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A Brief Overview of DNA Origami and Its Applications

Tatiana H. RiordanMath 89S Duke University

September 27th, 2016

A BRIEF OVERVIEW OF DNA ORIGAMI AND ITS APPLICATION

Introduction/Foundation of DNA Origami

Imagine injecting a minute robot into your body, it travelling harmlessly through your

bloodstream, until it comes into direct contact with a tumor, at which point it would open up and

attack the cancerous cells. DNA “origami” technology could make this magical robot a reality in

the foreseeable future. The idea of manipulating DNA to form new structures originated from

Ned Seeman, a nanotechnologist and crystallographer. Seeman’s inspiration came from his

desire to create DNA “prisons” that would be rigid and stable enough to hold proteins still so that

he could take X-Ray “mugshots” of them, a process that required the use of nanotechnology.

Seeman’s creation of various geometric forms of DNA showed that DNA doesn’t just have to be

in the shape of a double helix. More specifically, it showed the DNA could have joints, where

three strands of DNA double helices could come together and wind along one helix and jump to

another helix, as seen in Figure 1 (Rothemund, P. (2016)).

Figure 1. Ned Seeman’s Geometric Forms

This process of “joining, coupling or weaving two molecules together to produce a

structure of the same certainty enjoyed by a carpenter”, came to be known as DNA carpentry

(Seeman, N. Zhang, Y., Du, S. & Chen, J. (1995)). However, the process of DNA carpentry is

complex and very time consuming. In 2006, Paul W.K. Rothemund, a senior research fellow at

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the Computation and Neural System Department at Caltech came up with a faster and simpler

process of taking a long string of DNA and folding it into any shape or pattern, now known as

DNA origami.

What is DNA Origami?

DNA origami is the process of folding DNA to make systematic, two and three

dimensional nanoscale shapes. DNA is the ideal construction material due to its predictable and

programmable nature and the specificity of the connections between its base pairs. (Zadegan, R.

M., & Norton, M. L. (2012). DNA also has “well-known nanometer structural geometry”, has

combined “stiffness and flexibility”, and can easily be manipulated by commercially available

enzymes (Lin, C., Liu, Y., Rinker, S., & Yan, H. (2006).

Process of Folding DNA

In order to create DNA origami, one would need a natural source of a single long strand

of DNA. The current dominating scaffold strand in the field is M13mp18, a mass bred, relatively

affordable bacteriophage (Said, H., Schüller, V. J., Eber, F. J., Wege, C., Liedl, T., & Richert, C.

(2013)). Around this long strand, about 200 programmable short strands of DNA are placed, and

are there to act as “staples” (Rothemund, P. (2016)). Each of the staple strands have a left half

and a right half; because the double helix habitually forms in agreement with the complimentary

rule of the Watson-Crick base pairing – adenine (A) pairs with thymine (T) and that guanine (G)

pairs with cytosine (C) – the left half of the staple strand binds to one position on the scaffold

strand, while the right half of the staple strand binds to a distant position on the scaffold,

bringing the two distant points of the strand together. This creates a constraint or “crease” in the

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DNA origami, as seen in figure 2. The action of the staples pulling the long strands together

occurs after the DNA is mixed and heated, while it is cooling. The net action of the 200 staple

strands creating creases, folds the long strand into various shapes (Seeman, N. C., Zhang, Y., Du,

S. M., & Chen, J. (1995)).

Figure 2. Staple Strands Folding Long Scaffold Strand

In order to produce a desired shape or pattern, an image is drawn with a raster fill,

folding a long scaffold strand. Then a computer program calculates the placement of individual

staple strands. This is possible due to the well understood intermolecular interactions of DNA.

The first design Rothemund created was the smiley face nanostructures, as seen in Figure 3. This

process if often referred to as ‘bottom-up fabrication’, as opposed to Seeman’s top-down

method. This method of self-assembly offers an inexpensive, equivalent synthesis of

nanostructures under comparatively mild conditions (Rothemund, P. W. (2006)).

