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; &#181;M following b = a &#215; 103 / (molecular weight of the DNA strand). Adjust the amount of TE solution to make a 10 &#181;M DNA stock solution.</li>
	<li>Use T4 DNA ligase to connect the 3&#39; and 5&#39; ends of 5&#39;-phosphorylated linear DNA templates ( Figure 1) of c64nt, c74nt and c84nt provided from commercial companies directly.</li>
	<li>Mix the 5&#39;-phosphorylated linear DNA strand (3.5 &#181;M) and its corresponding splint DNA strand (4.5 &#181;M) in 80 &#181;L TE buffer in a 200 &#181;L PCR test tube15. Incubate the tube in an open thermo bottle filled with 95 &#176;C water. Cool down the hot water to room temperature (25 &#176;C) for about 2-3 hours under the lab atmosphere.</li>
	<li>Add 10 &#181;L of 10x T4 buffer (660 mM Tris-HCl, 66 mM MgCl2, 100 mM DTT, 1 mM ATP) and 10 &#181;L of T4 ligase (300 U/&#181;L) to the mixture to a final volume of 100 &#181;L. Incubate the mixture in a thermocycler for 16 hours at 16 &#176;C. 
	NOTE: The above DNA concentrations and reaction volume of ~100 &#181;L are optimized for the high ligation efficiency and the high yield of correct oligo-monomer cyclization.&#160;Run two or more tubes of ligation reactions in the same time according to the experimental design. An alternative incubation procedure for ligation replacing step 1.6 is 4 hours at 25 &#176;C.</li>
	<li>After the incubation, inactivate the T4 ligase in 95 &#176;C water for 5 minminutes. Then transfer the tube to an ice water bath and incubate for 5 minutes to cool down.</li>
	<li>Add 10 &#181;L of 10x Exonuclease I buffer and 10 &#181;L of Exonuclease I (5 U/&#181;L) to the quenched mixture and incubate the tube at 37 &#176;C in a water bath for 30 min to selectively digest the remaining linear DNAs and leave the circularized DNAs intact.</li>
	<li>Prepare a 10% denaturing PAGE gel.
	<ol>
		<li>Wear rubber gloves and goggles when preparing the PAGE gel in the fume hood. Add 6.67 mL of 30% (w/v) acrylamide/bisacrylamide solution (19:1), 2 mL of 10x TAE&#183;Mg buffer (40 mM Tris, 40 mM HAc, 1 mM EDTA, pH 8.0 ,12.5 mM Mg(Ac)2), 8.4 g of urea (7 M) and deionized water to a final volume of 20 mL in a 40 mL centrifuge tube. 
		&#8203;Caution: The 30% (w/v) acrylamide/bisacrylamide solution (19:1) solution is toxic.</li>
		<li>Add 20 &#181;L of tetramethylethylenediamine (TEMED) and 100 &#181;L of ammonium persulfate (APS, 10% w/v) to the 40 mL tube before transfer the gel solution to the electrophoresis system immediately. Insert a 1.5 mm thick and 10-well comb. Wait at least 20 min until the gel solution is solidified.</li>
		<li>Set a constant voltage of 80 V for a gel of 85 &#215; 80 &#215; 1.5 mm3 (width &#215; height &#215; thickness). Add 1x TAE&#183;Mg buffer to the electrophoresis system and prerun the gel for 20 min at 10 V/cm.</li>
	</ol>
	</li>
	<li>Put the PCR test tube from step 1.8 in 95 &#176;C water for 5 min to inactivate Exonuclease I. Then transfer the tube to an ice water bath and incubate for another 5 min.</li>
	<li>Add 20 &#181;L of formamide in the tube to a final volume of 140 &#181;L and mix the solution. Distribute the solution to 7-9 denaturing PAGE gel wells equally with 16-20 &#181;L for each gel well. Mix 2 &#181;L of loading dye (0.05% bromophenol blue, 0.05% xylene cyanol FF, 60% glycerol, 10 mM Tris-HCl, 60 mM EDTA, pH 7.6) with 8 &#181;L of the corresponding precursor linear DNA (10 &#181;M) and inject the mixture to a separate well for reference. 
	NOTE: The addition of formamide is to increase the density of the loading solution for sinking to the bottom of the PAGE gel well and also to disassociate some double stranded DNA residues.</li>
	<li>Run the gel for about 2 h at 10 V/cm and stop running when xylene cyanol FF is at the 2/3 position of the glass plate with the naked eye.</li>
	<li>Wear rubber gloves and goggles to protect skins and eyes. Take the gel out of the glass plate. Place the gel on a fluorescent TLC (thin layer chromatography) plate. Cut off the target gel bands by a razor blade as exactly as shadowed by UV irradiation (Figure 2). 
	Caution: The UV light is harmful to eyes and skins. 
	NOTE: Under UV irradiation at 254 nm, DNA will absorb the light and cast a shadow onto the TLC plate. The circular DNA ran slightly slower than its corresponding precursor linear DNA. Some undigested linear DNAs and unwanted circular DNAs in small amounts which surround the shadowed target band will pollute the circular DNA if they are collected.</li>
	<li>Transfer the gel bands into a 2.0 mL microcentrifuge tube. Air dry or blow dry the gel bands and then mash the gels into a paste by a spatula or flattened glass rod. Make sure the gel has been completely crushed into a paste, which is critical to the high yield of circular DNA. Add twice of the gel volume deionized water into the tube and shake the tube at room temperature overnight.</li>
