Name: Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles (Video) | JoVE Journal
Uploaded: 2015-05-08T13:00:00.000+00:00
Duration: 10 min 23 s
Description: Learn DNA nanostructure self-assembly using single-stranded DNA tiles with annealing, gel electrophoresis, and atomic force microscopy verification. This protocol helps researchers construct programmable nanoscale shapes.

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5 minutes

This work was funded by the Office of Naval Research Young Investigator Program Award N000141110914, Office of Naval Research Grant N000141010827, NSF CAREER Award CCF1054898, NIH Director&#8217;s New Innovator Award 1DP2OD007292 and a Wyss Institute for Biologically Inspired Engineering Faculty Startup Fund (to P.Y.) and Tsinghua-Peking Center for Life Sciences Startup Fund (to B. W.).

1. DNA Sequence Design
<ol>
	<li>Use UNIQUIMER software27 to design a SST-finite structure by specifying the number of double helices, lengths of top and bottom helix for each double helix, and the crossover pattern to create a 24H &#215; 28T canvas. After defining these parameters, the overall architecture (strand composition and the complementarity arrangement) is illustrated graphically&#160;in the program.</li>
	<li>Generate sequences for the strands of the specified structure to meet the complementarity arrangement and additional requirements (if any). Design DNA sequences by minimizing the sequence symmetry28 (for most of the structures).
	<ol>
		<li>Generate nucleotides (A, C, G and T) randomly one by one.</li>
		<li>Match nucleotides complementary to those generated following the base-pairing rule: A to T and vice versa; C to G and vice versa.</li>
		<li>Do not use repeating segments beyond eight or nine nucleotides. When such repeating segments emerge during design, mutate the most recently generated nucleotides until the repeating-segment requirement is satisfied.</li>
		<li>Do not use four consecutive A, C, G or T bases.</li>
		<li>Use pre-specified nucleotides at the single-stranded linkage points (for example T and G as the twenty-first and twenty-second nucleotides, respectively, for most of the strands) to avoid sliding bases around the linkage points (e.g., if both twenty-first and twenty-second nucleotides were T, it was possible for the twenty-first nucleotide to bind to the nucleotide that the twenty-second nucleotide is supposed to bind to).</li>
	</ol>
	</li>
	<li>Modify DNA sequences manually for streptavidin labeling, the transformation of rectangles into tubes, the formation of different rectangles and tubes across different scales, and to prevent aggregation caused by exposed domains on SST.
	<ol>
		<li>Replace the exposed domains on the boundary of the molecular canvas with poly-T tracts.</li>
		<li>For streptavidin labeling, design the handle segments (the extra segment appended to the component strand that can bind to a complementary counterpart such as an anti-handle strand) such that they can accommodate an anti-handle strand with 3&#39; biotin modification.</li>
		<li>For the conversion of a rectangle into a tube, remove the top and bottom rows from the rectangle and insert a new row whose SSTs have specific complementarity to the corresponding tiles on the second to top row and the second to bottom row.</li>
		<li>For an exposed single stranded domain of different shapes, replace with poly-T segments of 10-11 nucleotides length, or cover by edge protectors that are complementary to the exposed domain and terminate with a 10-11 nucleotide long poly-T segments.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Molecular Canvas
<ol>
	<li>Obtain DNA component strands of specified sequences dissolved in RNase-free water from an oligonucleotide manufacturer in V bottom 96 well plates with oligonucleotides synthesized at a 10 nmol scale with standard desalting. Centrifuge the 96 well plates with the DNA strands for roughly 10 s at 600 x g.
	<ol>
		<li>Pipette 1 &#956;l from each of the 362 wells with DNA strands for the 24H &#215; 28T rectangle (Figure 3A) at 100 &#956;M per strand (manufacturer specification) into a 2 ml test tube. Add 138 &#956;l of distilled deionized H2O to the 2 ml test tube to make a stock solution of the strand mixture at a concentration of 200 nM per strand.</li>
	</ol>
	</li>
