The authors thank S. Voutilainen for her assistance with CD spectrometer. The authors acknowledge the provision of facilities and technical support by Aalto University at OtaNano - Nanomicroscopy Center (Aalto-NMC). This work was supported by the Academy of Finland (grant 308992) and the European Union&#39;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement No. 71364.

1. Design of the DNA origami
<ol>
	<li>Identify the desired relative spatial arrangement of AuNRs and the suitable shape of the DNA origami template (Figure 1A). Estimate the structural parameters of the AuNRs and the origami templates. Locate the approximate positions of staples that need further modification (Figure 1B).</li>
	<li>Download and install caDNAno18 to design a DNA origami template. In caDNAno, route the scaffold and staple strands according to the desired shape of the template and generate the staple strands sequence by clicking Seq Tool. Click Paint Tool and mark the staple strands that require further modification (Figure 1C).</li>
	<li>Click Export Tool to export the DNA staple sequences (Figure 1C) to a .csv file.</li>
	<li>Design double-stranded locks to fix the angle &#920; between the two origami bundles. Depending on the relative orientation of the two bundles, the origami construct can adapt left- or right-handed (LH/RH) chiral spatial configuration (Figure 1B).</li>
	<li>Import the staples&#39; .csv file into a spreadsheet application. Add a polyA10 sequence at the end of the staples used for AuNR assembly (handles). Modify the staple strands on the designed lock sites with lock sequences. 
	NOTE: The assemblies in the representative results contain 36 handles protruding at the 3&#39; end of the staple strands, 18 on each DNA origami bundle, equally distributed on two parallel helices every 21 nt. The distance between the first and the last handle position is 168 nt, approximately 57 nm (see the attached caDNAno file).</li>
</ol>
2. Assembly of the DNA origami templates
<ol>
	<li>Prepare a working stock of staple strands (SM), including strands with handles and locks, by mixing equal amounts of concentration-normalized staple oligonucleotides (e.g., 100 &#181;M). 
	NOTE: Origami structures usually contain several hundreds of staple strands. Staples are typically purchased from vendors specializing in the chemical synthesis of DNA oligonucleotides in multiwell (e.g., 96-well) plates.</li>
	<li>For 500 &#181;L of 10 nM origami, mix 50 &#181;L of Tris-EDTA (TE, 10x), 100 &#181;L of MgCl2 (100 mM), 25 &#181;L of NaCl (100 mM), 175 &#181;L of H2O, 100 &#181;L of SM (0.5 &#181;M), 5 &#181;L of lock strands (5 &#181;M), and a 50 &#181;L scaffold (100 nM).</li>
	<li>Anneal the mixture in a thermocycler from 80 &#176;C to 20 &#176;C as described in Table 1.</li>
</ol>
3. DNA origami purification
NOTE: This section describes the protocol for agarose gel purification. DNA origami templates can also be purified using alternative approaches38,39.
<ol>
	<li>For 1% gel, dissolve 1 g of agarose in 100 mL of Tris-borate-EDTA (TBE, 0.5x) by heating the mixture in a microwave oven. Add 10 &#181;L of 10,000x DNA stain according to the stain specification. To minimize the exposure to UV light at the extraction step (step 3.6), use a DNA stain that can be visualized under blue excitation.</li>
	<li>Cool the solution to approximately 40 &#176;C and slowly add 1 mL of MgCl2 (1.3 M) while shaking. Cast gel and incubate for 30 min at room temperature.</li>
	<li>Set the electrophoresis device and pour cold (4 &#176;C) running buffer (0.5x TBE with 11 mM MgCl2) into the gel box. Place the gel box in an ice water bath.</li>
	<li>Add loading buffer to the origami samples (6x loading buffer contains 15% polysucrose 400 and 0.25% bromophenol blue in water). Load the samples into the wells with a proper volume according to the comb used (e.g., 50 &#181;L for an 8-well comb of 1.5 mm in thickness).</li>
	<li>Run the electrophoresis for 2 h at 80 V. 
	NOTE: To characterize the origami and separate the open and closed structure, use 2% gel instead of 1% and prolong the running time to 4 h.</li>
	<li>Image the gel with the gel imager (Figure 2). Use a blue light transilluminator to visualize the bands, cut the origami band, smash the gel on a parafilm, and extract the liquid. The recovery yield is approximately 40%.</li>