	<li>Filter the mixture to collect the supernatants in a 2.0 mL microcentrifuge tube. Recover any residual DNA by rinsing with small volume of deionized water and filter again to combine the supernatants.&#160;</li>
	<li>Extract the eluent with equal volume of n-butanol. Repeat this procedure until the aqueous volume is reduced to about 200 &#181;L.</li>
	<li>Add 500 &#181;L of absolute ethanol (&#8722;20 &#176;C) and 20 &#181;L of 3 M NaOAc (pH 5.1) to the mixture for DNA precipitation. Store the tube at &#8722;20 &#176;C for 30 min.</li>
	<li>Centrifuge the tube at 12,000 &#215; g for 10 min at 4 &#176;C, discard the supernatant. The remaining DNA precipitate is about 100 &#181;L in the tube.</li>
	<li>Add 600 &#181;L of 75% ethanol (&#8722;20 &#176;C) to the tube and store it at &#8722;20 &#176;C for 30 min. Then centrifuge at 12,000 &#215; g for 10 min at 4 &#176;C, discard the supernatant. The remaining DNA precipitate is about 100 &#181;L solution in the tube again. Repeat the procedure twice.</li>
	<li>After the last centrifugation, discard the supernatant as much as possible. Dry the remains with a vacuum concentrator.</li>
	<li>Store the purified circular DNA in the tube at &#8722;20 &#176;C.</li>
	<li>Resuspend the circular DNA in TE buffer to make a 10 &#181;M circular DNA stock solution according to step 1.3 when needed.</li>
</ol>
2. Annealing of Assembly Solutions
<ol>
	<li>Prepare each tile or nanostructural assembly solution of c64bp, c84bp, HJ-c64nt, aHJ-c64nt, HJ-c84nt, tHJ-c84nt, cDAO-c64nt, acDAO-c64nt, cDAO-c84nt, c64nt, c84nt, and acDAO-c64nt-E with a designed set of linear and circular DNAs for one-pot annealing.
	<ol>
		<li>For each assembly solution, mix 2 &#181;L of 10x TAE&#183;Mg buffer, 1 &#181;L of each DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional TE buffer in a 200 &#181;L PCR test tube to a final concentration of each strand at 0.5 &#181;M and a final volume of 20 &#181;L. Anneal the assembly solution in a thermo bottle from 95 &#176;C to 25 &#176;C over 48 h.</li>
	</ol>
	</li>
	<li>Prepare each assembly solution of 2D infinite lattices of cDAO-c64nt-E, cDAO-c64nt-O, cDAO-c84nt-E, and cDAO-c84nt-O with a designed set of linear and circular DNAs for two-step annealing.
	<ol>
		<li>Prepare each precursor sub-tile assembly solution by mixing 2 &#181;L of 10x TAE&#183;Mg buffer, 1 &#181;L of each DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional TE buffer in a 200 &#181;L PCR test tube to a final concentration of 0.5 &#181;M for each strand and a final volume of 20 &#181;L. 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 &#176;C to 25 &#176;C over 2.5 h.</li>
		<li>Prepare each assembly solution of the above four infinite lattices by mixing&#160;10 &#181;L of each of the two corresponding sub-tile solutions together to a final concentration of 0.25 &#181;M for each strand. In the second lattice annealing step, anneal the 20 &#181;L mixture in a PCR thermocycler using a slow cooling method of staying at 50 &#176;C for 2 h and cooling down at a rate of 0.1 &#176;C per 5 minutes to 20 &#176;C, about 24 h in total. 
		NOTE: Two parallel experiments can be run simultaneously or subsequently in the second annealing step for each 2D infinite lattice.</li>
	</ol>
	</li>
	<li>Prepare assembly solutions of two finite rectangle assemblies of 5 &#215; 6 cDAO-c64nt-O and 5 &#215; 6 cDAO-c74&#38;c84nt-O for two-step annealing.
	<ol>
		<li>Prepare each precursor sub-tile assembly solution by mixing 1 &#181;L of 10x TAE&#183;Mg buffer, 0.5 &#181;L of each DNA stock solution of a set of linear and circular strands in equal molar ratios, and additional TE buffer in a 200 &#181;L PCR test tube to a final concentration of 0.5 &#181;M for each strand and a final volume of 10 &#181;L. In the first annealing step, anneal each precursor sub-tile solution in a PCR thermocycler using a fast-linear cooling method from 95 &#176;C to 25 &#176;C over 2.5 h.</li>
		<li>Prepare each finite rectangle lattice assembly solution by mixing 32 x (2 &#181;L of each sub-tile solution) together in a 200 &#181;L PCR test tube to a final concentration of ~15 nM for each sub-tile and a final volume of 64 &#181;L. In the second annealing step, anneal the 64 &#181;L mixture in a PCR thermocycler using a slow cooling method of staying at 50 &#176;C for 2 h and cooling down at a rate of 0.1 &#176;C per 5 min to 20 &#176;C, about 24 hours in total. 
		NOTE: Five parallel experiments can be run simultaneously or subsequently in the second annealing step for each finite rectangle assembly.</li>
	</ol>
	</li>
</ol>
3. Native PAGE Analysis
<ol>