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Figure 3. Nanoscale Smiley Face DNA origami

Observing DNA Origami Structure and its Intracellular Interactions

The nanostructures created by DNA origami are so miniscule that 70 of these structures

would fit across the width of just one blood cell. Because of their minuteness, the nanostructures

cannot be observed through normal light microscopy. The main method used to measure these

molecules is through atomic force microscopy, a method of measuring molecules invented in the

eighties. This process begins by immobilizing the molecules on a hard substrate. As seen in

Figure 4, they are stuck down firmly on a rectangular platform. Then a very fine needle is

dragged back and forth over the surface with a laser beam shining at the tip of the needle. As the

needle bumps up and down the platform, a computer measures the deflection of the needle,

recording a white pixel when the needle goes up and a black pixel when the needle goes own.

The image formed on the computer shows the structure of the folded DNA.

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Figure 4. Atomic Force Microscopy

Applications

As of now, DNA origami is still in the experimentation process, with researchers creating

microscopic smiley faces and three dimensional cubes. However, as the technology progresses

DNA origami has many potential applications, particularly with specifically constructed DNA

lockboxes – a cage or basket that protects a fragile, toxic, or precious payload – that could be

used to deliver drugs directly to tumors.

DNA lockboxes are being heavily researched due to their ability to attack target cells and

leave other cells alone. This technology is possible due to the “locks” on the DNA origami

boxes. The locks are made of double helix DNA strands that are designed to be complementary

to tumorous cells. Because of this, the DNA strands remain attached, closing the lid of the box,

until it’s in contact with a tumorous cell, where it will unwind and open up the box. This same

technology can also be used to create jails for viruses or be used as a way to immobilize enzymes

(Kean, S. (2016, September)). Another major application for DNA origami is attaching

conductors to the DNA and creating tiny computer chips. Below is an overview of the current

research done on these possible applications (“DNA 'origami' could help build faster, cheaper

computer chips” - American Chemical Society. (2016, March 13)).

Researchers at the Hansjörg Wyss Institute for Biologically Inspired Engineering at

Harvard University created cell-targeted, payload-delivering DNA nanorobots. Ido Bachelet, one

of the researchers in the study stated that "the nanorobot we designed and fabricated is a machine

that can be programmed to autonomously recognize target cells and deliver payloads to those

cells". The nanorobot they created looks like an open ended barrel, as seen in figure 5, that has

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anchor strands and linker strands attached to the ends, which are computed to be complementary

to the payload and connects the barrel to the payload.

Figure 5. Visual of Nanorobot

The locks on the lockboxes are DNA double helices. In the absence of the key, the

helixes are held strong enough to maintain the structure in a closed position. However, when the

key is present, the DNA lock is designed to recognize that key and switch to bind to the key,

unzipping the double helices and opening the box. Their findings showed that a "nanorobot can

recognize a small population of target cells within a larger population of bystander cells, which

should be left alone". This is because while all cells share the same drug target that they wanted

to attack, only the target cells possessed the proper set of keys to open the lock and therefore

only they will be attacked by the nanorobot and the drug. The nanorobot they designed was

bearing a functional payload, so it had antibodies fragments that were able to communicate with

a cell and induce it to apoptosis (DNA Nanorobot [Video file]. (2012)). The first models the

researchers used were leukemia and lymphoma (Garde, D. (2012, May 15)).

In another study conducted by bioengineers at the Hansjörg Wyss Institute for

Biologically Inspired Engineering at Harvard University, collaborated with Bar-Ilan University

to fabricate “nanoscale robots that are capable of dynamically interacting with each other in a

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living animal” (Amir, Ben-Ishay). The nanorobots are able to perform similar operations to a

silicon-based computer inserted into a living animal. To prove their concept, the researchers

injected cockroaches with various nanorobots and measured the nanorobots’ diffusion. The DNA

“computers” traveled around the cockroaches’ bodies and interacted with each other and with the

insects’ cells. The team of researchers then injected a variety of nanorobots into the cockroaches

to analyze how “different robot combinations affect where substances are delivered”

(Spickernell, S. (2014, April 8)). They found that the more nanorobots they injected, the more

complex the logic operations became, and that the accuracy of the delivery and control of the

nanorobot is equivalent to that of a computer system. They also stated that they could potentially

increase the computing power in the cockroach to match that of an 8-bit computer (Amir, Y.,

Ben-Ishay, E., Levner, D., Ittah, S., Abu-Horowitz, A., & Bachelet, I. (2014).)