	<li>Pipette the liquid into a centrifugal filter unit and spin at 3,000 x g for 5 min. Measure the absorption of the origami solution at 260 nm with a UV-visible (UV-VIS) spectrometer. Estimate the concentration of origami using an extinction coefficient of 1.3 x 108 M-1&#183;cm-1. 
	NOTE: The typical concentration of origami solution after agarose gel purification is 1-2 nM.</li>
	<li>Store the purified origami templates at 4 &#176;C for later use.</li>
</ol>
4. Synthesis of gold nanorods
NOTE: The protocol for AuNR synthesis is adapted from previous literature40 with minor modifications.
<ol>
	<li>Wash all glassware with aqua regia for 5 min, rinse it with water, sonicate it with ultrapure water, and dry it before use.</li>
	<li>Prepare 0.2 M hexadecyltrimethylammonium bromide (CTAB), 1 mM HAuCl4, 4 mM AgNO3, 64 mM L(+)-ascorbic acid, and 6 mM NaBH4. Use cold water (4 &#176;C) to dissolve NaBH4 and keep it in a fridge at 4 &#176;C. Ascorbic acid solution has to be freshly prepared. 
	CAUTION: CTAB is hazardous in case of skin contact (irritant), eye contact (irritant), ingestion, and inhalation. Wear suitable protective clothing. In case of insufficient ventilation, wear suitable respiratory equipment. NaBH4 is extremely hazardous in case of skin contact (irritant), eye contact (irritant), ingestion, and inhalation. Wear splash goggles, a lab coat, gloves, and a vapor and dust respirator. Be sure to use an approved/certified respirator or equivalent.</li>
	<li>Prepare Au seeds.
	<ol>
		<li>Add 500 &#181;L of CTAB (0.2 M), 250 &#181;L of ultrapure water, and 250 &#181;L of HAuCl4 (1 mM) into a glass vial. Stir at 450 rpm at room temperature for 5 min.</li>
		<li>Increase the stirring rate to 1,200 rpm. Add 100 &#181;L of cold NaBH4 solution (6 mM, 4 &#176;C). After 2 min, stop the stirring and incubate the solution in a water bath at 30 &#176;C for 30 min before use.</li>
	</ol>
	</li>
	<li>Prepare AuNRs.
	<ol>
		<li>Dissolve 0.55 g of CTAB and 0.037 g of 2,6-dihydroxybenzoic acid in 15 mL of warm water (60-65 &#176;C) in a round-bottom flask. Cool down the solution to 30 &#176;C, add 600 &#181;L of AgNO3 (4 mM), and stir at 450 rpm for 2 min. Then, leave the solution undisturbed for 15 min at 30 &#176;C.</li>
		<li>Add 15 mL of HAuCl4 (1 mM) to the solution, and stir at 450 rpm for 15 min. Add 120 &#181;L of L(+)-ascorbic acid (64 mM), and then, immediately, stir at 1,200 rpm for 30 s. Add 12 &#181;L of Au seeds, and keep stirring at 1,200 rpm for 30 s.</li>
		<li>Incubate the solution in a water bath at 30 &#176;C for 18 h. Do not disturb the solution and use a cap to close the flask.</li>
		<li>Transfer the resultant solution to centrifuge tubes, and centrifuge at 9,500 x g for 12 min at 20 &#176;C. Discard the supernatant, disperse the pellet in 20 mL of ultrapure water, and perform one more centrifugation step.</li>
		<li>Disperse the final pellet in 3 mL of distilled water. Estimate the concentration of AuNRs from a UV-VIS absorption measurement using the extinction coefficient for the longitudinal plasmon resonance. The extinction coefficient can be predicted using AuNR shape parameters41. Store the AuNRs at 4 &#176;C for further use.</li>
	</ol>
	</li>
</ol>
5. Functionalization of gold nanorods with single-stranded DNA
NOTE: This section describes the protocol for AuNR functionalization with single-stranded DNA (ssDNA), following the so-called low pH route adapted from previous literature42. The AuNRs covered with DNA are purified by centrifugation; alternatively, the purification can be performed using agarose gel electrophoresis.
<ol>
	<li>Incubate 20 &#181;L of thiol-functionalized polyT DNA strands (1 mM) with 20 &#181;L of freshly prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 14 mM) for 1 h to reduce disulfide bonds. 
	NOTE: The thiol groups form bonds with AuNRs, and the polyT sequence hybridizes with the polyA10 handle on the origami, in which too many or too few base pairs may lead to a malfunction or an unstable assembly. 
	CAUTION: TCEP can cause severe skin burns and eye damage. Wear protective gloves/protective clothing/eye protection/face protection.</li>
	<li>Mix 150 &#181;L of AuNRs (10 nM) and 40 &#181;L of TCEP-treated thiol-DNA (0.5 mM). Add 1% sodium dodecyl sulfate (SDS) to the AuNR solution to reach a final SDS concentration of 0.05%. Adjust the pH to 2.5-3 with ~1 &#181;L of HCl (1 M).</li>
	<li>Incubate for 2 h while shaking at 70 rpm. 
