get a flat surface and place a specimen disc onto the imaging stage of the microscope.

Add 40 microliters of one x and kneeling buffer A followed by five microliters of the purified sample of the 24 HELOCs by 28 turns rectangle to the freshly cleaved Micah surface. Allow the mixture to settle for approximately two minutes. Install an A FM silicon nitrite cantilever chip onto a cantilever holder image the sample in the fluid tapping mode using a scan size of two micrometers 1024 lines for the resolution.

And a scan rate of 0.5 to one hertz for internal labeling attached to three prime 17 nucleotide segment to eight internal tiles and six boundary tiles of the rectangle. Mix the 28 strands with handles with the rest of the component strands of the 24 HELOCs by 28 turns rectangle to make a 200 NANOMOLAR stock solution for boundary labeling. Mix the 14 strands with handles with the rest of the component strands of the 24 heel seas by 28 turns rectangle to obtain a 200 Nanomolar stock solution for internal labeling.

For the boundary labeling, add 50 microliters of the 200 Nanomolar stock solution. 60 microliters of the 100 micromolar biotin modified anti handle strands. 10 microliters of 10 x annealing buffer A and 34 microliters of distilled deionized water to A PCR test tube.

For internal labeling, add 50 microliters of the 200 nanomolar stock solution. Three microliters of the internal anti handle biotin modified strand at 100 micromolar. 10 microliters of 10 x annealing buffer A and 37 microliters of distilled deionized water to a PCR test tube.

Next weigh 0.06 grams of urinal formate in three milliliters of distilled water. To prepare a 2%aqueous urinal formate stain solution, filter the stain solution using a 0.2 microliter filter attached to the syringe. Add five microliters of five normal sodium hydroxide to one milliliter of the filtered stain solution.

After briefly vortexing the solution centrifuge at 20, 000 G glow. Discharge the carbon coated grids with the coated side facing up using the following settings When finished, pipette 3.5 microliters of sample onto the glow discharge treated grid. After four minutes, use a piece of filter paper to wick off the sample by bringing the filter paper in contact with the grid from the side.

Following this immediately add 3.5 microliters of the stain solution onto the grid. After one minute, wick off the stain and hold the filter paper against the grid for one to two minutes. Transfer the grid to A TEM specimen holder.

An image using A-J-E-O-L GM 1400, operated at 80 kilovolts with magnification ranging from 10 K to 80 K.Once the shape has been identified, select the strands that correspond to the shape on the molecular canvas. Replace the exposed domain with a poly T segment, 10 to 11 nucleotides long, or add an edge protector that is complimentary to the exposed domain and terminate it with a 10 to 11 nucleotide long poly T segment. To prevent aggregation, create a strand library for the 310 pixel molecular canvas, which includes the corset.

Set one star set two star set, three star and set four star to give a total of 1, 344 edge protectors. After centrifuging the 96 well plates for the different sets, pipette one microliter of 206 strands from the core set, zero from set one star zero from set, two star zero from set, three star and 24 strands from set four star into a two milliliter centrifuge tube. Add 20 microliters of distilled deionized water to the tube to make a 400 nanomolar stock solution of the DNA strands.

Next, add 50 microliters of the 400 nanomolar stock solution. 10 microliters of 10 x annealing buffer B and 40 microliters of distilled deionized water to a 0.2 milliliter PCR tube ane the mixture in the thermal cycler for 17 hours as described previously. After purifying the sample and measuring the DNA concentration image, the sample using a FM in the same manner as before four finally construct different sized rectangles by simply altering the number of parallel heoc and the number of helical turns.

The self-assembly of single stranded tiles will yield a 24 HELOCs by 28 turns rectangle DNA sequences for the different single stranded tiles can be modified and optimized to enable strep divide and labeling the transformation of a rectangle into a tube. The programmable self-assembly of single stranded tiles to form tubes and rectangles of varying sizes and the construction of two dimensional arbitrary shapes using the molecular canvas Domain substitution design and edge protector design were tested as solutions to aggregation along exposed domains of arbitrary shapes. Both designs have comparable gel yield and structural integrity, but the edge protector design is more cost effective since it requires fewer auxiliary species.

After watching this video, you should have a good understanding of how to make complex DNA nanostructures based on single strand tiles.