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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Rev. 40 (12), 5636-5646 (2011).</li><li>Wei, B., Dai, M., Yin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+Shapes+self-assembled+from+single+stranded+DNA+tiles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uniquimer:+software+of+de+novo+DNA+sequence+generation+for+DNA+self-assembly:+an+introduction+and+the+related+applications+in+DNA+self-assembly.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+design+of+sequences+for+nucleic+acid+structural+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+binding+to+mica+correlates+with+cationic+radius:+assay+by+atomic+force+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-free+nucleic+acid+logic+circuits.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+biomolecular+self-assembly+pathways.">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 border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNA Strands&#160;</td>
			<td style="width:183px;">Integrated DNA Technology</td>
			<td style="width:119px;"></td>
			<td>Section 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR Safe DNA gel stain</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">S33102</td>
			<td>Section 3.4.2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Freeze&#39;N Squeeze DNA Gel Extraction Spin Columns</td>
			<td style="width:183px;">BIO-RAD</td>
			<td style="width:119px;">731-6166</td>
			<td>Section 3.6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bruker&#39;s Sharp Nitride Lever Probes</td>
			<td style="width:183px;">Bruker AFM Probes</td>
			<td style="width:119px;">SNL10</td>
			<td>Section 4.3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Safe Imager 2.0 Blue Light Transilluminator</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">G6600</td>
			<td>Section 3.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge 5430R</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:119px;">5428 000.414</td>
			<td>Section 3.6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission Electron Microscope&#160;</td>
			<td style="width:183px;">Jeol</td>
			<td style="width:119px;">Jem 1400</td>
			<td>Section 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multimode 8</td>
			<td style="width:183px;">Veeco</td>
			<td style="width:119px;"></td>
			<td>Section 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Typhoon FLA 9000 Laser Scanner</td>
			<td style="width:183px;">GE Heathcare Life Sciences</td>
			<td style="width:119px;">28-9558-08</td>
			<td>Section 3.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure Distilled water</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">10977-023</td>
			<td>Section 3.7.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mica disk</td>
			<td style="width:183px;">SPI Supplies</td>
			<td style="width:119px;">12001-26-2</td>
			<td>Section 4.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Steel mounting disk</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:119px;">16218</td>
			<td>Section 4.1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">carbon coated copper grid for TEM</td>
			<td style="width:183px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">FCF400-Cu</td>
			<td>Section 7.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tweezers</td>
			<td style="width:183px;">Dumont</td>
			<td>0203-N5AC-PO</td>
			<td>Section 7.31</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glow discharge system</td>
			<td style="width:183px;">Quorum Technologies</td>
			<td style="width:119px;">K100X</td>
			<td>Section 7.2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNA Engine Tetrad 2 Peltier Thermal Cycler</td>
			<td style="width:183px;">BIO-RAD</td>
			<td>PTC&#8211;0240G</td>
			<td>Section 3.3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Owl Easycast B2 Mini Gel Electrophoresis Systems</td>
			<td style="width:183px;">ThermoScientific</td>
			<td>B2</td>
			<td>Section 3.4.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Seekam LE Agarose 500G</td>
			<td style="width:183px;">Lonza</td>
			<td style="width:119px;">50004</td>
			<td>Section 3.4.1</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GeneRuler 1kb Plus DNA Ladder, Ready-To-Use 75-20000bp</td>
			<td style="width:183px;">ThermoScientific</td>
			<td style="width:119px;">SM1333</td>
			<td>Section 3.4.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nanodrop 2000c UV-vis Spectrophotometer</td>
			<td style="width:183px;">ThermoScientific</td>
			<td style="width:119px;"></td>
			<td>Section 3.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 um filter</td>
			<td style="width:183px;">Corning Inc.</td>
			<td style="width:119px;">431219</td>
			<td>Section 7.1.2</td>
		</tr>
	</tbody>
</table>

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 &#215; 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 t...

Watch this Scientific Journal Video about Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles at JoVE.com

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

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 ...

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11.5K Views.  Tsinghua University. 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.

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

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

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.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

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

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

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).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

Synthesis of a Water-soluble Metal&amp;#8211;Organic Complex Array

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Negative Additive Manufacturing of Complex Shaped Boron Carbides

Boron carbide (B4C) is one of the hardest materials in existence. However, this attractive property also limits its machineability into complex shapes for high wear, high hardness, and lightweight material applications such as armors. To overcome this challenge, negative additive manufacturing (AM) is employed to produce complex geometries of boron carbides at various length scales. Negative AM first involves gelcasting a suspension into a 3D-printed plastic mold. The mold is then dissolved away, leaving behind a green body as a negative copy. Resorcinol-formaldehyde (RF) is used as a novel gelling agent because unlike traditional hydrogels, there is little to no shrinkage, which allows for extremely complex molds to be used. Furthermore, this gelling agent can be pyrolyzed to leave behind ~50 wt% carbon, which is a highly effective sintering aid for B4C. Due to this highly homogenous distribution of in situ carbon within the B4C matrix, less than 2% porosity can be achieved after sintering. This protocol highlights in detail the methodology for creating near fully dense boron carbide parts with highly complex geometries.

A method called negative additive manufacturing is used to produce near fully dense complex shaped boron carbide parts of various length scales. This technique is possible via the formulation of a novel suspension involving resorcinol-formaldehyde as a unique gelling agent that leaves behind a homogenous carbon sintering aid after pyrolysis.

Boron carbide (B4C) is one of the hardest materials in existence. However, this attractive property also limits its machineability into complex shapes for ...

Exfoliation and Analysis of Large-area, Air-Sensitive Two-Dimensional Materials

We describe methods for producing and analyzing large, thin flakes of air-sensitive two-dimensional materials. Thin flakes of layered or van der Waals crystals are produced using mechanical exfoliation, in which layers are peeled off a bulk crystal using adhesive tape. This method produces high-quality flakes, but they are often small and can be hard to find, particularly for materials with relatively high cleavage energies such as black phosphorus. By heating the substrate and the tape, two-dimensional material adhesion to the substrate is promoted, and the flake yield can be increased by up to a factor of ten. After exfoliation, it is necessary to image or otherwise analyze these flakes but some two-dimensional materials are sensitive to oxygen or water and will degrade when exposed air. We have designed and tested a hermetic transfer cell to temporarily maintain the inert environment of a glovebox so that air-sensitive flakes can be imaged and analyzed with minimal degradation. The compact design of the transfer cell is such that optical analysis of sensitive materials can be performed outside of a glovebox without specialized equipment or modifications to existing equipment.

A method for exfoliating large thin flakes of air sensitive two-dimensional materials and safely transporting them for analysis outside of a glovebox is presented.

We describe methods for producing and analyzing large, thin flakes of air-sensitive two-dimensional materials. Thin flakes of layered or van der Waals ...

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

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

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.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...