Name: stable dna motifs, 1d and 2d nanostructures constructed from small circular dna molecules
Uploaded: 2019-04-12T13:30:00
Description: Watch this Scientific Journal Video about Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules at JoVE.com

We are grateful for financial support from the NSFC (grants no. 91753134 and 21571100), and the State Key Laboratory of Bioelectronics of Southeast University.

1. Preparation of Circular DNAs
<ol>
	<li>Use all linear DNAs provided by commercial companies directly without further purification.</li>
	<li>Centrifuge the DNA samples at 5,000 &#215; g for 5 min to collect all DNA pellets at the bottom of the tubes. Add an appropriate volume of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to dissolve the DNA.</li>
	<li>Measure the concentration of &#34;a&#34; ng/&#181;L for each ss-DNA solution using a micro UV spectrometer at 260 nm. Convert &#34;a&#34; ng/&#181;L to &#34;b&#34; &#18...

<ol>
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The protocols presented in this article focus on the synthesis of small circular DNA molecules and the assembly of DNA nanostructures. Most of randomly-sequenced DNA designs can be used in this protocol. The purity of circular DNAs is critical for the success of DNA assemblies. The production yield of cyclization can be improved by lowering the concentration of 5'-phosphorylated linear DNA; however, this will increase the workload to produce the same amounts of circular DNAs. The length of splint DNA strands also affects...

DNA molecules have been used to build many kinds of nanostructures over decades. Typical motifs include DAE (double crossover, antiparallel, even half-turns) and DAO tiles1,2,3, star tiles4,5,6,7, single stranded (ss) tiles8,9,10, and DNA origami11,12,13. These DNA motifs and lattices are assembled from linear ss-DNAs. Recently, others and we have reported the use of circular ss-oligonucleotides as scaffolds to build motifs, 1D nanotubes, and 2D lattices14,15,16,17. By inserting a Holliday junction (HJ)18,19,20,21 at the center of c64nt, a pair of two coupled DAO tiles can be formed17. This new cDAO motif and its derivatives are stable and rigid enough to assemble 2D DNA lattices up to 3 &#215; 5 &#181;m2. In this paper, we use a term of &#34;circular tile&#34;, which is defined as a stable DNA complex molecule constructed with one circular scaffold and other linear staples of ss-oligonucleotides, and another term of &#34;linear tile&#34;, which is built from a full set of linear ss-oligonucleotides.
This protocol demonstrates how to construct five kinds of DNA nanostructures with small circular DNA molecules as scaffolds: 1) infinite 1D c64nt and c84nt nanowires, 2) infinite 2D cDAO-c64nt-O and cDAO-c64nt-E (-O represents an odd number&#160;of 5 half-turns and -E represents an even number&#160;of 4 half-turns) lattices, 3) infinite 2D cDAO-c84nt-O and cDAO-c84nt-E lattices, 4) finite 2D 5 &#215; 6 cDAO-c64nt-O and 5 &#215; 6 cDAO-c74&#38;84nt-O rectangles, 5) infinite 1D acDAO-c64nt-E nanorings and nanospirals (please refer to Figure&#160;3-5 for the schematic drawings and images of the above five kinds of DNA nanostructures). The 1D c64nt and c84nt nanowires are assembled from each c64nt and c84nt scaffold associated with two linear staples respectively. Each circular tile of cDAO-c64nt, acDAO-c64nt, cDAO-c74nt, or&#160;cDAO-c84nt is annealed from its corresponding scaffold of c64nt, c74nt, or c84nt with four linear staples respectively. The infinite 2D lattices are assembled from the same type of two circular tiles with different sequences. The two finite 2D rectangle lattices are assembled from two&#160;sets of 32 circular sub-tiles respectively. To save money, only one-sequenced c64nt, c74nt, and c84nt is used as the respective scaffold while different overhangs are used to anneal the 32 cDAO-c64nt, 12 cDAO-c74nt, and 20 cDAO-c84nt circular sub-tiles respectively in the first sub-tile annealing step, then mix the corresponding 32 circular sub-tiles together and apply the second lattice annealing step to assemble the finite 5 &#215; 6 cDAO-c64nt-O and 5 &#215; 6 cDAO-c74&#38;84nt-O lattices, respectively. Definitely, differently-sequenced circular scaffolds can be adopted to assemble a variety of finite size nanostructures, however it will cost more money and labors. The infinite 1D acDAO-c64nt-E nanorings and nanospirals are annealed from one-sequenced asymmetric acDAO-c64nt tiles with linear connections of an even number of 4 half-turns. There are two approaches to assemble infinite 2D lattices from circular tiles of cDAO-c64nt and cDAO-c84nt, which are distinguished by the intertile distances of an even number of 4 and an odd number of 5 half-turns respectively. The former requires all tiles to be aligned identically; the latter requires alternation of the faces of two neighboring tiles along the helical axes. If the tile is rigid and planar, such as cDAO-c64nt, both approaches will generate planar nanoribbons; if the tile is curved towards one direction, such as cDAO-c84nt, the intertile connection of an even number of 4 half turns will generate nanotubes, whereas the intertile connection of an odd number of 5 half turns will produce planar nanoribbons due to elimination of curvature-biased growth by alternate&#160;alignment of curved tiles. The successful assembly of 1D and 2D DNA nanostructures from circular tiles indicates several advantages of this new approach: enforced stability and rigidity of circular tiles over linear tiles, chiral tiles for assembly of asymmetrical nanostructures such as nanorings and nanoribbons, new visions on understanding the DNA mechanics and molecular structures, etc.

