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...

<ol>
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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 ...

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

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11.4K visualizações.  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.