	<li>Prepare a 10% native PAGE following step 1.9 except for the urea component.</li>
	<li>For control samples of c64bp and c84bp, add 2 &#181;L of each assembly solution and 1 &#181;L of loading dye to 3 &#181;L of TE buffer as controls separately. For each of other assemblies listed in Figure 3, add 1 &#181;L of each assembly solution and 1 &#181;L of loading dye to 4 &#181;L of TE buffer.</li>
	<li>Inject each of the above solutions to a gel well. Add 5 &#181;L of DNA marker (25-500 bp) to a separate well for reference.</li>
	<li>Run the gel for about 4 hours at 10 V/cm in an ice water bath. The xylene cyanol FF will run out of the glass plate.</li>
	<li>Wear rubber gloves and goggles to protect skins and eyes. Take the gel out of the glass plate and immerse it in 100 mL of deionized water with 10 &#181;L of a nucleic acid gel stain for 30 min. 
	Caution: The nucleic acid gel stain solution is toxic.</li>
	<li>Observe the gel under an UV imaging system and take a picture of the stained gel (Figure 3).</li>
</ol>
4. Purification of Finite Lattices
<ol>
	<li>Prepare a native 2% agarose gel for electrophoresis purification of finite lattices.
	<ol>
		<li>Mix 1 g of agarose powder, 5 mL of 10x TBE&#183;Mg buffer (89 mM Tris, 89 mM Boric Acid, 2 mM EDTA, pH 8.0, 12.5 mM Mg(Ac)2), 2 &#181;L of nucleic acid gel stain and 47.5 mL of deionized water in a 250 mL triangle bottle. Boil the mixture until the bubbles become small and dense. Add hot water in the bottle to a total volume of 50 mL and a concentration of 2% agarose.</li>
		<li>Wear thick gloves. Pour the gel solution into a plastic gel box of 7 x 10 x 1 cm3 (width &#215; length &#215; thickness). Insert a 1.5 mm thick and 12-well gel comb. Wait for the gel to cool down to room temperature. If necessary, put the gel in a 4 &#176;C fridge. 
		Caution: Be aware of scalding because the solution is very hot.</li>
		<li>Add 0.5x TBE&#183;Mg buffer in the electrophoresis system. Prerun the gel at 5 V/cm for 20 min in an ice water bath at a constant voltage of 50 V.</li>
		<li>Inject 20 &#181;L of the finite lattice solution prepared in step 2.3 in each agarose gel well and add 5 &#181;L of a DNA marker (100-3,000 bp) to a separate well. Run the gel for 2 h at 5 V/cm in an ice water bath.</li>
		<li>Wear rubber gloves and goggles to protect skins and eyes. Cut the target gel bands with a position similar to the 1,000 bp marker under UV light and slice them into fine pieces and place the crushed gels into a filter column8.</li>
		<li>Centrifuge the column at 2,000 x g for 5 min at 4 &#176;C. Extract the sample solution through the column. Collect the solution for AFM imaging.</li>
	</ol>
	</li>
	<li>Purify finite lattices by PEG precipitation.
	<ol>
		<li>Mix 50 &#181;L of the finite lattice solution prepared in step 2.3 with an equal volume of 15% PEG8000 (w/v) buffer (20 mM Mg(Ac)2, 5 mM Tris, 1 mM EDTA and 505 mM NaCl)22. Centrifuge the solution at 18,000 &#215; g at 4 &#176;C for 30 min.</li>
		<li>Remove the supernatant and add 100 &#181;L of PEG buffer. Repeat the centrifugation.</li>
		<li>After removing the supernatant, dissolve the pellet in 1x TAE&#183;Mg buffer for AFM imaging. 
		NOTE: Purification is necessary for the finite lattices of 5 &#215; 6 cDAO-c64nt-O and 5 &#215; 6 cDAO-c74&#38;c84nt-O for AFM imaging and other applications. If the yield is too low, increase the centrifugation speed. If the product is not pure enough, repeat step 4.2.2.</li>
	</ol>
	</li>
</ol>
5. AFM Imaging
<ol>
	<li>For c64nt nanowire, c84nt nanowire, acDAO-c64nt-E, infinite 2D lattices of cDAO-c64nt-E(O), and cDAO-c84nt-E(O), deposit 2 &#956;L of annealed sample on a freshly cleaved mica surface. Leave for 2 min for adsorption of DNA lattices to the mica surface. Wash the surface with 100 &#181;L of deionized water twice and dry it by compressed air.</li>
	<li>Obtain AFM images of infinite DNA lattices in air by scanning the mica surface with triangular AFM probes under tapping mode. Set the following parameters for scanning: scan size of 0.5~5 &#181;m, scan resolution of 512 lines, and scan rate of 3.5 Hz (Figure 4).</li>
	<li>For finite DNA lattices of 5 &#215; 6 cDAO-c64nt-O and 5 &#215; 6 cDAO-c74&#38;c84nt-O,&#160;deposit 80 &#181;L of 1x TAE&#183;Mg buffer on a freshly cleaved mica surface. Then add 5 &#181;L of finite samples into the buffer. Leave for 2 min for adsorption of DNA lattices to the mica surface. Add another 50 &#181;L of 1x TAE&#183;Mg buffer on the AFM probe.</li>
	<li>Obtain AFM images of finite DNA lattices in fluid by scanning the mica surface with triangular AFM probes under tapping mode. Set the following parameters for scanning: scan size of 0.5-1 &#181;m, scan resolution of 256 lines, and scan rate of 1.5 Hz (Figure 5).</li>
</ol>