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			<td height="21" style="height:21px;width:216px;">T4 ligase</td>
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			<td height="21" style="height:21px;width:216px;">T4 buffer</td>
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			<td style="width:131px;">2011A</td>
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			<td height="21" style="height:21px;width:216px;">Exonuclease I buffer</td>
			<td style="width:171px;">TaKaRa</td>
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			<td height="21" style="height:21px;width:216px;">DNA Marker (25~500 bp)</td>
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			<td>0.22 &#181;M</td>
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			<td>GelRed</td>
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			<td height="21" style="height:21px;width:216px;">Agarose</td>
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			<td height="42" style="height:42px;width:216px;">Agarose electrophoresis systerm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TBE premix podwer&#160;</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B540024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter column</td>
			<td style="width:171px;">Bio-Rad</td>
			<td style="width:131px;">7326165</td>
			<td>Freeze &#39;N Squeeze column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AFM</td>
			<td style="width:171px;">Bruker</td>
			<td style="width:131px;">Dimension FastScan</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEG8000</td>
			<td style="width:171px;">BBI</td>
			<td style="width:131px;">A100159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mica</td>
			<td style="width:171px;">Ted Pella</td>
			<td style="width:131px;">BP50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">triangular AFM probe in air</td>
			<td style="width:171px;">Bruker</td>
			<td style="width:131px;">FastScan-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">triangular AFM probe in fulid</td>
			<td style="width:171px;">Bruker</td>
			<td style="width:131px;">ScanAsyst-fluid+</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNA strands</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The circular DNA moves slightly slower than its precursor linear DNA in denaturing PAGE (Figure 2) because the pore inside the circular DNA is penetrated and retarded by gel fibers23,24,25. The correct ligation reaction efficiency for oligo-monomer cyclization depends on the substrate sequence and concentration, reaction temperature, time, etc. As the concen...

Watch this Scientific Journal Video about Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules at JoVE.com

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

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 ...