	NOTE: The AuNR-to-DNA ratio should be in the order of 1:5,000-15,000, depending on the size of the rods. For the AuNRs (70 nm x 30 nm) prepared following the protocol described in section 4, a 13,000 excess of thiol-DNA is recommended.</li>
	<li>Add NaCl to reach a final NaCl concertation of 0.5 M and incubate for 4 h at room temperature while shaking at 70 rpm. 
	NOTE: A color change at this step may indicate a failed DNA functionalization.</li>
	<li>Adjust the pH to ~8.5 with TBE buffer (10x) and incubate overnight.</li>
	<li>Wash the DNA-AuNRs 4x by mixing the samples with 1 mL of washing buffer (0.5x TBE with 0.1% SDS), and centrifuge at 7,000 x g for 30 min. Remove the supernatant and resuspend the DNA-AuNRs in the remaining liquid (~40 &#181;L). Estimate the concentration of DNA-AuNRs from a UV-VIS absorption measurement as described in step 4.4.5. 
	NOTE: The solution might become slightly &#39;cloudy&#39; at steps 5.3-5.4 due to the CTAB replacement from the surface of the AuNRs by thiol-DNA. The solution should become clear upon warming up to ~35 &#176;C for 5 min.</li>
</ol>
6. Assembly of gold nanorods on DNA origami templates
<ol>
	<li>Add MgCl2 to the solution of purified DNA-AuNRs, to a final concentration of 10 mM. Mix the purified DNA-AuNRs and origami to a 10:1 ratio. 
	NOTE: A lower ratio may decrease the product yield43.</li>
	<li>Anneal the mixture in a mixer with a temperature control from 40 &#176;C to 20 &#176;C while shaking at 400 rpm, following the procedure in Table 2. 
	NOTE: For CD characterization, the sample can be measured after this step without further purification.</li>
	<li>Use 0.7% agarose gel electrophoresis (3.5 h at 80 V) to purify the final origami-AuNR structures.</li>
	<li>Use a white light transilluminator for imaging. Cut the product band (origami-AuNR dimer) (Figure 3), smash the gel on a parafilm, and extract the liquid. Pipette the liquid into a centrifugal filter unit and spin at 3,000 x g for 5 min. Resuspend the origami-AuNRs in the solution. The recovery yield from the gel is approximately 50%.</li>
	<li>Estimate the concentration of the origami-AuNR structures from a UV-VIS absorption measurement as described in step 4.4.5.</li>
</ol>
7. Transmission electron microscopy imaging
NOTE: This uranyl formate (UFo) staining protocol is adapted from previous literature44.
<ol>
	<li>Mix 200 &#181;L of UFo solution (0.75%) and 1 &#181;L of NaOH (5 M) and vortex immediately for 2-3 min. Centrifuge the stain solution for 3-4 min at 14,000 x g. Protect the stain from light exposure (e.g., by wrapping it in aluminum foil). 
	CAUTION: UFo is toxic if inhaled or swallowed and can cause eye irritation. In the case of brief exposure or low pollution, use a respiratory filter device. In the case of intensive or longer exposure, use a self-contained respiratory protective device. Wear gloves. The glove material has to be impermeable and resistant to UFo and its solutions. Wear tightly sealed goggles.</li>
	<li>Glow-discharge carbon/formvar-coated TEM grids for 6 s just before use to increase hydrophilicity and promote the sticking of the structures. Pipette 5 &#181;L sample drops on the TEM grid, incubate for 5-8 min, and remove the drop by gently touching a filter paper with the edge of the grid.</li>
	<li>Pipette one big (~20 &#181;L) and one small (~10 &#181;L) drop of the stain solution on a parafilm.Put the grid on the small stain solution drop and dry immediately by touching the filter paper with the edge of the grid. Then, put it on the big stain solution drop for 30 s.</li>
	<li>Remove the liquid on the grid by touching the filter paper with the edge of the grid. Place the grid in the grid holder. Wait for the grid to dry for at least 10 min.</li>
	<li>Characterize the samples of origami (Figure 4), AuNRs (Figure 5), and origami-AuNRs (Figure 6) by TEM.</li>
</ol>
8. Circular dichroism measurement
<ol>
	<li>Purge the CD spectrometer with N2 for 20 min. 
	NOTE: Most of the CD spectrometers require purging with N2 before lamp ignition. Check the CD spectrometer manual.</li>
	<li>Set the bandwidth, scanning range, and acquisition step. 
	NOTE: The scanning range depends on the optical properties of AuNRs, which depend on the size of the AuNRs.</li>
	<li>Measure blank CD with buffer.</li>
	<li>Measure the CD spectra of origami-AuNR samples (Figure 7). 
	NOTE: Use quarts or glass cuvettes for CD measurement. Plastic cuvettes are unsuitable for CD spectroscopy. Also, most CD spectrometers allow the simultaneous acquisition of absorption and CD data.</li>
</ol>