<ol>
	<li>Tsu-Ju, F., 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=DNA+double-crossover+molecules.">DNA double-crossover molecules.</a> Biochemistry. 32 (13), 3211-3220 (1993).</li><li>Winfree, E., Liu, F., Wenzler, L. A., 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=Design+and+self-assembly+of+two-dimensional+DNA+crystals.">Design and self-assembly of two-dimensional DNA crystals.</a> Nature. 394 (6693), 539-544 (1998).</li><li>Liu, F., Sha, R., 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=Modifying+the+surface+features+of+two-dimensional+DNA+crystals.">Modifying the surface features of two-dimensional DNA crystals.</a> Journal of the American Chemical Society. 121 (5), 917-922 (1999).</li><li>Yan, H., Park, S. H., Finkelstein, G., Reif, J. H., LaBean, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-templated+self-assembly+of+protein+arrays+and+highly+conductive+nanowires.">DNA-templated self-assembly of protein arrays and highly conductive nanowires.</a> Science. 301 (5641), 1882-1884 (2003).</li><li>Liu, D., Wang, M., Deng, Z., Walulu, R., Mao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensegrity:+Construction+of+rigid+DNA+triangles+with+flexible+four-arm+DNA+junctions.">Tensegrity: Construction of rigid DNA triangles with flexible four-arm DNA junctions.</a> Journal of the American Chemical Society. 126 (8), 2324-2325 (2004).</li><li>Tian, C., Li, X., Liu, Z., Jiang, W., Wang, G., Mao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+DNA+tiles+into+complex+nanocages.">Directed self-assembly of DNA tiles into complex nanocages.</a> Angewandte Chemie: International Edition. 53 (31), 8041-8044 (2014).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrosynthetic+analysis-guided+breaking+tile+symmetry+for+the+assembly+of+complex+DNA+nanostructures.">Retrosynthetic analysis-guided breaking tile symmetry for the assembly of complex DNA nanostructures.</a> Journal of the American Chemical Society. 138 (41), 13579-13585 (2016).</li><li>Ke, Y., Ong, L. L., Shih, W. 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=Three-dimensional+structures+self-assembled+from+DNA+bricks.">Three-dimensional structures self-assembled from DNA bricks.</a> Science. 338 (6111), 1177-1183 (2012).</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>Ke, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+brick+crystals+with+prescribed+depths.">DNA brick crystals with prescribed depths.</a> Nature Chemistry. 6 (11), 994-1002 (2014).</li><li>Rothemund, P. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+to+create+nanoscale+shapes+and+patterns.">Folding DNA to create nanoscale shapes and patterns.</a> Nature. 440 (7082), 297-302 (2006).</li><li>Douglas, S. M., Dietz, H., Liedl, T., H&#246;gberg, B., Graf, F., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+DNA+into+nanoscale+three-dimensional+shapes.">Self-assembly of DNA into nanoscale three-dimensional shapes.</a> Nature. 459 (7245), 414-418 (2009).</li><li>Dietz, H., Douglas, S. M., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+into+twisted+and+curved+nanoscale+shapes.">Folding DNA into twisted and curved nanoscale shapes.</a> Science. 325 (5941), 725-730 (2009).</li><li>Ackermann, D., Schmidt, T. L., Hannam, J. S., Purohit, C. S., Heckel, A., Famulok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+double-stranded+DNA+rotaxane.">A double-stranded DNA rotaxane.</a> Nature Nanotechnology. 5 (6), 436-442 (2010).</li><li>Zheng, H., Xiao, M., Yan, Q., Ma, Y., Xiao, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+circular+DNA+molecules+act+as+rigid+motifs+to+build+DNA+nanotubes.">Small circular DNA molecules act as rigid motifs to build DNA nanotubes.</a> Journal of the American Chemical Society. 136 (29), 10194-10197 (2014).</li><li>Wang, M., Huang, H., Zhang, Z., Xiao, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+DNA+lattices+constructed+from+two-tile+DAE-O+systems+possessing+circular+central+strands.">2D DNA lattices constructed from two-tile DAE-O systems possessing circular central strands.</a> Nanoscale. 8 (45), 18870-18875 (2016).</li><li>Guo, X., Wang, X. M., Wei, S., Xiao, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+holliday+junction+in+small+circular+DNA+molecules+for+stable+motifs+and+two-dimensional+lattices.">Construction of a holliday junction in small circular DNA molecules for stable motifs and two-dimensional lattices.</a> ChemBioChem. 19 (13), 1379-1385 (2018).</li><li>Holliday, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+gene+conversion+in+fungi.">A mechanism for gene conversion in fungi.</a> Genet. Res. 5 (2), 282-304 (1964).</li><li>Duckett, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+Holliday+junction.">The structure of the Holliday junction.</a> Structure and Methods, Human Genome Initiative and DNA Recombination. 1 (1), 157-181 (1990).</li><li>Ariyoshi, M., Vassylyev, D. G., Iwasaki, H., Nakamura, H., Shinagawa, H., Morikawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+structure+of+the+RuvC+resolvase:+A+holliday+junction-specific+endonuclease+from+E.+coli.">Atomic structure of the RuvC resolvase: A holliday junction-specific endonuclease from E. coli.</a> Cell. 78 (6), 1063-1072 (1994).</li><li>Eichman, B. F., Vargason, J. M., Mooers, B. H., Ho, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Holliday+junction+in+an+inverted+repeat+DNA+sequence:+sequence+effects+on+the+structure+of+four-way+junctions.">The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (8), 3971-3976 (2000).</li><li>Stahl, E., Martin, T. G., Praetorius, F., Dietz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+and+scalable+preparation+of+pure+and+dense+DNA+origami+solutions.">Facile and scalable preparation of pure and dense DNA origami solutions.</a> Angewandte Chemie: International Edition. 53 (47), 12735-12740 (2014).</li><li>de Gennes, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reptation+of+a+polymer+chain+in+the+presence+of+fixed+obstacles.">Reptation of a polymer chain in the presence of fixed obstacles.</a> The Journal of Chemical Physics. 55 (2), 572-579 (1971).</li><li>Slater, G. W., Noolandi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+biased+reptation+model+for+charged+polymers.">New biased reptation model for charged polymers.</a> Physical Review Letters. 55 (15), 1579 (1985).</li><li>Lilley, D. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+branched+nucleic+acid+structure+using+comparative+gel+electrophoresis.">Analysis of branched nucleic acid structure using comparative gel electrophoresis.</a> Quarterly Reviews of Biophysics. 41 (1), 1-39 (2008).</li><li>Pfreundschuh, M., Martinez-Martin, D., Mulvihill, E., Wegmann, S., Muller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+high-resolution+imaging+of+native+proteins+by+force-distance+curve-based+AFM.">Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.</a> Nature Protocols. 9 (5), 1113-1130 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