	<li>Add 50 &#956;l of the 200 nM stock solution of strand mixture, 10 &#956;l of 10X annealing buffer A (50 mM Tris, pH 7.9, 10 mM EDTA, 250 mM MgCl2), and 40 &#956;l of distilled deionized H2O to a 0.2 ml PCR tube. This makes a 100 &#956;l sample of the molecular canvas in 1X annealing buffer A (5 mM Tris, pH 7.9, 1 mM EDTA, 25 mM MgCl2).</li>
	<li>Anneal the sample for 17 hr in a thermal cycler cooling from 90 &#176;C to 25 &#176;C. Program the thermal cycler as follows: ramp down from 90 &#176;C to 61 &#176;C at a constant speed of 5 min per &#176;C; ramp down from 60 &#176;C to 25 &#176;C at a constant speed of 20 min per &#176;C; and incubate at 4 &#176;C until the sample can be taken out of the thermal cycler.</li>
	<li>Prepare a native 2% agarose gel.
	<ol>
		<li>Measure 2.4 g of agarose into a 600 ml beaker. Add 120 ml of 0.5X TBE buffer (44.5 mM Tris-Borate, pH 8.3, 1 mM EDTA) and 30 ml of distilled deionized water to the beaker.</li>
		<li>Microwave for 3 min (or 40 - 60 sec more after boiling). Replenish the water lost during evaporation by addition of 30 ml of water, which helps to maintain the concentration of agarose in the gel at 2%.</li>
		<li>Swirl the contents of&#160;the beaker for 1 min in a cold water bath. Add 1 ml of 1.2 M MgCl2 (to make a 10 mM MgCl2 concentration in the gel) and pre-stain the gel with 6 &#956;l of SYBR Safe dye (1:20,000).</li>
		<li>Pour the gel solution into a dry gel box and insert a 1.5 mm thick 12 well gel electrophoresis comb. Let the solution solidify for 15 - 30 min on an even platform.</li>
		<li>Pour gel running buffer (44.5 mM Tris-Borate, pH 8.3, 1 mM EDTA, and 10 mM MgCl2) into the gel box. Load 2 - 3 &#956;l of 1 kb DNA ladder into a well of the agarose gel. Mix 4 &#956;l of 6X bromophenol blue dye (0.25% bromophenol blue, 5 mM Tris, 1 mM EDTA, 10 mM MgCl2 and 30% glycerol) with 20 &#956;l of the sample of the molecular canvas and load the solution into another well of the agarose gel.</li>
	</ol>
	</li>
	<li>Run the native 2% agarose for 2 hr at 100 V in an ice water bath (bromophenol blue, shown in blue, runs faster than component strands so it can serve as an indicator that if 2 hr running time is appropriate).</li>
	<li>Image the gel using a gel scanner with an appropriate filter for the SYBR safe stain (Figure 2B).</li>
	<li>On a blue light transilluminator, excise the dominant band with similar mobility to the band of 1,500 base pairs of the 1 kb DNA ladder using a sharp clean razor blade. Place the desired gel piece(s) in a spin column and then crush the gel into fine pieces using a microtube pestle. Centrifuge at 438 x g for 3 min at 4 &#176;C.</li>
	<li>Measure the concentration of the DNA using an ultra violet spectrophotometer at 260 nm.
	<ol>
		<li>Use 2 &#956;l of ultrapure DNase/RNase-Free distilled water as the blank to calibrate the machine.</li>
		<li>Use an equivalent volume of the collected sample from the spin column to get an estimate of the DNA concentration. The measured concentration value (&#8220;a&#8221; = ng/&#956;l) obtained at a UV absorption of 260 nm will help determine the dilution factor for AFM and TEM imaging.</li>
		<li>Convert the measured concentration from &#8220;a&#8221; (ng/&#956;l) to &#8220;b&#8221; (nM) following b = a &#215; 106 / (330 &#215; number of nucleotides). 
		NOTE: The molecular weight of a nucleotide is 330 g/mol. The calculated molar concentration value will determine the dilution factor for AFM and TEM imaging. As for the case of a 24H &#215; 28T rectangle, the common measurement for the purified sample is around 10 - 60 ng/&#956;l. As the number of nucleotides for such a structure is 14616 Da, the molar concentration is approximately 2 - 12 nM. The final concentration is determined by the collection yield (~20 - 50%) from centrifugation and the volume of the gel band excised out (the higher the volume, the more dilute the purified sample).</li>
	</ol>
	</li>
</ol>
3. Atomic Force Microscopy Imaging
<ol>
	<li>Peel off mica attached to a specimen metallic disc using scotch tape to get a flat surface and place the specimen disc onto the imaging stage.</li>
	<li>Add 40 &#956;l of 1X annealing buffer A followed by 5 &#956;l (2 - 5 nM) of the purified sample of the 24H &#215; 28T rectangle to the freshly cleaved mica surface. Allow the mixture to settle for approximately 2 min.</li>
	<li>Install AFM silicon nitride cantilever chip onto a cantilever holder. Use a C triangular tip (resonant frequency, f0 = 40 - 75 kHz; spring constant, k = 0.24 Nm-1) for imaging.</li>