The authors have nothing to disclose.

The protocol introduces the whole workflow of design, assembly, purification, and characterization of DNA origami-based chiral assemblies of AuNRs. The DNA origami templates used in the protocol are particularly suitable for the fabrication of stimuli-responsive assemblies. Various types of responses and functionalizes can be incorporated into the lock strands that define the chiral state of the origami template (Figure 1B)24,25,26,31. For static assemblies, simpler block-shaped templates are often sufficient14,45,46,47.
The DNA origami-based approach to the fabrication of plasmonic nanostructure inherits limitations of the DNA origami technique48. The size of the origami templates is typically limited by the size of the scaffold strand. The stability of DNA structures is reduced under law-salt conditions. The cost of synthetic staple strands remains rather high. However, recent developments in the field of structural DNA nanotechnology are expected to overcome these limitations49,50,51,52,53,54,55.
Compared to other molecular-based approaches for generating chiral assemblies of AuNRs34,35,36,37, DNA origami provides a high level of spatial precision and programmability.
For achieving reliable and reproducible optical responses of chiral assemblies, we strongly recommend adapting the protocols for AuNR synthesis40, since the quality and optical properties of commercial products may vary between batches. Additional annealing (step 6.2) is often crucial for ensuring the correct attachment of AuNRs to DNA origami templates (Figure 6).
Finally, the protocol described here is not limited to chiral assemblies. DNA origami provides a very flexible platform for the fabrication of complex plasmonic nanostructures9,10.

DNA nanostructures, DNA origami in particular, have been widely used to arrange molecules and other nanoscale components (e.g., proteins and nanoparticles [NPs]), with nanometer precision into almost arbitrary geometries1,2,3,4,5. The ability to arrange metal NPs on DNA origami templates with a high yield and accuracy enables the fabrication of plasmonic structures with novel optical properties6,7,8,9,10. DNA origami technique is especially useful for the generation of chiral plasmonic structures, which require genuinely three-dimensional architectures11,12,13,14,15,16,17,18,19,20.
This protocol describes in detail the entire process of the fabrication of DNA origami-templated chiral assemblies of AuNRs. The software used for the design21 and structure prediction22,23 of DNA origami is intuitive and freely available. The origami fabrication and AuNR synthesis use common biochemistry lab equipment (e.g., thermocyclers, gel electrophoresis, hot plates, centrifuges). The structures are characterized using standard TEM and CD spectroscopy.
The fabrication of similar plasmonic nanostructures with top-down methods (e.g., electron beam lithography) would require rather complicated and expensive equipment. In addition, DNA origami templates provide the possibility to incorporate structural reconfigurability in plasmonic assemblies24,25,26,27,28,29,30,31,32,33, which is extremely challenging for structures fabricated with lithography techniques. Compared to other molecular-based approaches34,35,36,37, DNA origami-based fabrication provides a high level of spatial precision and programmability.