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 the correct ligation reaction, it is optimized to be around 20 nucleotides long for both c64nt and c84nt. 
An appropriate concentration of magnesium cations (e.g., 12.5 mM) in the solution during and after assembly is very important for the formation and maintenance of DNA nanostructures. Thus, a magnesium cation concentration of 12.5 mM is always kept during the processes of annealing, native PAGE, agarose gel electrophoresis and PEG buffer centrifugation purifications, AFM imaging, etc. 
For finite rectangle assemblies of 5 &#215; 6 cDAO-c64nt-O and 5 &#215; 6 cDAO-c74&amp;84nt-O, the agarose gel purification does not affect the texture details, while the PEG buffer centrifugation purification harms the texture details of DNA nanostructures in the high-resolution AFM images.
Benefit from the stability and rigidity of circular tiles, it is much easier to produce well-organized and large-size single crystalline 2D lattices from circular modules than from linear tiles and scaffolded origami9,11, although extra work is needed to synthesize and purify the circular DNA molecules. With only one circular DNA as the same core structure, many circular modules can be generated with different overhangs; by means of specific sticky end cohesions of overhangs finite nanostructures can be built; this strategy reduces the workload and cost of finite nanostructures. One significant advantage of circular DNA nanostructures is the resolution of secondary and tertiary structures of DNA molecules and their key elements such as Holliday junction from the texture details of single crystalline lattices. The 1D, 2D and 3D nanostructures built from circular modules and their potential applications in biology, medicine, and nano-engineering will become a new family member of DNA nanotechnology in the future.