This method extends DNA nanotechnology from the source materials of linear oligonucleotide, DAS, into some more circular DAS. The main advantages of these technical are that it is a simple, robust and affordable for most labs. For circular DNA purification, add 20 microliters of formamide to a solution of freshly prepared, intact circular DNA, to a final volume of 140 microliters with mixing.Load 16 to 20 microliters of circular DNA Solution to each of seven to nine denaturing PAGE gel wells. Mix two microliters of loading dye with eight microliters of the corresponding precursor linear DNA, and inject the mixture into a separate reference well. Then run the gel for about two hours at 10 volts per centimeter, stopping the run when the xylene cyanol FF can be observed about two-thirds down the gel.Wearing rubber gloves and goggles, transfer the gel onto a fluorescent thin-layer chromatography plate, and use a razor blade to cut off the target gel bands, as shadowed by the UV irradiation. Place the gel bands into a two milliliter microcentrifuge tube for drying, then mash the dried gels into a paste, and add de-ionized water at twice the volume of gel, for overnight shaking at room temperature. The next morning, filter the mixture into a two milliliter microcentrifuge tube, rinsing the sides of the first tube with a small volume of de-ionized water to recover any residual DNA, and filtering again to combine the supernates.Then purify the circular DNA via ethanol precipitation, and store the DNA at minus 20 degrees Celsius. For one pot annealing, mix two microliters of 10-fold Tris acetate EDTA magnesium buffer, one microliter of each 10 micromolar DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional Tris EDTA buffer in a 200 microliter PCR test tube, to a final volume of 20 microliters. Then anneal the assembly solution in a thermal bottle from 95 to 25 degrees Celsius, over 48 hours.For two-step annealing of infinite lattices, mix two microliters of 10-fold Tris acetate EDTA magnesium buffer, one microliter of each 10 micromolar DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional Tris EDTA buffer in a 200 microliter PCR test tube, to a final volume of 20 microliters. In the first sub-tile annealing step, anneal each precursor sub-tile solution in a PCR thermocycler, using a fast linear cooling method from 95 to 25 degrees Celsius, over two and a half hours. To prepare the assembly solution, for each of the four infinite lattices, mix 10 microliters of each of the two corresponding sub-tile solutions together, to a final concentration of 0.25 micromolar for each strand.In the second lattice annealing step, anneal the 20 microliter mixture in a PCR thermocycler, using a slow cooling method. For two step annealing of two finite rectangle lattices, mix one microliter of 10 fold Tris acetate EDTA magnesium buffer, 0.5 microliters of each 10 micromolar DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional Tris EDTA buffer in a 200 microliter PCR test tube, to a final volume of 10 microliters. In the first annealing step, anneal each precursor sub-tile solution in the PCR thermocycler, using the fast linear cooling method, as demonstrated, from 95 to 25 degrees Celsius, over two and a half hours, and mix the annealed 32 sub-tile solutions, each with 2 microliters, together in a 200 microliter PCR test tube, to a final volume of 64 microliters.Then, in the second annealing step, anneal the 64 microliter mixture in the thermocycler, using the slow cooling method, as demonstrated. For electrophoresis purification of the finite lattices, prepare a native 2%agarose gel and load 20 microliters of the finite lattice solution into each well, along with the addition of 5 microliters of a 100 to 3000 base-pair DNA ladder, in a separate well. After running the gel for two hours at 5 volts per centimeter in an ice water bath, cut the target gel bands at about the level of the 1000 base pair marker under UV light, as demonstrated, and slice the gel bands into fine pieces.Place the crushed gels into a filter column, and centrifuge the column to extract the lattices. Then, collect the solution for atomic force microscopy imaging. For finite lattice purification by polyethalene glycol, or PEG, mix 50 microliters of the finite lattice solution with an equal volume of 15%PEG 8000 buffer, centrifuge the solution, and re suspend the pellet in 100 microliters of PEG buffer, for an additional centrifugation.After the second centrifugation, re suspend the pellet in Tris acetate EDTA magnesium buffer, for atomic force microscopy imaging. For infinite 2D lattice atomic force microscope imaging, cleave off a fresh layer of mica, and deposit two microliters of the annealed sample onto the clean surface. Allow the DNA lattices two minutes to absorb to the mica surface, before washing the surface two times, with 100 microliters of de-ionized water per wash, drying the surface with compressed air after the second wash.To obtain atomic force microscope images of the infinite DNA lattices in air, open the tapping mode in the imaging software, and set the scan size to 0.5 to five micrometers, the scan resolution to 512 lines, and the scan rate between one to 3.5 Hertz. Then, scan the mica surface with triangular atomic force microscope probes. For finite DNA lattices imaging, cleave off a fresh layer of mica, as demonstrated, and deposit 80 microliters of one fold Tris acetate EDTA magnesium buffer onto the clean mica surface.Next, add five microliters of finite samples into the buffer, and allow the DNA lattices two minutes to absorb to the mica surface, before adding another 50 microliters of Tris acetate EDTA magnesium buffer to the probe. Then, set the scan size to 0.5 to one micrometer, the scan resolution to 256 lines, and the scan rate to 1.5 Hertz, before scanning the surface with the triangular probes, as demonstrated. The circular DNA moves slightly slower than it's precursor linear DNA in the denaturing PAGE, because the pore inside the circular DNA is penetrated and retarded by the gel fibers.The circular 64 nucleotide assembly families have only one clear and clean band for each assembly, confirming the stability of their monomer motifs. The circular 84 nucleotide assembly families of tiles, however, have smears around their main bands, indicating the presence of minor by-products. Regardless of the minor by products of incorrect associates, excellent lattices with high yields can be assembled from the target monomer motifs.Each assembly has it's own morphological features in the micrometer scale, such as nanowires, nanotubes, nanospirals, nanoribbons, and nanorectangles. While attempting this procedure, it is important to remember to avoid, particularly, debris contamination during or off the steps. After it's development, this technique paved the way for researches in the field of DNA nanotechnology to explore new family members of some more circular DNA nanotechnology.

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