<table><tbody><tr><td>2,6-Dihydroxybenzoic acid</td><td>Sigma-Aldrich</td><td>D109606-25</td><td>98+%</td></tr><tr><td>AgNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Alfa Aesar</td><td>AA1141414</td><td>99.90%</td></tr><tr><td>Blue light transilluminator&amp;nbsp;</td><td>Nippon Genetics</td><td>FG-06</td><td>FastGene LED Transilluminator</td></tr><tr><td>Bromophenol Blue</td><td>Acros Organics</td><td>403160050</td><td>For agarorose gel&amp;nbsp; loading buffer</td></tr><tr><td>Centrifugal filter units</td><td>Merck Millipore</td><td>42600</td><td>DNA extraction from agarose</td></tr><tr><td>Chirascan CD spectrometer&amp;nbsp;</td><td>Applied Photophysics</td><td></td><td></td></tr><tr><td>Cuvette&amp;nbsp;</td><td>Hellma&amp;nbsp;</td><td>105-202-85-40</td><td>Quartz SUPRASIL</td></tr><tr><td>DNA lobind tubes</td><td>Eppendorf</td><td>30108051</td><td></td></tr><tr><td>Eppendorf Biospectrometer&amp;nbsp;</td><td>Eppendorf</td><td>6135000904</td><td></td></tr><tr><td>Eppendorf ThermoMixer C</td><td>Eppendorf</td><td>5382000015</td><td></td></tr><tr><td>Ficoll 400</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>BP525-10</td><td>Polysucrose 400 (For agarorose gel&amp;nbsp; loading buffer)</td></tr><tr><td>Gel electrophoresis sets</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Gel imager</td><td>Bio-Rad</td><td></td><td>Gel Doc XR+ System&amp;nbsp;</td></tr><tr><td>HAuCl&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Alfa Aesar</td><td>AA3640006</td><td>99.99%</td></tr><tr><td>HCl&amp;nbsp;</td><td>Scharlau</td><td>AC07441000</td><td>1M</td></tr><tr><td>Hexadecyltrimethylammonium bromide (CTAB)</td><td>Sigma-Aldrich</td><td>H9151-100</td><td>BioXtra, 98+%</td></tr><tr><td>L(+)-ascorbic acid</td><td>Acros Organics</td><td>401471000</td><td>99+%</td></tr><tr><td>M13p7560 scaffold strand</td><td>Tilibit nanosystems</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M2670-500</td><td>BioXtra, 99+%</td></tr><tr><td>NaBH&lt;sub&gt;4&lt;/sub&gt;</td><td>Acros Organics</td><td>200050250</td><td>99%</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653-500</td><td>BioXtra, 99.5+%</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S8045-500</td><td>BioXtra, 98+%</td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>P7668-1EA</td><td>PARAFILM M</td></tr><tr><td>PBS buffer (10X)</td><td>Thermo Fisher Scientific</td><td>BP3991</td><td>Molecular Biology</td></tr><tr><td>ProFlex PCR System</td><td>Thermo Fisher Scientific</td><td>4484073</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>74255-250</td><td>99+%</td></tr><tr><td>Staple strands</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sybr Safe</td><td>Invitrogen&amp;nbsp;</td><td>S33102</td><td>For DNA stain</td></tr><tr><td>TBE buffer (10X)</td><td>Invitrogen</td><td>15581-044</td><td>Molecular Biology</td></tr><tr><td>TE buffer (10X)</td><td>Thermo Fisher Scientific</td><td>BP24771</td><td>Molecular Biology</td></tr><tr><td>TEM</td><td>FEI</td><td></td><td>FEI Tecnai F12</td></tr><tr><td>Thiol-functionalized&amp;nbsp; ssDNA&amp;nbsp;</td><td>Biomers.net</td><td></td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl)</td><td>Thermo Fisher Scientific</td><td>PI20491</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen&amp;nbsp;</td><td>16500-100</td><td></td></tr><tr><td>Ultrapure water (Type 1)</td><td></td><td></td><td>Milli-Q Direct 8 system</td></tr><tr><td>Uranyl Formate</td><td>Tebu-bio</td><td>24762-1</td><td></td></tr><tr><td>White light transilluminator</td><td>UVP</td><td>TW-26</td><td></td></tr></tbody></table>

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.

