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

1. Design of the DNA origami

1. 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).
2. 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).
3. Click Export Tool to export the DNA staple sequences (Figure 1C) to a .csv file.
4. Design double-stranded locks to fix the angle Θ 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).
5. Import the staples' .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' 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).

2. Assembly of the DNA origami templates

1. 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 µM).
   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.
2. For 500 µL of 10 nM origami, mix 50 µL of Tris-EDTA (TE, 10x), 100 µL of MgCl₂ (100 mM), 25 µL of NaCl (100 mM), 175 µL of H₂O, 100 µL of SM (0.5 µM), 5 µL of lock strands (5 µM), and a 50 µL scaffold (100 nM).
3. Anneal the mixture in a thermocycler from 80 °C to 20 °C as described in Table 1.

3. DNA origami purification

NOTE: This section describes the protocol for agarose gel purification. DNA origami templates can also be purified using alternative approaches^38,39.

1. 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 µL 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.
2. Cool the solution to approximately 40 °C and slowly add 1 mL of MgCl₂ (1.3 M) while shaking. Cast gel and incubate for 30 min at room temperature.
3. Set the electrophoresis device and pour cold (4 °C) running buffer (0.5x TBE with 11 mM MgCl₂) into the gel box. Place the gel box in an ice water bath.
4. 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 µL for an 8-well comb of 1.5 mm in thickness).
5. 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.
6. 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%.
7. 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·cm^-1.
   NOTE: The typical concentration of origami solution after agarose gel purification is 1-2 nM.
8. Store the purified origami templates at 4 °C for later use.

4. Synthesis of gold nanorods

NOTE: The protocol for AuNR synthesis is adapted from previous literature⁴⁰ with minor modifications.

1. Wash all glassware with aqua regia for 5 min, rinse it with water, sonicate it with ultrapure water, and dry it before use.
2. 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 °C) to dissolve NaBH₄ and keep it in a fridge at 4 °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.
3. Prepare Au seeds.
   1. Add 500 µL of CTAB (0.2 M), 250 µL of ultrapure water, and 250 µL of HAuCl₄ (1 mM) into a glass vial. Stir at 450 rpm at room temperature for 5 min.
   2. Increase the stirring rate to 1,200 rpm. Add 100 µL of cold NaBH₄ solution (6 mM, 4 °C). After 2 min, stop the stirring and incubate the solution in a water bath at 30 °C for 30 min before use.
4. Prepare AuNRs.
   1. Dissolve 0.55 g of CTAB and 0.037 g of 2,6-dihydroxybenzoic acid in 15 mL of warm water (60-65 °C) in a round-bottom flask. Cool down the solution to 30 °C, add 600 µL of AgNO₃ (4 mM), and stir at 450 rpm for 2 min. Then, leave the solution undisturbed for 15 min at 30 °C.
   2. Add 15 mL of HAuCl₄ (1 mM) to the solution, and stir at 450 rpm for 15 min. Add 120 µL of L(+)-ascorbic acid (64 mM), and then, immediately, stir at 1,200 rpm for 30 s. Add 12 µL of Au seeds, and keep stirring at 1,200 rpm for 30 s.
   3. Incubate the solution in a water bath at 30 °C for 18 h. Do not disturb the solution and use a cap to close the flask.
   4. Transfer the resultant solution to centrifuge tubes, and centrifuge at 9,500 x g for 12 min at 20 °C. Discard the supernatant, disperse the pellet in 20 mL of ultrapure water, and perform one more centrifugation step.
   5. 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 °C for further use.

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.

1. Incubate 20 µL of thiol-functionalized polyT DNA strands (1 mM) with 20 µL 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.
2. Mix 150 µL of AuNRs (10 nM) and 40 µL 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 µL of HCl (1 M).
3. 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.
4. 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.
5. Adjust the pH to ~8.5 with TBE buffer (10x) and incubate overnight.
6. 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 µL). 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 'cloudy' 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 °C for 5 min.

6. Assembly of gold nanorods on DNA origami templates

1. 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.
2. Anneal the mixture in a mixer with a temperature control from 40 °C to 20 °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.
3. Use 0.7% agarose gel electrophoresis (3.5 h at 80 V) to purify the final origami-AuNR structures.
4. 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%.
5. Estimate the concentration of the origami-AuNR structures from a UV-VIS absorption measurement as described in step 4.4.5.

7. Transmission electron microscopy imaging

NOTE: This uranyl formate (UFo) staining protocol is adapted from previous literature⁴⁴.

1. Mix 200 µL of UFo solution (0.75%) and 1 µL 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.
2. 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 µL 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.
3. Pipette one big (~20 µL) and one small (~10 µL) 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.
4. 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.
5. Characterize the samples of origami (Figure 4), AuNRs (Figure 5), and origami-AuNRs (Figure 6) by TEM.

8. Circular dichroism measurement

1. 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.
2. 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.
3. Measure blank CD with buffer.
4. 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.