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.

<table><tbody><tr><td>T4 ligase</td><td>TaKaRa</td><td>2011A</td><td></td></tr><tr><td>T4 buffer</td><td>TaKaRa</td><td>2011A</td><td></td></tr><tr><td>TE buffer</td><td>Sangon</td><td>B548106</td><td></td></tr><tr><td>Thermo bottle</td><td>Thermos</td><td>SK-3000</td><td></td></tr><tr><td>Thermo cycler</td><td>Bio Gener</td><td>GE4852T</td><td></td></tr><tr><td>Exonuclease I</td><td>TaKaRa</td><td>2650A</td><td></td></tr><tr><td>Exonuclease I buffer</td><td>TaKaRa</td><td>2650A</td><td></td></tr><tr><td>30% (w/v) Acryl/Bis solution (19:1)</td><td>Sangon</td><td>B546016</td><td></td></tr><tr><td>TAE premix podwer</td><td>Sangon</td><td>B540023</td><td></td></tr><tr><td>Mg(Ac)2&amp;middot;4H2O</td><td>Nanjing Chemical Reagent</td><td>C0190550223</td><td></td></tr><tr><td>Urea</td><td>Sangon</td><td>A510907</td><td></td></tr><tr><td>TEMED</td><td>BBI</td><td>A100761</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Nanjing Chemical Reagent</td><td>13041920295</td><td></td></tr><tr><td>Power supply</td><td>Beijing Liuyi</td><td>DYY-8C</td><td></td></tr><tr><td>Water bath</td><td>Sumsung</td><td>DK-S12</td><td></td></tr><tr><td>Formamide</td><td>BBI</td><td>A100314</td><td></td></tr><tr><td>DNA Marker (25~500 bp)</td><td>Sangon</td><td>B600303</td><td></td></tr><tr><td>DNA Marker (100~3000 bp)</td><td>Sangon</td><td>B500347</td><td></td></tr><tr><td>Loading buffer</td><td>Sangon</td><td>B548313</td><td></td></tr><tr><td>PAGE electrophoresis systerm</td><td>Beijing Liuyi</td><td>24DN</td><td></td></tr><tr><td>Filter</td><td>ASD</td><td>5010-2225</td><td>0.22 &amp;micro;M</td></tr><tr><td>UV imaging System</td><td>Tanon</td><td>2500R</td><td></td></tr><tr><td>n-butanol</td><td>Sangon</td><td>A501800</td><td></td></tr><tr><td>Absolute Ethanol</td><td>SCR</td><td>10009257</td><td></td></tr><tr><td>NaOAc</td><td>Nanjing Chemical Reagent</td><td>12032610459</td><td></td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>Centrifuge 5424R</td><td></td></tr><tr><td>Vacuum concentrator</td><td>CHRIST</td><td>RVC 2-18</td><td></td></tr><tr><td>Ultraviolet spectrum</td><td>Allsheng</td><td>Nano-100</td><td></td></tr><tr><td>nucleic acid stain</td><td>Biotium</td><td>16G1010</td><td>GelRed</td></tr><tr><td>Agarose</td><td>Biowest</td><td>G-10</td><td></td></tr><tr><td>Agarose electrophoresis systerm</td><td>Beijing Liuyi</td><td>DYCP-31CN</td><td></td></tr><tr><td>Heating Plate</td><td>Jiangsu Jintan</td><td>DB-1</td><td></td></tr><tr><td>TBE premix podwer&amp;nbsp;</td><td>Sangon</td><td>B540024</td><td></td></tr><tr><td>filter column</td><td>Bio-Rad</td><td>7326165</td><td>Freeze &#039;N Squeeze column</td></tr><tr><td>AFM</td><td>Bruker</td><td>Dimension FastScan</td><td></td></tr><tr><td>PEG8000</td><td>BBI</td><td>A100159</td><td></td></tr><tr><td>Mica</td><td>Ted Pella</td><td>BP50</td><td></td></tr><tr><td>triangular AFM probe in air</td><td>Bruker</td><td>FastScan-C</td><td></td></tr><tr><td>triangular AFM probe in fulid</td><td>Bruker</td><td>ScanAsyst-fluid+</td><td></td></tr><tr><td>DNA strands</td><td>Sangon</td><td></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 concentration of a precursor linear DNA is high enough at around 3.5 &#181;M, the cyclization products of c64nt (or c84nt) and the precursor reference in this protocol can be directly shadowed as bands in the TLC plate under UV light without dying. If the bands of circular DNA are vague or invisible, indicating a failure of the ligation reaction or a much low product yield. Sometimes there are two bands above the linear precursor band, referring an additional oligo-dimer ring except for the correct oligo-monomer ring. Just leave the higher bands alone and collect the lower ones. The purified circular DNA products can be seen as white powder in the tube after vacuum drying. Except for the denaturing PAGE, the DNA purity can also be measured by UV spectrometer. The absorption peak of DNA is at 260 nm. Two standard criteria for the DNA purity are the absorption ratio of 280 nm/260 nm at 1.8 and that of 280 nm/230 nm commonly in the range of 2.0-2.2. If the above two ratios deviate from the standard values, the remains should be extracted again by following steps 1.18-1.20. The yields of c64nt and c84nt for the correct oligo-monomer cyclization are measured at the range of 30-60% according to this protocol.
