1 DNA Origami Folding

1.1 6HB Folding Reaction

Materials:

  • DNA LoBind eppendorf tubes
  • Folding buffer (12 mM MgCl2 + 5 mM TE)
  • MiliQ water
  1. DNA Origami folding protocol was derived from Gür et. al. to fold the 6HB design.
  2. 8064 bp long scaffold strand along with staple strands were used in a ratio of 1:10.
  3. These staple strands included:
    • Core staple strands
    • Handle sequences for the attachment of liposomes
    • For attachment to magnetic beads: biotinylated oligo
  4. The following mixture was prepared for the folding of 6HB (400 µl) and placed in a thermal cycler:
         
    MiliQ H2O TE buffer MgCl2 Scaffold Handle Staples Core Staples Capture Oligo
    Stock Concentration - 50 mM (10x) 120 mM 100 nM 4.16 µM 704 nM 1000 nM
    Final Concentration - 5 mM (1x) 12 mM 10 nM 100 nM 100 nM 100 nM
    Volume (μL) 173.6 40 40 40 9.6 56.8 40

1.2 Annealing protocol

Materials:

  • DNA LoBind eppendorf tubes
  • Thermal cycler (Biorad C1000 Touch)

Procedure

  1. Heating up to 80°C
  2. Cooling to 65°C at the rate of 1°C per minute
  3. Once at 65°C, lower the temperature to 20°C at the rate of 1°C per 20 minutes
  4. Cool down to 4°C
  5. The DNA origami can be stored at this temperature in DNA LoBind tubes

2 Modification of 6HB

Materials needed:

  • 1.5 mL Eppendorf tubes
  • Pipettes
  • Pipette Tips
  • 1M tris(2-carboxyethyl)phosphine (TCEP)
  • Single-stranded DNA 5'ThioMC6-D/TTTTTTTCTTTGTTTCTTT
  • Liquid nitrogen
  • Biotin-maleimide
  • MilliQ Water
  • N-Methyl-2-pyrrolidone (NMP)
  • 0.1 mM KH2PO4 (Potassium Dihydrogen Phosphate) buffer
  • Vortex mixer
  • Centrifuge
  • Sample Rotator
  • Ultra Performance Liquid Chromatography (UPLC) machine
  • Mass Spectrometer
  • Speed vacuum

Procedure:

  1. Ensure that all Eppendorf tubes and pipette tips are all low-bind and low-retention respectively
  2. Pipette 200 μL of 100 μM ssDNA for modification
  3. Prepare a three-fold excess of 1 M TCEP
  4. Dilute the TCEP sample to 1:100
  5. Add 6 μL of TCEP to the DNA
  6. Let the sample incubate at room temperature for 30 minutes
  7. Dissolve 1 mg of biotin-maleimide in 1 mL NMP
  8. Add 90.3 μL of dissolved biotin-maleimide and add it to the DNA
  9. Check the pH of the sample and make sure it is approximately 7.2
  10. Add 10 μL of 0.1 mM potassium dihydrogen phosphate buffer at a time until pH reaches 7.2
  11. Place sample in a rotator overnight
  12. Run UPLC and Mass Spectrometry on a 10μL aliquot of the sample
  13. If product is obtained, purify the sample using High Performance Liquid Chromatography (HPLC) and place the sample in a speed vacuum set at 0.1 mbar

3 Purification

3.1 Agarose Gel Electrophoresis

Materials:

  • 1 kb ladder (GeneRuler 1 kb Plus DNA Ladder, Thermo Scientific)
  • DNA grade agarose
  • SYBR® Safe DNA Gel stain (Life technologies)
  • 10x TBE

Procedure:

  1. 0.75 % Agarose gel was prepared for the purification of DNA-origami
  2. Take 0.9 g of Agarose in a Sybr-safe beaker
  3. Add 6 ml of 10x TBE buffer
  4. Add remaining water to make the total volume 120 ml
  5. Microwave for 3-3:30 min
  6. Add 1.2 ml of 1.2 mM MgCl2 (Tip: Try to pour it on the sides of the beaker to minimize the temperature difference)
  7. Use 12 µl of the dye (Sybr Safe) and add it to the beaker (in a spiral fashion)
  8. Slowly pour the solution in the cast with a comb and use a pipetting tip to move any bubbles aside
  9. Let the gel cool for about 20-30 minutes

3.2 Amicon Filtration

Materials:

  • 1x Folding Buffer (12 mM MgCl2, 1x TE)
  • 100 kD Amicon filter

Procedure:

  1. Amicon filtration was performed to get rid of the excess staples after the folding reaction
  2. Incubate amicon filters with 1xFB overnight
  3. Wash with 1xFB twice at 14x G for 2 minutes
  4. Add 50 µl sample + 400 µl 1xFB, centrifuge for 2 minutes at 14x G
  5. Wash 6 times with 450 µl FB
  6. Invert and run at 1x G for 2 minutes
  7. High concentration was achieved and almost no staple strands were remaining after purification

3.3 Magnetic Bead Purification

Materials:

  • Pierce Streptavidin Magnetic Beads (Thermo Fisher-Pierce)
  • DynaMag™-2 Magnet (Thermo Fisher-Scientific)
  • Washing buffer (1x PBS, 12 mM MgCl2), 0.5% TWEEN

Procedure:

  1. Biotinylated staple strands were used to bind the DNA-origami to streptavidin functionalized magnetic beads
  2. This method was used to remove excess staples and streptavidin in the 6HB solution
  3. Mix together the magnetic beads and biotinylated 6HB with impurities
  4. Let the mixture incubate overnight in a shaker
  5. Wash the beads after placing them on a magnetic stand to remover the unbounded impurities using the washing buffer
  6. Add 200 nM of the displacement staples and incubate for one hour while mixing gently
  7. Recover the 6HB by placing the eppendorf on the magnetic stand

4 Quantification of DNA-Origami

  1. The concentrations of the purified DNA origami structures were determined by measuring the absorbance at 260 nm using a spectrophotometer (Implen NanoPhotometer® P360)
  2. Value generated by photometer: mass concentration (µg/ml)
  3. Molar concentration of the sample can be measured by knowledge of the scaffold length (8064 bp in this case) and mass of one bp (660 g/mol)
  4. Thus, molar concentration = mass concentration (µg/ml) / (8064 * 660 g/mol)

5 Imaging

5.1 TEM/SEM Imaging

  1. Plasma etching (SPI Plasma Prep™ III Plasma Etcher) was performed on carbon-coated copper grids (Plano GmbH)
  2. 10 µL of sample was placed on each grid for 5-10 min and then absorbed on kimtech™ wipes
  3. The samples containing DNA were stained with 4 µl of 2% uranyl acetate (+ 25 mM sodium hydroxide) for 90 seconds before removing it again.
  4. The grids were allowed to dry for 15 minutes and placed in the grid holder for storage

5.2 Cryo-TEM

  1. 2µl of sample in 1x FB was deposited on a holey carbon support film attached to a copper grid.
  2. After rapid freezing, the sample was loaded to a Gatan cryo transfer holder at -130°C to prevent ice crystallization.