Experimental Process

Methods, rationale, and protocols. The nitty gritty stuff.

Creating the DNA origami

We began with two sizes of our DNA origami platform, which were a 60x60nm and 80x80nm hollow square, shown in Figures 1a,b below. Cholesterol strands were extended from the 3’ ends of the Teal strands shown in Figure 1a and Red strands shown in Figure 1b. In both figures the 3’ ends are also circled in red. Twist correction techniques were vital for flattening out the structure, as shown in Figure 2a,b to avoid creating tensile stresses on the liposomes from the rigid origami structure. Twist correction is performed by periodically skipping bases in the design. For flattening the structure, skips were added every 48 bases across the structure. Some of these areas are shown, for example, in Figure 1b circled in green.

The hollow square design anticipates release of aqueous drug from each of the liposomes. Inspired by the antipathic properties of the structure, the small window should enable release therapeutic loads to vortex together for better mixing. Furthermore, as there is no suitable way of controlling the orientation of the anchor strands (i.e into the page or out of the page), removing the strands in that area lower risks of entanglement. Additionally, using fewer DNA lowers the hydrohilicity of the structure, thereby allowing the origami structure to bind with fewer cholesterols, saving cost, which we have also considered to be a design constraint in drug delivery vehicles.

Figure 1a) 60x60 DNA origami design #1 for parameter testing

Figure 1b) 80x80 DNA origami design for parameter testing

Figure 1c) 60x60 DNA origami design #2 for parameter testing

Figure 2. a) CanDo simulation of 80x80nm cadnano design with twist correction techniques applied. b) Same design without twist correction techniques. Exhibits large fluctuations that could cause stresses on liposomes.

Our experiments determined that we would encounter a phenomenon called “stacking”, which occurs with the edge strands of the origami structure, called “sticky ends”, attracting and linking together with the sticky ends of other origami structures. These experiments are evaluated in our Discussion of Results. The resulting structure would be linked and chained together in this form. As an initial compromise, we omitted the edge strands of the 60x60 design shown in Figure 1a, and then compensated for this change permanently in our secondary design, shown in Figure 1c.

In design #2, we also decided to add more stability along the vertical axis of our structure. It is visible in Figures 1a, b above compared to Figure 1c. In Figures 1a, b. staple strands span farther horizontally, and few crossovers are made vertically, while in Figure 1c, we aligned many more crossovers is the vertical direction.

Folding DNA origami

DNA is folded by a thermocycling annealing reaction. This protocol is generalized for our 60x60 No Edge Design 2 structure, which is what we used for our final composite structure. See the Lab Book section under Experimental Process for more information on how this decision was developed.

(+) Full step-by-step protocol (click to expand)

a. Resuspension of Staple Strands

A 384-well plate of custom, dried staple strands (oligonucleotides) were resuspended in 1xTE buffer to a concentration of 100 µM in each well.

  1. Centrifuge the 384-well plate at 2000 RPM for 7 minutes to ensure that all dried staple strands are brought down to the bottom of the wells.
  2. Using a multi-channel pipette, dispense appropriate volumes of 1xTE buffer into each well to make up a concentration of 100 µM. Let the plate sit on ice for approximately 10 minutes to let the TE buffer soak into the staple strands.
  3. Resuspend the staple strands in each well and transfer them to individually labeled 0.2 µL PCR tubes for easier preparation of DNA origami pre-stocks.
  4. Store staple strands at -20°C.

b. Preparation of Pre-Stocks

Each DNA Origami Pre-stock contains the staple strands needed to form a certain section of the DNA Origami. In this case, there are five sections: “Anchor”, “Left Body”, “Right Body”, “Left Edge” and “Right Edge”.
Note: Each Pre-stock tube will contain different volumes, depending on the number of staple strands needed to form a particular section.

  1. Transfer 10 µL of staple strand solution from each individually labelled PCR tube into a pre-stock tube. Each pre-stock tube should correspond to one section of the DNA Origami (i.e., pool together staple strand solutions of “Anchor”, “Left Body”, “Right Body”, “Left Edge” and “Right Edge” sections into respective pre-stock tubes).
  2. Mix the contents of each tube by pipetting up and down.

c. Preparation of Working Stock

The working stock is a combination of the staple strand solutions from all pre-stocks. It contains a certain ratio of all the staple strands needed to form the whole DNA-origami structure.

