Purification of DNA Origami Nanoparticles

The main purpose of this undergraduate research project is to find a more efficient and viable method of purifying DNA origami nanoparticles with the intent of increasing its product yield. Based on studies such as “DNA Origami Presenting the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Response” by Veneziano et al., the most optimal method for purification currently is to use filtration-based ultracentrifugation. The filters, AmiconUltra, assist in separating the desired nanoparticle structures from residual particles, oligonucleotides, or byproducts. However, this method of purification often leads to a low yield of DNA nanoparticles, which is not optimal to reproduce within a clinical or industrial environment. Due to this matter, the objective of the project is to test multiple purification methods and identify the best method (including the current method). These methods include the usage of filters with different membrane compositions, column-based bead purification, and others. The expectation set for this project is to observe and analyze the results of purification from each method, and depict the best method based on the yield.

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Mahin Chowdhury: Alright, Hello! My name is Mahin Chowdhury, and this and this is ‘The Purification of DNA Origami Nanoparticles’. Firstly, I would like to thank Dr. Remi Veneziano for helping me with with this project from start to finish, and I would also like to thank the Oscar program for providing me with this wonderful opportunity.

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Mahin Chowdhury: So the overview of the project: DNA origami nanoparticles is a new vaccination method that represents the next generation of vaccine delivery to fight infectious diseases. The current method towards purifying these nanoparticles relies upon ultracentrifugation, using Amicon Ultra filters which typically result in a low yield

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Mahin Chowdhury: In a laboratory environment this is-

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Mahin Chowdhury: -standard, pretty all right.

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Mahin Chowdhury: However, if you were to scale this up to the industrial level, it becomes a lot more complicated, as this will increase costs to manufacture and produce the nanoparticles. So the main objective is to find a way that that that could help reduce the cost or

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Mahin Chowdhury: purify the the structures as much as possible

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Mahin Chowdhury: The methods that we will be using: So we will be using 3 different DNA nanoparticles of

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Mahin Chowdhury: 3 different sizes, basically a 6-Helix Bundle, which is referred to as 6HB. Pentagonal Bipyramidal (PB), and Pentakis Dodecahedron (PD). As I mentioned, before, these particles are different than one another.

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Mahin Chowdhury: They have different properties, shaped differently. There are some similarities between 6HB and PB in terms of size (base pairs).

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Mahin Chowdhury: Pentakis Dodecahedron, on the other hand, is a lot bigger.

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Mahin Chowdhury: So we will be trying to purify these 3 types of nanoparticles, using the methods shown below

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Mahin Chowdhury: The first method we use is the regenerated cellulose membrane filter. As you can see, we have the experimental concentrations retrieved from the trials and the theoretical that was calculated, based on the volume of a retrieved at the end of the trial.

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Mahin Chowdhury: As you can see, there are some differences between the theoretical and the experimental concentrations. I encourage you that you please pause and look through these diagrams.

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Mahin Chowdhury: These diagrams / data would be compared to the agrose gel that we collected via a gel electrophoresis.

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Mahin Chowdhury: These bright bands are the nanoparticles. These bands below are the staple strands, or any byproducts that may come off from purifying the actual nanoparticles.

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Mahin Chowdhury: These are what we’re trying to get rid of essentially, as you can see.

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Mahin Chowdhury: The brightness indicates the concentration of both the nanoparticles and the staple strands.

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Mahin Chowdhury: So the brighter it is, the more concentrated it is. Meaning, there’s more of it; there’s a lot in the actual solution.

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Mahin Chowdhury: As you can see the 30kDa, based on the each structure. You can see that each band becomes fainter and fainter as you go up the scale for the filters, which indicates that the filters

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Mahin Chowdhury: do purify the staple strands or byproducts as much as possible.

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Mahin Chowdhury: Moving onto trial 2.

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Mahin Chowdhury: We can see that it is also a little bit consistent in comparison to trial one, and

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Mahin Chowdhury: if you compare it to the gel from electrophoresis

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Mahin Chowdhury: you can see that these bands become faint or barely visible, which shows that it has become pretty pure, and

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Mahin Chowdhury: could often indicate that some based on the comparison with the theoretical and the experimental, we can determine whether or not we lost the majority of the nanoparticles, or we retain them, in which case some we lose a lot, and some we aren’t able to purify as much

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Mahin Chowdhury: Moving on the Spin X polyethersulfone filters.

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Mahin Chowdhury: These filters are used in the similar method with the Cellulose filters. As you can see-

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Mahin Chowdhury: -you have the 6HB. Which is similar in the theoretical and experimental. However, if you were to compare it to the agarose gel, you have a lot of stable strands, so you can’t always rely upon the numerical data / figure. You have to use both the data and the visual gel.

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Mahin Chowdhury: In which case you can see that many for, trial one at least, many of these are not that pure

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Mahin Chowdhury: Moving onto trial 2, using the same gel, it’s pretty consistent, not as pure as it would seem.

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Mahin Chowdhury: Especially for PD, since the bands are very bright.

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Mahin Chowdhury: The Spin Kleen columns by Bio-Rad. They have a very high theoretical concentration, but a low experimental, which typically indicates that we we lost a lot more of the nanoparticles, and, as you can see here.

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Mahin Chowdhury: we actually have a a lot more nanoparticles

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Mahin Chowdhury: band of nanoparticles, but not a band for the staple strand, which means that we were able to purify it. However, at the cost we lost a lot of product.

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Mahin Chowdhury: The same is consistent with trial, 2, in which case trial 2 also had some consist of the same consistency as trial one. We lost a lot more of the nanoparticles, but we were able to purify as much as we could.

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Mahin Chowdhury: Moving on to this Zbba spin desalting columns, we could see that the experimental concentration is really high compared to the theoretical concentration, and this is also backed up by the bands. The staple strand bands that we see here, especially for PD. Which are pretty bright, indicating that these are not as pure as it should be.

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Mahin Chowdhury: The same thing goes with the trial 2 same consistency.

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Mahin Chowdhury: It just shows that these are these columns are not a viable method towards purifying the nanoparticles.

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Mahin Chowdhury: So the conclusions

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Mahin Chowdhury: Based on the data, many different nanoparticles are best purified by a specific method. PD, for instance, is very well purified, using the 100kDa cellulose membrane filter

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Mahin Chowdhury: There is still some access that you may have, but

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Mahin Chowdhury: you still have a good amount of nanoparticles. The same goes with the other nanoparticles. They all have a different type of purification yield, depending on whether or not the method, the method, whether filter or column, can purify, based on its structure and size.

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Mahin Chowdhury: so as they as dated for some methods would just result in a setting pure structure, but with a high yield. Other structures

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Mahin Chowdhury: have a very pure structure, but a low you.

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Mahin Chowdhury: So is this optimal for industrialization?

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Mahin Chowdhury: Not as much.

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Mahin Chowdhury: It could be better, but it is mainly because we just don’t have a very viable method at this point right now. The current method is still the best method.

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Mahin Chowdhury: On the other hand, we were able to find that many different structures are purified better with different methods.

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Mahin Chowdhury: better way to purify each structure based on a different method that we can use.

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Mahin Chowdhury: So what’s next? The next? The next step is to continue searching for a better better solution. This is not the end. This is merely the beginning of finding a better way to purify these nanoparticles.

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Mahin Chowdhury: depending on the size or shape. we just have to find something that works for that specific nanoparticle.

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Mahin Chowdhury: Thank you so much for listening to my presentation. Please leave any questions if you have any. Once again. Thank you so much to Dr. Veneziano and the Oscar program, and thank you again for listening to my presentation. I hope you have a great one.