Lab Book


The DNA Origami Chronicles

This documents all the experimentation performed to arrive at our final 60x60 NE Design 2 structure without cholesterol anchors. The first few experiments investigate 60x60 and 80x80 Design 1 structures, as well as the 60x60 with Edges (E) and 60x60 with No Edge (NE) structures.

Experiment 1: In the Beginning

60x60 with Edge (E) and 60x60 No Edge (NE)

Purpose: To determine whether the DNA Origami protocol yields the formation of desired structures, via gel electrophoresis and AFM; To determine whether the 60x60 E (edge) will cause any stacking due to sticky end interactions.

(+) Experimental Parameters (click to expand)

Folding Reaction:

  • ​1:5 ratio of scaffold at 42.6 nM to staple strands.
  • Thermally anneal by holding the samples at 90 C for 4 minutes, then ramping down -1 C/min to 25 C using a thermocycler.

Gel Electrophoresis:

  • A 1% agarose gel was run for 1.5 hours at 60V in a cold room and post-stained with SYBRsafe. The gel was left to rest for 30 minutes to allow staining to take place.

Purification: 100 kDa Amicon Centrifuge Filters:

  • 50 µL of origami sample in 450 µL of TE/Mg buffer in an Amicon Centrifuge filter were centrifuged at 14000G for 4 minutes. This was performed three times at room temperature, ensuring every time that there was at least 500 µL in the tube.
  • To elute, the centrifuge filters were turned upside down, placed into a new collection tube and centrifuged at 2000G for 2 minutes.

AFM Imaging:

  • Around 2-5µL of DNA origami samples in TE solution were placed onto freshly cleaved mica​ and incubated at room temperature for around 5 minutes before washing with DI water.
  • Samples were left to dry overnight before imaging with the AFM.

Results

Figure 1: Gel image of 60x60 E and 60x60 NE, filtered and unfiltered.


  • AFM image is inconclusive since no clear structures are imaged.
  • Not all samples were imaged due to AFM malfunction.
  • Samples may have been washed out during the sample preparation.
Figure 2: NanoSurf EasyScan 2 AFM image of 60x60 NE. filtered.


  • The faint bands show that the gel staining was not very effective, and it may be better to either stain for longer, change the stain used or run the gel for longer that 1.5 hours.
  • The DNA ladder does not show very clear separation.
  • All the filtered samples seem to have either run off or been filtered out, since they do not show up on the gel. It is possible that the samples were centrifuged for too fast (14000G) and that performing this at room temperature destabilized the DNA structures.
  • No conclusion can be made based on this gel.

Experiment 2: Chol(esterol) Me Maybe

60x60 with Edge (E), 60x60 no edge (NE) and 80x80 all with cholesterol

Purpose: To determine whether the DNA Origami with cholesterol-TEG-modified anchors are forming; To determine if modifying the purification conditions, gel electrophoresis conditions, and AFM sample preparation will improve results.

(+) Experimental Parameters (click to expand)

Folding Reaction:

  • ​1:5 ratio of scaffold at 42.6 nM to staple strands.
  • Thermally anneal by holding the samples at 90 C for 4 minutes, then ramping down -1 C/min to 25 C using a thermocycler.

Gel Electrophoresis:

  • Parameters changed:
    • The gel was run at room temperature.
    • The gel was post-stained with SYBR Gold.
  • The gel was still a 1% agarose gel that ran for 1.5 hours at 60V.
  • Rationale: SYBR Gold is supposed to be a more sensitive dye than SYBR Safe, and according to research running the gel at room temperature is a more standard procedure

Purification: 100 kDa Amicon Centrifuge Filters:

  • Parameters changed:
    • Centrifuged five times at 2000G for 15 minutes at 4 C.
    • To elute, filters were centrifuged at 2000G for 5 minutes.
  • Rationale: Slower centrifugation at a much lower temperature will stabilize the samples more and minimizes losses due to high speed.

AFM Imaging:

  • Parameters changed:
    • TE-NiCl2 solution was added to the freshly cleaved mica to promote DNA origami adhesion.
    • TE-MgCl2 was used to wash away any excess staples once the DNA origami samples were placed on the mica.
  • Rationale: Based on P. Rothemund’s paper, NiCl2 will help the negatively charged DNA adhere stronger to the positively charged mica.
Figure 3: Gel image using SYBR gold post-stain, run for 1.5 hours at 70 V. All DNA origami samples were folded with cholesterol-TEG-modified anchors.