  1. Take around 10% of each pre-stock and combine them in a working stock tube. The concentration of each pre-stock in the working-stock tube is around 102 µM. This concentration depends on how many staples are need to form the structure.
  2. Mix working stock by pipetting up and down or vortex for 5 seconds.
  3. Centrifuge at 1000 RPM for a couple of seconds to bring down working stock to the bottom of the tube.

d. Preparation of Folding Reactions

The folding reaction consists of a 10:1 ratio of staple strands to scaffold strand (M13mp18). The table below shows the specific ratios, volumes of solutions and buffers used. These reactions were prepared in 0.2 µL PCR tubes.

Components Volumes
M13mp18 Scaffold @ 52.5 nM 19.1
Custom Oligonucleotide Staple Strands @ 1 µM 10
1x TE buffer 50
MgCl2 solution @ 0.25 M 5
Nuclease-free ddH20 15.9
Total 100

e. Annealing Reaction

To form the DNA Origami structure, the PCR tube containing the folding reaction was placed in a thermocycler for approximately 2 hours. The thermocycler was programmed to hold for 4 minutes at 90°C and then to ramp down at -0.5°C/min to 25°C.

f. PEG Precipitation of DNA Origami

PEG (Polyethylene Glycol) precipitation of DNA origami was performed to purify the DNA and get rid of excess staple strands. 6% PEG solution with 250 mM NaCl and 10 mM Mg was used as a precipitation buffer.

  1. Mix 50 µL of DNA origami sample with 100 µL of TE/Mg buffer in a 500 µL DNA low-bind tube. The DNA low-bind tube is used to minimize any possible losses from sample absorption into the tube walls.
  2. Resuspend the solution with 150uL of 15% PEG solution (1x TE; 20mM MgCl2; 500mM NaCl).
  3. Incubate the samples for 10 minutes at room temperature.
  4. Centrifuge the samples for 16 minutes at 10,000 G.
  5. Carefully remove the supernatant using a pipette, and transfer this to another 500 µL DNA low-bind tube.
  6. Resuspend the tube containing the “DNA-origami pellet” with 50 µL TE/Mg buffer and let the solution equilibrate for 10 minutes.
  7. Repeat PEG precipitation for 2-3 cycles, or as needed. Incubating at either room temperature overnight or at 37 C for around 4 hours after each cycle will yield more ideal results.

To determine if purification was successful, the NanoDrop Spectrophotometer at lambda=260 was used. Pipette around 10 µL of purified DNA origami sample onto the calibrated NanoDrop and measure absorbance readings. Successful purification shows an absorbance ratio of greater than or equal to 1.5.

Gel Electrophoresis

Agarose gel electrophoresis is performed to determine whether or not the DNA Origami structure has formed. This is determined by comparison to a 1 kb DNA ladder showing the theoretical positions of the different molecular weights. Ideally, the molecular weight of the DNA origami (~5500 bp) should be greater than both the scaffold (3500 bp) and the staple strands (~2000 bp for the 60x60); therefore, its band on the gel will be positioned higher.

(+) Full step-by-step protocol (click to expand)

a. Gel Preparation

  1. Weigh 0.8 g agarose ultra-pure (Invitrogen) using Analytical Balance and add to an Erlenmeyer flask
  2. Add 100 mL of Tris-EDTA (TE) Buffer with 12.5 mM MgCl2 to the flask.
  3. Microwave Erlenmeyer Flask for 1 minute, with additional 20 second increments if necessary, until agarose is dissolved.
  4. Allow mixture to cool until warm to the touch.
  5. Pour mixture onto the opposite side of the com on the gel mold. Make sure to take away any bubbles between comb teeth with a pipette tip.
  6. Allow the gel to solidify for approximately 20 minutes at room temperature, then remove comb.

b. Sample Preparation & Gel Loading

  1. Mix 10 µL of sample with 2 µL of glycerol loading dye. This helps the sample fall to bottom of well when loaded.
  2. Place gel mold inside gel box and carefully add loading buffer.

c. Running the Gel

  1. Connect electrodes from gel box to power supply and set to run for 3 hours at 70 V.

d. Observing Results

  1. Mix 20 µL of SYBRsafe with 20 mL TE/Mg buffer in a container.
  2. Transfer the gel into the container and let sit for around 30 minutes.
  3. View gel under ultraviolet light and observe results.