	<li>Image a sample in the fluid-tapping mode. Use the following imaging parameters for scanning: scan size of 2 &#956;m, 1024 lines for the resolution, and a scan rate of 0.5 - 1 Hz (Figure 2C).</li>
	<li>If necessary, add 10 &#956;l or more of 10 mM NiCl2 solution to increase the strength of DNA-mica binding29.</li>
</ol>
4. Sample Preparation for Streptavidin Labeling
<ol>
	<li>Manually design the 24H &#215; 28T rectangle such that&#160;handle strands at specific locations&#160;are incorporated to be complementary to the anti-handle strands with biotin modification, which in turn are able to bind to streptavidin specifically.
	<ol>
		<li>Modify the molecular canvas by attaching a 3&#39; 17-nucleotide segment that consists of a two-nucleotide sequence (TT) as a spacer and a 15-nucleotide handle (GGAAGGGATGGAGGA) to the 14 tiles on the top row and 14 tiles on the bottom row of the rectangle (Figure 3A) to label the boundary. This sequence is complementary to a 3&#39; biotin modified anti-handle strand (TCCTCCATCCCTTCC-biotin).</li>
	</ol>
	</li>
	<li>For internal labeling, attach the 3&#39; 17 nucleotide segment to 8 internal tiles and 6 boundary tiles of the rectangle (Figure 4A).
	<ol>
		<li>Mix the 28 strands with handles (boundary labeling) with the rest of the component strands of the 24H &#215; 28T rectangle (334 strands) to make a 200 nM stock solution. Mix the 14 strands with handles (internal labeling) with the rest of the component strands of the 24H &#215; 28T rectangle (348 strands) to obtain a 200 nM stock solution.</li>
	</ol>
	</li>
	<li>For boundary labeling, add 50 &#956;l of the 200 nM stock solution of the strands, 6 &#956;l of the 100 &#956;M biotin modified anti-handle strands, 10 &#956;l of the 10X annealing buffer A, and 34 &#956;l of distilled deionized water to a 0.1 - 0.2 ml PCR test tube. The final concentration of the component strand is 100 nM and the final concentration of the anti-handle biotin modified strand is 6,000 nM. Note that 28 molecules of the anti-handle biotin modified strand bind to a 24H &#215; 28T rectangle so the anti-handle biotin modified strand is in excess of 100% to make the binding favorable.</li>
	<li>For internal labeling, add 50 &#956;l of the 200 nM stock solution of the strands, 3 &#956;l of the internal anti-handle biotin modified strand at 100 &#956;M, 10 &#956;l of the 10X annealing buffer A, 37 &#956;l of distilled deionized water to a 0.1 - 0.2 ml PCR test tube. The final concentration of the component strand is 100 nM and the final concentration of the anti-handle biotin modified strand is 3,000 nM. Note that 14 molecules of the anti-handle biotin modified strand bind to a 24H &#215; 28T rectangle so anti-handle biotin modified strand is in excess of 100% to make the binding favorable.</li>
	<li>Anneal both samples in a thermal cycler for 17 hr using the steps described in 2.3.</li>
	<li>Prepare a native 2% agarose gel as described in steps 2.4.</li>
	<li>Purify both samples as described in steps 2.5.</li>
	<li>Measure the concentrations of the desired DNA structures as described in steps 2.6.</li>
</ol>
5. Atomic Force Microscopy For Streptavidin Labeling
<ol>
	<li>Image each sample using an AFM as described in step 3 (Figures 3B and 4B).</li>
	<li>After the first round of imaging, add 40 &#956;l of 1X annealing buffer A followed by 1 &#956;l of streptavidin at 10 mg/ml to the sample. Allow the mixture to settle for approximately 2 min before reimaging (Figures 3C and 4C).</li>
</ol>
6. Converting A Rectangle Into A Tube
<ol>
	<li>In order to design a tube based on the rectangle, keep all of the component SSTs in place except the top and bottom rows. Introduce a new row whose SSTs are designed by concatenating the top and bottom half-tiles of their respective columns to cyclize the original rectangle into a tube conformation (Figure 5A).</li>
	<li>Mix the new row of strands (14 strands) with the component strands of the 24H &#215; 28T rectangle excluding the top and bottom rows (336 strands) to obtain a 200 nM stock solution.</li>
	<li>Follow the gel purification steps 2.2 - 2.7 (Figure 5B).</li>
</ol>
7. Transmission Electron Microscope Imaging
<ol>
	<li>Weigh 0.06 g of uranyl formate in 3 ml of distilled water to prepare a 2% aqueous uranyl formate stain solution. Filter the 2% aqueous uranyl formate stain solution using a 0.2 &#181;m filter attached to a syringe.
	<ol>
		<li>Add 5 &#181;l of 5 N NaOH to the 1 ml of filtered stain solution. Briefly vortex the solution and centrifuge at 20,000 x g.</li>
	</ol>
