XIE/Enzyme:2025/Dec

12.08

• HR-DNA Origami（NO） Folding
• Materials

Material	Volume（100 μL）
Staples (500 nM)	25 μL
P8064 (150 nM)	10 μL
10X PBS	10 μL
H2O	55 μL

• Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h.

• Purification (12.09): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add UP Water to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with UP Water, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate.

12.09

• HR-DNA Origami（NO） Folding
• Materials

Material	Volume（100 μL）
Staples (500 nM)	25 μL
P8064 (150 nM)	10 μL
10X PBS	10 μL
H2O	55 μL

• Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h.

• Purification (12.10): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add 1X PBS to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with 1X PBS, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate.

12.10

• Agarose Gel Electrophoresis
• Gel Preparation

1. Recipe: 2.4 g agarose in 120 mL 0.5× TBE
2. Heat in a microwave to dissolve, cool slightly, adjust volume with 0.5× TBE to 120 ul, then add 1.2 mL of 1 M MgCl2 and 12 µL GoldView DNA stain.
3. Insert comb and waite for the gel to cool and solidify.

• Sample preparation

1. Origami (15 nM): sample:loading buffer = 5:1.
2. Scaffold: take 1 µL scaffold, dilute to 10 µL (final concentration 15 nM); scaffold:loading buffer = 5:1.

• Electrophoresis

1. Run at 90 V for 120 min in 0.5× TBE with 10 mM MgCl2
2. Marker、Scaffold（1X）、Origami（10.08-未洗）、Origami（10.08—清洗）、Origami（10.09-未洗）、Origami（10.09-清洗）、Scaffold（10X）、Marker

Origami(NO)

12.11

• TEM

Negative staining (uranyl acetate)

• Grid activation: glow‑discharge copper grids for 30 s.
• Sample staining

1. apply 4.5 µL of sample onto the grid.
2. Blot off the sample, add buffer to the grid, then blot off the buffer.
3. Prepare parafilm and place three drops of uranyl acetate (pH adjusted) on the parafilm.
4. Quickly transfer the grid into a staining droplet, blot off excess, and incubate for 30 s.
5. Air‑dry the grid.

TEM_Origami(NO)

12.12

• S2 conjugated G5

Incubation

Stock Solution: 1 mM

1. Measure G5 stock (3 mg/mL, 46 μM). Dilute 10 μL of the stock to 100 μL (4.6 μM); aliquot and store at -80°C as the storage sample.
2. Take one storage aliquot and add 10 μL DBCO stock (10 mM). Divide evenly into two groups (ultrafiltration and non-ultrafiltration). For the non-ultrafiltration group, aliquot 16 μL into a PCR tube (designated M1) and incubate on ice at 4°C for 4 h.
3. For the ultrafiltration group, transfer the sample into a 10 kDa centrifugal filter unit, dilute to 500 μL with 1× PBS, and centrifuge at 10,000 × g, 4°C for 30 min. Concentrate the sample to approximately 100 μL, then spin at 8,000 × g for 2 min to collect. Aliquot 16 μL into a PCR tube (designated M2).
4. Add 1 μL S1 (500 μM) to both the ultrafiltered and non-ultrafiltered samples and incubate on ice at 4°C for 4 h. Label the non-ultrafiltered sample N-U and the ultrafiltered sample U.
5. Retain the −80°C storage aliquot as control C.

Native PAGE

• Gel: 7.5% native gel.
• Sample preparation: Mix 16 μL of each sample (C, M1, M2, N-U, U) with 4 μL loading buffer and mix thoroughly.
• Electrophoresis: Run at 120 V for 100 min in 1× TBE.

12.13

• S2 conjugated G5

Incubation

1. Prepare two aliquots of G5 stock, labeled A and B.
2. Add 1 µL DBCO to each of A and B and incubate for 4 h.
3. For A: remove 16 µL as sample M1; add 10 µL S2 to the remaining A (named O1) and incubate for 4 h.
4. For B: dilute the sample to 500 µL, transfer to a 10 kDa centrifugal ultrafiltration tube and centrifuge at 10,000 g for 30 min; concentrate to 100 µL, remove 16 µL as sample M2; add 10 µL S2 to the remaining B (named O2) and incubate for 4 h