Atomic Force Microscopy (AFM)

We image DNA origami samples with tapping mode in air on a Nanosurf Easyscan 2 with a cantilever of Spring Constant 50N/m at a resonant frequency of 200kHz, and later an Asylum MFP-3D with force constant 0.47N/m at a resonant frequency of 73kHz. After washing a freshly cleaved mica disc with imaging buffer (TE-NiCl2, for binding smaller structures) and drying with compressed air, DNA is deposited and incubated in room temperature. The sample is washed again with a less positively ionic imaging buffer (TE-MgCl2, for washing away staples), such that staples do not agglomerate on the mica surface, and the sample is then dried with compressed air.

(+) Full step-by-step protocol (click to expand)

  1. Use double sided tape or masking tape to cleave a mica disc. Place the mica on the tape and then gently peel it off.
  2. Clean the mica surface with 200uL MilliQ water.
  3. Wash the mica disc with 100uL TE-NiCl2 imaging buffer to promote adhesion of smaller DNA origami structures.
  4. Dry with nitrogen gas or compressed air.
  5. Incubate 4uL of DNA origami after purification or thermocycling reaction.
  6. Wash with 100uL TE-MgCl2 to wash away any excess staples.
  7. Dry with nitrogen gas or compressed air.
  8. Image samples on Nanosurf Easyscan 2 in a 5um window size at a resolution of 512 pixels per line.

Forming the DNA Origami-Liposome Complex

It is important to verify the binding of DNA origami to the liposomes via cholesterol-TEG-modified anchors. We have two DNA origami designs which involve DNA folded with cholesterol-TEG-modified anchors and anchoring into the liposome as ssDNA; and also DNA origami folded with clean anchors, then incorporated into the cholesterol-TEG-modified oligonucleotides anchored onto the liposome for a dsDNA bond. For this experiment, only the second design was tested due to time constraints. An anion exchange column is used to verify the formation of this complex. ​

(+) Full step-by-step protocol (click to expand)

a. Prepare Liposome Solution

  1. Dilute the liposomes in 10% sucrose. Sucrose maintains osmotic pressure of the liposomes and is a cryo-protectant.
  2. Store at 4 C. Ensure that the liposomes do not freeze.

b. Form the DNA origami-liposome complex

  1. Combine the cholesterol-TEG-modified anchors in 1.6 times excess with the DNA origami structures in a 500 µL low-bind tube.
  2. Incubate at 37 C and mix the solution with a pipette every 30 minutes, for a total of 2 hours.
  3. Add liposomes in a 1:1 ratio with the DNA origami and cholesterol-TEG-modified anchors. Liposomes are not added in excess because this might cause aggregation.
  4. Incubate at 37 C and mix the solution with a pipette every 30 minutes, for a total of 4 hours. At 37 C, there is a higher probability of interaction due to increased brownian motion. Ideally, the solution can be left overnight at 37 C in a rotating incubator.

c. Select the DNA origami-liposome complex via Anion Exchange Column​

​For this step, ensure that there is always at least 100 µL of washing buffer in the tube containing the resin. This prevents drying of the resin.

  1. Add 10x volume of the DEAE Sepharose CL-6B Resin with 250 µL of washing buffer (20 mM Tris-HCl (pH 7.5); 100 mM of NaCl) in a 0.5 mL Lo-Bind tube.
  2. Centrifuge the tube at 400G for 4 minutes.
  3. Discard supernatant and add 250 µL of the washing buffer to the tube again.
  4. Repeat steps 2-3 four times. This ensures that the resin is washed with a total volume of 1 mL of the washing buffer.
  5. Load 50 µL of the DNA origami-liposome solution into the tube and add 250 µL of washing buffer.
  6. Centrifuge the tube at 400G for 4 minutes.
  7. Measure absorbance readings at lambda=260 nm using the NanoDrop and record results.
    • If anchoring is successful, DNA-liposome complexes will bind to the anion exchange resin due to interactions between the negatively charged DNA and the positively charged resin ligand. When the complex is eluted with a NaCl gradient, the liposomes bound to the DNA origami will come out.
    • If the DNA origami-liposome anchoring is not successful, it will not bind to the anion exchange resin, and when eluted, all of the liposomes will come out before the DNA.

Gold Nanoparticle (AuNP)-DNA Origami Complex

The gold nanoparticles (AuNP) bind to the DNA origami-liposomes complex via electrostatic interactions with the negative phosphate groups in the DNA. Absorption spectrophotometry is carried out to confirm if the gold nanoparticles with a permanent positive charge will form a strong electrostatic interaction with the DNA origami frame.