	</li>
	<li>Glow discharge the carbon-coated grids (coated side facing up) with the following settings: 25 mA, 45 s, 0.1 mBar, and negative High Tension polarity.</li>
	<li>Use self-closing forceps to grab a glow discharger treated grid from the edge.</li>
	<li>Pipette 3.5 &#181;l of sample onto the grid for 4 min. Use a piece of filter paper to wick off the sample by bringing the filter paper in contact with the grid from the side.</li>
	<li>Immediately add 3.5 &#181;l of the stain solution onto the grid for 1 min.</li>
	<li>Wick off the stain as in 7.4 and hold the filter paper against the grid for 1 - 2 min.</li>
	<li>Transfer the grid to a TEM specimen holder and image using a JEOL JEM-1400 operated at 80 kV with magnification ranging from 10 K to 80 K (Figure 5C).</li>
</ol>
8. Constructing Arbitrary Shapes Using the Molecular Canvas
<ol>
	<li>Identify a shape and select the strands that correspond to the shape on the molecular canvas. For a triangle shape for example, select 206 out 362 strands for the molecular canvas.</li>
	<li>Replace the exposed domain (that is, along the hypotenuse of the triangle) with a poly-T segment 10 - 11 nucleotides long (design 1 in Figure 6A), or add an edge protector that is complementary to the exposed domain and terminate it with a 10 - 11 nucleotide long poly-T segment (design 2 in Figure 6A) to prevent aggregation. 
	NOTE: Simply annealing the SSTs that correspond to the pixels of the triangle (without using protector strands) will lead to aggregation and no distinct product band formation on a gel. Use the edge protector design for various shapes as it is more resource efficient; it requires only four additional sets of strands (Figure 6C) as compared to 14 additional sets in replacement design (Figure 6B).</li>
	<li>Create a strand library for the 310-pixel molecular canvas which includes the core set (the 362 SST canvas),&#160;Set 1* (edge protectors that bind to domain 1 of a SST), Set 2* (edge protectors that bind to domain 2 of a SST), Set 3* (edge protectors that bind to domain 3 of a SST), and&#160;Set 4* (edge protectors that bind to domain 4 of a SST) to give a total of 1344 edge protectors.</li>
	<li>Centrifuge the 96 well plates for the different sets for roughly 10 s. Pipet out the desired strands from the plates in order to make a stock solution for a certain shape.
	<ol>
		<li>For the shape of a triangle with edge protectors, pipette 1 &#956;l of 206 strands from the core set, 0 from set 1*, 0 from set 2*, 0 from set 3* and 24 strands from set 4* into a 2 ml centrifuge tube. Add 20 &#956;l of distilled deionized water to the tube to make a 400 nM stock solution of the DNA strands.</li>
	</ol>
	</li>
	<li>Add 50 &#956;l of the 400 nM stock solution of DNA strands, 10 &#956;l of the 10X annealing buffer B (50 mM Tris, pH 7.9, 10 mM EDTA, 125 mM MgCl2) stock solution and 40 &#956;l distilled deionized H2O to a 0.2 ml PCR tube. This makes a 100 &#956;l sample of the triangle in 1X annealing buffer B (5 mM Tris, pH 7.9, 1 mM EDTA, 12.5 mM MgCl2) with strands at a concentration of approximately 200 nM per strand.</li>
	<li>Anneal the mixture in a thermal cycler for 17 hr as described in step 2.3.</li>
	<li>Prepare a native 2% agarose gel as described in step 2.4.</li>
	<li>Purify the sample as described in step 2.5.</li>
	<li>Measure the concentration of the DNA as described in step 2.6.</li>
	<li>Image the sample under AFM as described in step 3 (Figure 7C and 7D).</li>
	<li>Pick and mix strands for different shapes using the same strand library. Anneal the different solutions and combine purified samples of different shapes to save time during AFM imaging.</li>
</ol>
9. Optional: Robot Automation Of Shape Design and Liquid Mixing
<ol>
	<li>Use a custom MATLAB program to aid the design of complex shapes.</li>
	<li>Use the software to deliver instructions in the form of a pipetting sequence to a robot liquid handler. Use the robot liquid handler to select and mix the strands that constitute the target shape. 
	NOTE: Shape design and strand mixing was automated to reduce human error and make the construction less labor intensive.</li>
</ol>
10. Rectangles and Tubes Across Different Scales
<ol>
	<li>Construct different sized rectangles (Figure 10) by simply altering the number of parallel helices (H) and the number of helical turns (T).</li>
	<li>Follow Step 3 for a specific size rectangle.</li>
	<li>Follow Step 6 for a specific size tube.</li>
</ol>

