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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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 ligase</td>
			<td style="width:171px;">TaKaRa</td>
			<td style="width:131px;">2011A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 buffer</td>
			<td style="width:171px;">TaKaRa</td>
			<td style="width:131px;">2011A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TE buffer</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B548106</td>
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			<td height="21" style="height:21px;width:216px;">Thermo bottle</td>
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			<td style="width:131px;">SK-3000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermo cycler</td>
			<td style="width:171px;">Bio Gener</td>
			<td style="width:131px;">GE4852T</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Exonuclease I</td>
			<td style="width:171px;">TaKaRa</td>
			<td style="width:131px;">2650A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Exonuclease I buffer</td>
			<td style="width:171px;">TaKaRa</td>
			<td style="width:131px;">2650A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30% (w/v) Acryl/Bis solution (19:1)</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B546016</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TAE premix podwer</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B540023</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mg(Ac)2&#183;4H2O</td>
			<td style="width:171px;">Nanjing Chemical Reagent</td>
			<td style="width:131px;">C0190550223</td>
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			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">A510907</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:171px;">BBI</td>
			<td style="width:131px;">A100761</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium Persulfate</td>
			<td style="width:171px;">Nanjing Chemical Reagent</td>
			<td style="width:131px;">13041920295</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Power supply</td>
			<td style="width:171px;">Beijing Liuyi</td>
			<td style="width:131px;">DYY-8C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:171px;">Sumsung</td>
			<td style="width:131px;">DK-S12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formamide</td>
			<td style="width:171px;">BBI</td>
			<td style="width:131px;">A100314</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNA Marker (25~500 bp)</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B600303</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNA Marker (100~3000 bp)</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B500347</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Loading buffer</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">B548313</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PAGE electrophoresis systerm</td>
			<td style="width:171px;">Beijing Liuyi</td>
			<td style="width:131px;">24DN</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter</td>
			<td style="width:171px;">ASD</td>
			<td style="width:131px;">5010-2225</td>
			<td>0.22 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV imaging System</td>
			<td style="width:171px;">Tanon</td>
			<td style="width:131px;">2500R</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">n-butanol</td>
			<td style="width:171px;">Sangon</td>
			<td style="width:131px;">A501800</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Absolute Ethanol</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaOAc</td>
			<td style="width:171px;">Nanjing Chemical Reagent</td>
			<td style="width:131px;">12032610459</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:171px;">eppendorf</td>
			<td style="width:131px;">Centrifuge 5424R</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum concentrator</td>
			<td style="width:171px;">CHRIST</td>
			<td style="width:131px;">RVC 2-18</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultraviolet spectrum</td>
			<td style="width:171px;">Allsheng</td>
			<td style="width:131px;">Nano-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nucleic acid stain</td>
			<td style="width:171px;">Biotium</td>
			<td style="width:131px;">16G1010</td>
			<td>GelRed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:171px;">Biowest</td>
			<td style="width:131px;">G-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agarose electrophoresis systerm</td>
			<td style="width:171px;">Beijing Liuyi</td>
			<td style="width:131px;">DYCP-31CN</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating Plate</td>
			<td style="width:171px;">Jiangsu Jintan</td>
			<td style="width:131px;">DB-1</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>
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		<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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Research

JoVE Journal

Chemistry

6.0K vues.  Nanjing University. Cette méthode étend la nanotechnologie de l’ADN à partir des matériaux sources de l’oligonucléotide linéaire, DAS, dans un DAS plus circulaire. Les principaux avantages de ces techniques sont qu’il s’agit d’un simple, robuste et abordable pour la plupart des laboratoires. Pour la purification circulaire de l’ADN, ajouter 20 microlitres de formamide à une solution d’ADN circulaire fraîchement préparé et intact, à un volume final de 140 microlitres avec mélange....

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

Gyroid Nickel Nanostructures from Diblock Copolymer Supramolecules

Nanoporous metal foams possess a unique combination of properties - they are catalytically active, thermally and electrically conductive, and furthermore, have high porosity, high surface-to-volume and strength-to-weight ratio. Unfortunately, common approaches for preparation of metallic nanostructures render materials with highly disordered architecture, which might have an adverse effect on their mechanical properties. Block copolymers have the ability to self-assemble into ordered nanostructures and can be applied as templates for the preparation of well-ordered metal nanofoams. Here we describe the application of a block copolymer-based supramolecular complex - polystyrene-block-poly(4-vinylpyridine)(pentadecylphenol) PS-b-P4VP(PDP) - as a precursor for well-ordered nickel nanofoam. The supramolecular complexes exhibit a phase behavior similar to conventional block copolymers and can self-assemble into the bicontinuous gyroid morphology with two PS networks placed in a P4VP(PDP) matrix. PDP can be dissolved in ethanol leading to the formation of a porous structure that can be backfilled with metal. Using electroless plating technique, nickel can be inserted into the template&#39;s channels. Finally, the remaining polymer can be removed via pyrolysis from the polymer/inorganic nanohybrid resulting in nanoporous nickel foam with inverse gyroid morphology.

This article describes the preparation of well-ordered nickel nanofoams via electroless metal deposition onto nanoporous templates obtained from self-assembled diblock copolymer based supramolecules.

Nanoporous metal foams possess a unique combination of properties - they are catalytically active, thermally and electrically conductive, and furthermore, ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

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 ...

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.

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 ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.