Native PAGE analysis provides a lot of information about the motif&#39;s stability, purity, rigidity, assembly mode of monomer or polymer, etc (Figure 3). The c64nt assembly families of c64bp, HJ-c64nt, aHJ-c64nt, cDAO-c64nt, and acDAO-c64nt have only one clear and clean band for each assembly, representing they are stable monomer motifs. While the c84nt assembly families of HJ-c84nt, tHJ-c84nt, and cDAO-c84nt tiles have smears around their main bands, indicating minor by-products except for the target monomer motifs. Regardless of the minor byproducts of incorrect associates, excellent cDAO-c84nt-O (E) lattices with high yields can be assembled. To get a clear and clean electrophoresis image, the loading volume should be no more than 10 &#181;L and the quantity of DNA should be 0.01~0.02 &#181;g/mL.
The success of experiments is finally evaluated by 1D and 2D DNA nanostructures imaged by AFM (Figure 4 and Figure 5). Each assembly has its own morphological features in the micrometer scale such as nanowires, nanotubes, nanospirals, nanoribbons, etc. Moreover, the detailed textures of the DNA assemblies in the nanometer scale correlated to their theoretical circular tile sizes and organization modes very well respectively are the key for verification of successful and correct assembly. Therefore, both panoramic and high-resolution AFM images in micrometer and nanometer scales must be obtained. Choices of AFM methods and probes are crucial for the high-quality AFM images. The scan force should be adjusted as small as 50 pN26. If the scan force is too big, it would damage the DNA nanostructural patterns. The environmental cleanliness is another key parameter to get a clean and beautiful high-resolution AFM image in fluid. All buffers must be filtered by 0.22 &#181;m filter; the probe holder and tweezers must be washed by detergent and rinsed by deionized water. If the environment is polluted by particulate debris, the probe tip would be damaged or clung by particles in the buffer, thus affecting the quality of AFM images.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58744/58744fig1.jpg" /> 
Figure 1. Synthesis of circular DNA. The flow diagram represents how a long 5&#39;-phosphorylated linear oligonucleotide in blue evolves to a circular DNA molecule. The two short red strands represent the splint oligonucleotides. <a href="https://www.jove.com/files/ftp_upload/58744/58744fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58744/58744fig2.jpg" /> 
Figure 2. Denaturing PAGE photograph of DNA cyclization products under UV light without dying. A precursor linear 64nt DNA band is in the far-left lane and its cyclization products of circular 64nt (c64nt) DNA appear as 9 bands at the same horizontal level in other 9 lanes. The 9 bands of c64nt will be cut off for abstracting circular DNAs. <a href="https://www.jove.com/files/ftp_upload/58744/58744fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58744/58744fig3.jpg" /> 
Figure 3. Native PAGE photographs after dying and schematic double-helical models of circular tiles. Both polymers of c64nt nanowire and c84nt nanowire are represented by their simplest folding unit cells, in which the two aligned dots above and below each unit cell indicate the infinite alignment of unit cells vertically up and down, with equal distances between duplexes to form nanowires. Monomer circular tiles of c64bp, c84bp, HJ-c64nt, aHJ-c64nt, HJ-c84nt, and tHJ-c84nt in A) have no protruding overhangs out of their rings, whereas cDAO-c64nt, acDAO-c64nt, and cDAO-c84nt in B) have both blunt-ended 10 bp overhangs respectively. For sequences, please refer to the Table of DNA Sequences. This figure has been modified from a previously published figure17.&#160;<a href="https://www.jove.com/files/ftp_upload/58744/58744fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58744/58744fig4.jpg" /> 
Figure 4. AFM images of typical 1D and 2D infinite DNA assemblies scanned in air. A) c64nt nanowire, B) infinite acDAO-c64nt-E, C) infinite cDAO-c64nt-E, D) infinite cDAO-c64nt-O, E) infinite cDAO-c84nt-E, and F) infinite cDAO-c84nt-O are annealed by sticky end cohesion. All texture details in these AFM images are in line with the tile sizes and organization modes very well. For sequences, please refer to the Table of DNA Sequences. This figure has been modified from a previously published figure17.&#160;<a href="https://www.jove.com/files/ftp_upload/58744/58744fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58744/58744fig5.jpg" /> 
Figure 5. AFM images of finite rectangle assemblies scanned in fluid. The finite rectangle assembly of A) 5 &#215; 6 cDAO-c64nt-O is composed of 32 cDAO-c64nt sub-tiles, and B) 5 &#215; 6 cDAO-c74&#38;84nt-O is composed of 12 cDAO-c74nt and 20 cDAO-c84nt sub-tiles. For sequences, please refer to the Table of DNA Sequences. This figure has been modified from a previously published figure17.&#160;<a href="https://www.jove.com/files/ftp_upload/58744/58744fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table of DNA Sequences.&#160;<a href="https://www.jove.com/files/ftp_upload/58744/Table_of_DNA_Sequences.xls" target="_blank">Please click here to download this file.</a>