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<li>Voigt, N. V., Nangreave, J., Yan, H., Gothelf, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+origami%3A+a+quantum+leap+for+self-assembly+of+complex+structures%5BTitle%5D)+AND+%22Chem.+Soc.+Rev%22%5BJournal%5D)">DNA origami: a quantum leap for self-assembly of complex structures.</a> Chem. Soc. Rev. 40 (12), 5636-5646 (2011).</li>
<li>Wei, B., Dai, M., Yin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+Shapes+self-assembled+from+single+stranded+DNA+tiles%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Complex Shapes self-assembled from single stranded DNA tiles.</a> Nature. 485 (7400), 623-626 (2012).</li>
<li>Wei, B., Wang, Z., Mi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uniquimer%3A+software+of+de+novo+DNA+sequence+generation+for+DNA+self-assembly%3A+an+introduction+and+the+related+applications+in+DNA+self-assembly%5BTitle%5D)+AND+%22J.+Comput.+Theor.+Nanosci%22%5BJournal%5D)">Uniquimer: software of de novo DNA sequence generation for DNA self-assembly: an introduction and the related applications in DNA self-assembly.</a> J. Comput. Theor. Nanosci. 4 (1), 133-141 (2007).</li>
<li>Seeman, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+design+of+sequences+for+nucleic+acid+structural+engineering%5BTitle%5D)+AND+%22J.+Biomol.+Struct.+Dyn%22%5BJournal%5D)">De novo design of sequences for nucleic acid structural engineering.</a> J. Biomol. Struct. Dyn. 8 (3), 573-581 (1990).</li>
<li>Hansma, H. G., Laney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+binding+to+mica+correlates+with+cationic+radius%3A+assay+by+atomic+force+microscopy%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">DNA binding to mica correlates with cationic radius: assay by atomic force microscopy.</a> Biophys. J. 70 (4), 1933-1939 (1996).</li>
<li>Seelig, G., Soloveichik, D., Zhang, D. Y., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme-free+nucleic+acid+logic+circuits%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Enzyme-free nucleic acid logic circuits.</a> Science. 314 (5805), 1585-1588 (2006).</li>
<li>Yin, P., Choi, H. M. T., Calvert, C. R., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programming+biomolecular+self-assembly+pathways%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Programming biomolecular self-assembly pathways.</a> Nature. 451 (7176), 318-322 (2008).</li>
</ol>