  • The SYBR Gold post-stain seems to yield clearer and brighter bands.
  • The gel image over-all shows better DNA ladder separation, although there is still some streaking of samples.
  • The bands at 100 bp are most likely excess staple strands, since each structure has around 100-130 bp.
  • The 100 kDa and 30 kDa centrifuge filter probably filtered out all the samples, as no bands are showing up in the lanes while unpurified samples display clear bands. The structures may have been destroyed by centrifugation against the Amicon filter membrane, which allows DNA to be filtered out of the sample. This implies our structure may be less stable than other square-shaped DNA origami designs in the literature.
  • M13mp18 scaffold is supposed to have around 3500 bp, but the Figure 3 shows a band at around 1650 bp. The gel can be run longer next time to allow the samples to fully move along the gel.

There are no AFM images for these samples.

Experiment 3: PEG Saves the Project!

60x60 no edge (NE) with cholesterol

Purpose: To determine if PEG purification is more ideal than centrifuge filtration.

The only changes were the purification method and the concentration of the gel. A 7.5% and 10% PEG purification was performed, and a 0.8% Agarose Gel Electrophoresis. The rationale for this is that lower Agarose concentrations help structures with greater molecular weight move along the gel.

Figure 4: Gel image comparing the two different conditions: a) Hold at 80 C and ramp down to 65 C holding for 30 minutes before ramping down to 25 C and b) Hold at 90 C and ramp down -1 C/min to 25 C.


Results:

  • 7.5% PEG purification seemed to show clearer bands compared to the 10% PEG purification.
  • Faint or no bands are seen for the filtered samples giving further evidence that the centrifuge filters did not work as effectively as the PEG purification.
  • The DNA ladder bands show more distinct separation compared to previous gels run. It indicates that staple strands are at around 250 bp, while the DNA origami is at around 3000 bp or higher.
  • The bands at around 2000 bp are most likely scaffold strands, however no pure scaffold sample was run to verify this.
  • The scaffold strand should be at around 3500 bp, the DNA origami at around 5500 bp, and excess staple strands at around 2000 bp. Disparities in expected results are mostly likely due to some DNA degradation.
  • Smeared bands are generally observed. This is probably due to salt contamination from the PEG purification. Unfortunately, due to lack of time, ethanol precipitation to remove excess NaCl after purification was not performed.
Figure 5: AFM image of 60x60 NE samples


  • The AFM image is quite blurry. This is most likely due to contamination and improper sample preparation, which caused the DNA samples to disperse.

Experiment 4: Annealing Conditions

60x60 no edge (NE) and 60x60 Edge (E) - New DNA Origami Design

Purpose: To investigate the effect of two different annealing conditions on the formation of the DNA origami; To test out the new DNA origami designs, which do not include the cholesterol anchors; To verify the PEG purification results.

The new 60x60 DNA designs with no cholesterol anchors were annealed using two different conditions:

  1. Hold at 80 C for 5 minutes, ramp down -1 C/min to 65 C and hold for 30 minutes; Ramp down to 25 C for -1 C/min.
  2. Hold at 90 C for 5 minutes, ramp down -1 C/min to 25 C in 0.1 C increments.

7.5% PEG purification was still performed based on the success of the previous experiments, and the gel was run for 3 hours instead of 1.5 hours. The longer gel running time was expected to yield clearer separation and allow the samples to move further along the gel.

Figure 6: Gel image of 7.5% PEG purified 60x60 NE and 60x60 E using two different annealing conditions.


  • There is no significant difference between the two different annealing conditions, based on the gel image. Therefore, we decided to use the 90 C condition for future experiments to be consistent.
  • The 60x60 E samples do not seem to show up as clearly as the 60x60 NE samples. This may be due to some errors in preparing the samples or possible degradation of DNA.
  • Once more smudging is evident, and the bands are not positioned where they are expected to be.
  • Consistent with other gel results, there are distinct bands positioned higher than the scaffold strand. This indicates that there are some structures being formed. ​
Figure 7: The AFM image of the 60x60 E DNA Origami shows stacking of formed structures.


  • The AFM image shows stacking of formed structures. This confirms the initial suspicion of sticky end interactions of origami structures with edges.

Experiment 5: Annealing Conditions

60x60 no edge (NE) with cholesterol and 80x80 with cholesterol

Purpose: To determine if the DNA origami successfully anchored onto the liposome by running the eluted sample through the gel.

In addition to running DNA origami samples, origami-liposome samples are eluted through an anionic exchange column with aSepharose CL-6B resin. The origami-liposomes were eluted using varying NaCl concentrations are observed through gel electrophoresis. All other experimental parameters remained the same. See the Experimental Protocol for details on the Anion Exchange Column.