TEM images of DNA origami templates, AuNRs, and final origami-AuNR assemblies are shown in Figure 4, Figure 5, and Figure 6A, respectively. Due to their binding preference to TEM grids, origami-AuNR assemblies are usually seen as parallel origami bundles and rods (Figure 6A). Thermal annealing is required for the correct alignment of AuNRs on origami templates (Figure 6A,B). The protocol enables high yields of the assembly of AuNRs into chiral metamolecules with strong plasmonic CD responses (Figure 7).
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Temperature (&#176;C)</td>
			<td>Time&#160;</td>
		</tr>
		<tr>
			<td>80</td>
			<td>15 min</td>
		</tr>
		<tr>
			<td>79 - 71</td>
			<td>1 &#176;C / 1 min</td>
		</tr>
		<tr>
			<td>70 - 66</td>
			<td>1 &#176;C / 5 min</td>
		</tr>
		<tr>
			<td>65 - 60</td>
			<td>1 &#176;C / 30 min</td>
		</tr>
		<tr>
			<td>59 - 37</td>
			<td>1 &#176;C / 60 min</td>
		</tr>
		<tr>
			<td>36 - 30</td>
			<td>1 &#176;C / 15 min</td>
		</tr>
		<tr>
			<td>29 - 20</td>
			<td>1 &#176;C / 5 min</td>
		</tr>
		<tr>
			<td>20</td>
			<td>Hold</td>
		</tr>
	</tbody>
</table>
Table 1: Temperatures and rates for the thermal annealing of DNA origami templates.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Temperature (&#176;C)</td>
			<td>Time (min)</td>
		</tr>
		<tr>
			<td>40</td>
			<td>130</td>
		</tr>
		<tr>
			<td>36</td>
			<td>180</td>
		</tr>
		<tr>
			<td>32</td>
			<td>180</td>
		</tr>
		<tr>
			<td>22</td>
			<td>Hold</td>
		</tr>
	</tbody>
</table>
Table 2: Temperatures and holding times for the annealing of AuNRs and DNA origami templates. The cooling rate between the steps is set at 0.1 &#176;C/min. The DNA origami-AuNR samples are annealed while shaking at 400 rpm.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59280/59280fig1.jpg" /> 
Figure 1: Design of DNA origami-templated chiral metamolecules. (A) Identify the desired relative spatial arrangement of gold nanorods (AuNRs) and a suitable shape of the DNA origami template. (B) Estimate the structural parameters of the AuNRs (DAuNR, LAuNR) and the origami template (Worigami, Lorigami,&#920;). Locate the approximate positions of the staples that need further modification. (C) Design of DNA origami templates using caDNAno. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig1large.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/59280/59280fig2.jpg" /> 
Figure 2: The agarose gel electrophoresis of origami. (A) Purification with 1% agarose gel electrophoresis for 2 h at 80 V. (B) Characterization with 2% agarose gel electrophoresis for 4 h at 80 V. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig2large.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/59280/59280fig3.jpg" /> 
Figure 3: The agarose gel electrophoresis purification of origami-AuNRs. Gel (0.7%) was run for 3.5 h at 80 V for the samples prepared following the assembly procedure with different DNA-AuNR-to-origami ratios (20:1, 5:1) and samples (10:1 DNA-AuNRs-to-origami ratio) with/without annealing procedure. For TEM images of the samples in bands 1, 2, and 3, see Figure 6. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig3large.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/59280/59280fig4.jpg" /> 
Figure 4: Representative TEM image of the DNA origami templates. The origami structure consists of two 14-helix bundles (80 nm x 16 nm x 8 nm) linked together by the scaffold strand. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig4large.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/59280/59280fig5.jpg" /> 
Figure 5: Representative TEM image of the AuNRs. The average dimensions of synthesized AuNRs are 70 x 30 nm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59280/59280fig6.jpg" /> 
Figure 6: TEM images of origami-AuNR assemblies. (A) AuNR dimers on origami after annealing (band 1 in Figure 3). (B) AuNR dimers on origami without annealing (band 2 in Figure 3). (C) Origami-AuNR aggregates (band 3 in Figure 3). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59280/59280fig7.jpg" /> 
Figure 7: CD spectra of the origami-AuNR assemblies. The CD spectra of the closed structures (the origami templates fixed by lock strands into a right-handed configuration, with 50&#176; between two origami bundles) and the open structure (the origami templates without lock strands). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59280/59280fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates at JoVE.com

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

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 ...

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8.4K Views.  Aalto University School of Science, Aalto FI-00076. 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.

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

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

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 ...

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

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Engineering

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

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

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

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

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 ...

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 ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...

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 ...