The authors declare competing financial interests.

In the structure formation step, it is important to keep an appropriate concentration of magnesium cations (e.g., 15 mM) in the DNA strand mixture to self-assemble DNA nanostructures. Similarly, in the agarose gel characterization/purification step, it is important to keep an appropriate magnesium cation concentration (e.g., 10 mM) in the gel and the gel running buffer to retain the DNA nanostructures during electrophoresis. For the 24H&#215;28T rectangle structure, we tested annealing in different Mg<s...

Previous nucleic acid self-assembly work1-25 has led to the successful construction of a variety of complex structures, including DNA2&#8211;5,8,10&#8211;13,17,23 or RNA7,22 periodic3,4,7,22 and algorithmic5 two-dimensional lattices, ribbons10,12 and tubes4,12,13, 3D crystals17, polyhedra11 and finite, 2D shapes7,8. A particularly effective method is scaffolded DNA origami, whereby a single scaffold strand is folded by many short auxiliary staple strands to form a complex shape9,14&#8211;16,18&#8211;21,25.
We recently reported a method for constructing discrete nanostructures with prescribed 2D shapes using single-stranded tiles (SST), and demonstrated structures with complexity comparable to DNA origami26. This article is an adaptation of our earlier work26 and describes detailed protocols for arranging individually addressable SSTs into sophisticated finite 2D shapes with precisely prescribed dimensions (widths and lengths) and morphologies. One key advantage of the SST method is its modularity. Every component SST of a structure serves as a modular construction unit in the assembly, and different subsets of these SSTs produce distinct shapes. Thus, we established a general platform to construct nanostructures with prescribed sizes and shapes from short, synthetic DNA strands.
SSTs contain four domains, each 10 or 11 nucleotides long (Figure 1A). The SSTs bind such that their parallel helices create a DNA lattice held together by crossover linkages. Each crossover is the phosphate between domains 2 and 3. The phosphate is stretched artificially in the diagrams for visual clarity. The crossovers are spaced two helical turns (21 bases) apart (Figure 1B). The composite rectangles are referred to by their dimensions in the number of helices and helical turns. For example, a rectangle that is six helices wide and eight helical turns long is referenced as a 6H &#215; 8T rectangle. SSTs can be left out, added, or otherwise rearranged to create structures of arbitrary shapes and sizes (Figure 1C). For instance, a rectangular design can be rolled into a tube with a desired length and radius (Figure 1D).
Alternatively, the rectangular SST lattice can be viewed as a molecular canvas made up of SST pixels, each 3 nm by 7 nm. In this study, we use a molecular canvas of 310 full-length internal SSTs, 24 full-length SSTs making up the left and right boundaries, and 28 half-length SSTs forming the top and bottom boundaries. The canvas has 24 double helices linked by crossovers and each helix contains 28 helical turns (294 bases) and is therefore referred to as a 24H &#215; 28T rectangular canvas. The 24H &#215; 28T canvas has a molecular weight similar to that of a DNA origami structure created from an M13 phage scaffold.