(+) Full step-by-step protocol (click to expand)

a. Measure Absorbance Spectrum of uncomplexed DNA origami and uncomplexed AuNP

For this step, it is important to ensure that the sample matrices of both the DNA and AuNP solutions are replicable and identical to one another to account for any signal interference from the matrix. The absorbance spectrum for each sample should be measured at least twice.

  1. Calibrate the spectrophotometer with a blank solution of 200 µL PBS. Ensure that the microcuvette is clean and rinsed with DI water before proceeding.
  2. Discard the blank, rinse the cuvette and pipette 200µL of uncomplexed DNA origami sample into the cleaned microcuvette.
  3. Ensure there are no bubbles in the cuvette and that sides of the cuvette are smudge free. Use a Kimwipe to clean as necessary.
  4. Place the cuvette in the spectrophotometer and measure the absorbance spectrum of the solution.
  5. Discard the sample and rinse the micro cuvette with DI water.
  6. Repeat steps 1-5 for uncomplexed AuNP samples.

b. Sponsor Complexation of DNA Origami and Gold Nanoparticle

  1. Pipette 2 mL of AuNP solution into 5 labeled microcentrifuge tubes.
  2. Pipette 5 µL, 8µL, 9µL, 10 µL and 15 µL DNA origami solution into each respective tube. Each tube ensures that the entire DNA origami surface is covered by having a 200-300 times excess of AuNP to DNA origami.

c. Preparation of Working Stock

Repeat the procedure for part C-a) using the 5 AuNP-Origami containing microcentrifuge tubes. Compare the absorbance spectra with the uncomplexed DNA origami and uncomplexed AuNP. Note that it is best to measure the absorbance spectra of the 5 different AuNP-Origami solutions beginning with the least concentrated tube (i.e., the tube with 5 µL of DNA origami), and then going up in concentration.

Final DNA origami-Liposome-Gold Nanoparticle Composite Structure

This step confirms if the overall structure with gold nanoparticles, DNA origami, and lipid nanoparticles have formed a structure as predicted. Repeat step a) using the DNA origami-liposome-AuNP complex and observe the differences in absorbance spectrum. If complexation between the DNA origami-lipid nanoparticle structures and the AuNP’s has occurred, there should be a change in the absorbance spectrum.

Triggered Release via Near Infrared Light

Liposomes supplied by Avanti Polar Lipids are loaded with calcein to verify triggered release via near infrared light. The AuNPs are heated by photo excitation and disperse the heat to its surroundings. In response, the heat increases the permeability of the liposome membrane, disrupting it and thus allowing the release of calein 1.

Calcein is self-quenching and only gives off a visible fluorescent signature when its concentration in solution is rapidly reduced (i.e., when the liposome carrier breaks and releases dye into the general solution).The amount of calcein released can be quantitatively measured using a fluorometer with an excitation wavelength in the near infrared region (NIR), and qualitatively observed using a fluorescent microscope. This can give a good indication of how well the triggered mechanism works.

(+) Full step-by-step protocol (click to expand)

  1. Mix 30, 40, 50, 60, 120 uL of AuNP with 10 uL of calcein-loaded liposomes in 0.2 mL tubes.
  2. Excite the solution using infrared light at 800nm wavelength.
  3. Dilute the sample to approximately 2mL and place in the Cary Eclipse Fluorescence Spectrophotometer.
  4. Excite the AuNP and then excite the released calcein at 470nm and measure for emissions between 500nm and 550nm.

  1. Wu, G., Milkhailovsky, A., Khant, H., Fu, C., Chiu, W., & Zasadzinski, J. (2008). Remotely triggered liposome release by near-infrared light absorption via hollow gold nanoshells. Journal of the American Chemical Society,130(26), 8175-8175. doi:10.1021/ja802656d 

  2. Gopinath, A., & Rothemund, P. (2014). Optimized Assembly and Covalent Coupling of Single-Molecule DNA Origami Nanoarrays. ACS Nano, 12030-12040. 

  3. Rothemund, P. W. K. (2005). Design of DNA origami. Presented at Proceedings of the 2005 IEEE/ACM International conference on computer-aided design, 05/2005. 471-478. doi:10.1109/ICCAD.2005.1560114 

  4. Rothemund, P. W. K. (2006). Folding DNA to create nanoscale shapes and patterns. Nature, 440 (16): 297–302. 

  5. Woo, S., & Rothemund, P. W. K. (2011). Programmable molecular recognition based on the geometry of DNA nanostructures. Nature Chemistry, 3 (8): 620-627