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 ...

stable-dna-motifs-1d-2d-nanostructures-constructed-from-small

Nanyang Technological University

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Chemistry

6.3K Views.  Nanjing University. 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. 

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

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 ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

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

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Bioengineering

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 ...

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic nanostructures. The high spatial precision of a DNA origami-templated assembly allows controlling the coupling between plasmonic resonances of individual particles and enables tailoring optical properties of the constructed nanostructures. Recently, chiral plasmonic systems attracted a lot of attention due to the strong correlation between the spatial configuration of plasmonic assemblies and their optical responses (e.g., circular dichroism [CD]). In this protocol, we describe the whole workflow for the generation of DNA origami-based chiral assemblies of gold nanorods (AuNRs). The protocol includes a detailed description of the design principles and experimental procedures for the fabrication of DNA origami templates, the synthesis of AuNRs, and the assembly of origami-AuNR structures. In addition, the characterization of structures using transmission electron microscopy (TEM) and CD spectroscopy is included. The described protocol is not limited to chiral configurations and can be adapted for the construction of various plasmonic architectures.

We describe the detailed protocol for the DNA origami-based assembly of gold nanorods into chiral plasmonic metamolecules with strong chiroptical responses. The protocol is not limited to chiral configurations and can be easily adapted for the fabrication of various plasmonic architectures.

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic ...

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

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

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

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

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

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

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

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

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

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...