<table><tbody><tr><td>DNA Strands&amp;nbsp;</td><td>Integrated DNA Technology</td><td></td><td>Section 3.1</td></tr><tr><td>SYBR Safe DNA gel stain</td><td>Invitrogen</td><td>S33102</td><td>Section 3.4.2</td></tr><tr><td>Freeze&#039;N Squeeze DNA Gel Extraction Spin Columns</td><td>BIO-RAD</td><td>731-6166</td><td>Section 3.6</td></tr><tr><td>Bruker&#039;s Sharp Nitride Lever Probes</td><td>Bruker AFM Probes</td><td>SNL10</td><td>Section 4.3</td></tr><tr><td>Safe Imager 2.0 Blue Light Transilluminator</td><td>Invitrogen</td><td>G6600</td><td>Section 3.6</td></tr><tr><td>Centrifuge 5430R</td><td>Eppendorf</td><td>5428 000.414</td><td>Section 3.6</td></tr><tr><td>Transmission Electron Microscope&amp;nbsp;</td><td>Jeol</td><td>Jem 1400</td><td>Section 7.4</td></tr><tr><td>Multimode 8</td><td>Veeco</td><td></td><td>Section 4</td></tr><tr><td>Typhoon FLA 9000 Laser Scanner</td><td>GE Heathcare Life Sciences</td><td>28-9558-08</td><td>Section 3.5</td></tr><tr><td>Ultrapure Distilled water</td><td>Invitrogen</td><td>10977-023</td><td>Section 3.7.1</td></tr><tr><td>Mica disk</td><td>SPI Supplies</td><td>12001-26-2</td><td>Section 4.1</td></tr><tr><td>Steel mounting disk</td><td>Ted Pella, Inc.</td><td>16218</td><td>Section 4.1</td></tr><tr><td>carbon coated copper grid for TEM</td><td>Electron Microscopy Sciences</td><td>FCF400-Cu</td><td>Section 7.2</td></tr><tr><td>tweezers</td><td>Dumont</td><td>0203-N5AC-PO</td><td>Section 7.31</td></tr><tr><td>glow discharge system</td><td>Quorum Technologies</td><td>K100X</td><td>Section 7.2</td></tr><tr><td>DNA Engine Tetrad 2 Peltier Thermal Cycler</td><td>BIO-RAD</td><td>PTC&amp;ndash;0240G</td><td>Section 3.3</td></tr><tr><td>Owl Easycast B2 Mini Gel Electrophoresis Systems</td><td>ThermoScientific</td><td>B2</td><td>Section 3.4.3</td></tr><tr><td>Seekam LE Agarose 500G</td><td>Lonza</td><td>50004</td><td>Section 3.4.1</td></tr><tr><td>GeneRuler 1kb Plus DNA Ladder, Ready-To-Use 75-20000bp</td><td>ThermoScientific</td><td>SM1333</td><td>Section 3.4.4</td></tr><tr><td>Nanodrop 2000c UV-vis Spectrophotometer</td><td>ThermoScientific</td><td></td><td>Section 3.7</td></tr><tr><td>0.2 um filter</td><td>Corning Inc.</td><td>431219</td><td>Section 7.1.2</td></tr></tbody></table>

self-assembly of complex two-dimensional shapes from single-stranded dna tiles

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

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

The self-assembly of SSTs (Figure 1) will yield a 24H × 28T rectangle, as illustrated in Figure 2. DNA sequences for the different SSTs can be modified/optimized to enable streptavidin labeling (Figure 3 and 4), the transformation of a rectangle into a tube (Figure 5), the programmable self-assembly of SSTs to form tubes and rectangles of varying sizes (Figure 10), and the construction of 2D arbitrary shapes using the mo...

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

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.

self-assembly-complex-two-dimensional-shapes-from-single-stranded-dna

Research

JoVE Journal

Chemistry

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Designing a Bio-responsive Robot from DNA Origami

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

Folding and Characterization of a Bio-responsive Robot from DNA Origami

Here, an automated workflow to perform modular DNA "device" assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

Design and Synthesis of a Reconfigurable DNA Accordion Rack

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Simple, Affordable, and Modular Patterning of Cells using DNA

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

Learn DNA nanostructure self-assembly using single-stranded DNA tiles with annealing, gel electrophoresis, and atomic force microscopy verification. This protocol helps researchers construct programmable nanoscale shapes.

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

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