Results:

Figure 8: Gel image including origami-liposome samples eluted using an anion exchange column, and 7.5% PEG Purified 60x60 NE and E samples


  • The DNA origami-liposome complex most likely aggregated and did not stick to the resin. This explains why the eluent lane on the gel (named as “Ion Exchange”) is empty.
  • There are bright bands at the very top of the lane which likely indicate that there was aggregation of structures; these were not able to move along the gel.
  • Lane 10, liposome-DNA origami structure, is shown to have a smear of DNA that appears to be the DNA staples. They are most likely the ssDNA cholesterol anchors strands added in 1.6x excess during the assembly process, after PEG purification of the DNA origami structures.
Figure 9: Image using Asylum Research AFM showing possible structures forming at 50-60 nm.


Figure 10: Topographical Map of structure boxed in Figure 9. The big drop in the y-coordinate possibly corresponds to the "hole" in the origami design.


  • Images using the Asylum Research AFM show possible structures corresponding to the rectangular origami design with a hole in the middle.
  • The topographical map in Figure 10 shows that there is a drop in the y-coordinate from around 6 nm to -6 nm which can indicate the presence of the “hole”. The reference point for this image was not properly set to the AFM platform, but still clearly shows a dip in the height of the structure.
  • Higher-resolution imaging should be performed to achieve more conclusive results.
Figure 11: NanoDrop absorbance readings of 60x60 E origami-liposome samples eluted with varying NaCl concentrations.


Figure 12: NanoDrop absorbance readings of 60x60 NE origami-liposome samples eluted with varying NaCl concentrations.


  • The expected absorbance ratio of wavelength 260 nm/280 nm should be greater than or equal to 1.5 to determine purity of the DNA. However, the absorbance ratio for both the 60x60 NE and E origami attached to liposome is at around 0.4-1.4. Currently, there we do not have empirical data available to show what a liposome to origami absorbance ratio is, but we expect it to be higher than 1.5.
  • The very low absorbance readings show that the origami-liposome did not bind to the resin. Instead, they were washed out sometime within the first few washes because the concentration of DNA was lower than expected. In the future, we could perform the washing gradient from approximately 0 to 100 mM NaCl to ensure that the samples are not washed out.
  • The liposomes could have also aggregated causing very anomalous absorbance readings.

Peter’s Perfect Purification!!!

After several failed trials, we finally purified DNA! Absorbance readings for previous trials were not the most ideal, so only the best 6% PEG purification performed is shown. These DNA samples were used for the Cryo-EM imaging of the whole origami-gold nanoparticle-liposome complex. The first purification cycle was incubate overnight at room temperature. The second cycle was incubated at 37 C for around 4 hours. This incubation is important to facilitate proper mixing of samples before measuring absorbance.

Figure 13: NanoDrop absorbance readings of 6% PEG purified 60x60 NE DNA origami (3 trials).


  • The average absorbance ratio of the samples at wavelength 260nm/280 nm is at around 1.5, which shows that the 6% PEG was successful in purifying the DNA structures.

Let’s Get Excited In Here!

Experiment 1: Annealing Conditions: Testing Triggered Release

Purpose: To determine whether or not the presence of the DNA origami structure was in fact necessary in order for triggered release of calcein from the liposomes.

Control: Calcein loaded liposomes combined with gold nanoparticles

Results:

Figure 14: Emission spectrum of calcein loaded liposomes in the absence of gold nanoparticles after exposure to 800 nm subsequent excitation of calcein at 470 nm.


Figure 15: Emission spectrum of calcein loaded liposomes in the presence of gold nanoparticles after exposure to 800 nm subsequent excitation of calcein at 470 nm.


  • Excitation of gold nanoparticles at 800 nm and then excitation of the released calcein at 470nm showed a minimal release of calcein into the surrounding solution.
  • Unfortunately the calcein loaded liposomes provided by Avanti were still suspended in a solution containing calcein. While gold nanoparticles absorbed some of the emitted radiation from the calcein, other noise could be removed from our measurements by measuring and subtracting a baseline

Experiment 2: Testing Triggered Release from Final Composite Structure

Purpose: To test the efficacy of our final composite structure while determining whether triggered release improves with the presence of the DNA origami structure to act as a tether to the liposome.

Results:

Figure 16: Emission spectrum of completed composite structure after exposure to 800 nm and subsequent excitation of calcein at 470 nm.


  • Unfortunately, the concentration of our final composite structure was to low to draw conclusive evidence from our results. Considering that our yields of DNA origami were quite low to begin with, it would appear that during the anion exchange portion of the experiment, our complex was eluted prematurely and the majority of it was lost.
  • As seen in the emission spectrum of the completed structure, it woud appear that calcein was in fact released from the structures, although due to the very low concentration that the structure was it, the intensity of emission was very low