Scientists from iNANO center and CDNA Centerin Aarhus University developed an

18x18x24nm3 hollow DNA origami box with a switchable lid. They noticed that there was a

“higher density of the projected stained DNA helices of boxes lying on the side”. This outcome

is probably due to a bigger surface reaction between negatively charged DNA with the positively

charged surface. The origami box had a unique “reclosing mechanism”, which permitted the box

to repeatedly open and close when exposed to DNA and RNA keys, making the box reusable.

The box can potentially be used for “controlling the function of single molecules, controlled drug

delivery, and molecular computing” (Zadegan, R. M., Jepsen, M. D., Thomsen, K. E., Okholm,

A. H., Schaffert, D. H., Andersen, E. S., . . . Kjems, J. (2012)).

Researchers in the National Center for Nanoscience and Technology in Beijing and

Arizona State University non-covalently attached Doxorubicin, a well-recognized anti-cancer

drug, to DNA origami nanostructures. This was achieved through intercalation, and resulted in

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high loading efficiency to typically doxorubicin-resistant cancer cells, “inducing a remarkable

reversal of phenotype resistance”. With this new technology, endocytosis of the cell was

increased, which amplified cell-killing activity to doxorubicin-resistant cells. The results suggest

“DNA origami has immense potential as an efficient biocompatible drug carrier and delivery

vehicle in the treatment of cancer” (Jiang, Q., Song, C., Nangreave, J., Liu, X., Lin, L., Qiu,

D., . . . Ding, B. (2012)).

One issue with the functionality of DNA origami structures, observed by group of

researchers at the Wyss Institute at Harvard University, are their susceptibility to nuclease

degradation, and their capability to trigger an inflammatory immune response. Addressing this

issue, the researchers created a structure that imitated a virus’ phospholipid coating to avoid

detection by the immune system, as seen in figure 6. To do this, they strictly controlled the

density of the attached lipid conjugates. When inserted into mice, the nanoparticles with the

phospholipid coating were able to last in the mice’s bloodstream for hours while the uncoated

nanoparticles were rapidly broken down. This designed strategy provided a “platform for the

engineering of sophisticated, translation-ready DNA nanodevices” (Perrault, S. D., & Shih, W.

M. (2014, April 2)).

Figure 6. DNA Nanostructure Mimicking the Structure of a Virus

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Conclusion

DNA Origami is still a new, progressing concept but has already proven to be a very

promising technology. The combination of chemistry, biology and computer science has led to a

new advancement that can ultimately save millions of lives. Through further research it seems

probable that DNA origami can be used to target specific cells, diagnose particular issues

without affecting unrelated cells, and to act as a miniature computer that is viable in the human

body.

Works Cited

Amir, Y., Ben-Ishay, E., Levner, D., Ittah, S., Abu-Horowitz, A., & Bachelet, I. (2014).

Universal computing by DNA origami robots in a living animal. Nature Nanotech Nature

Nanotechnology, 9(5), 353-357. doi:10.1038/nnano.2014.58

DNA 'origami' could help build faster, cheaper computer chips - American Chemical Society.

(2016, March 13). Retrieved September 26, 2016, from

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https://www.acs.org/content/acs/en/pressroom/newsreleases/2016/march/dna-

origami.html

DNA Nanorobot [Video file]. (2012). In Vimeo.

Garde, D. (2012, May 15). DNA origami could allow for 'autonomous' delivery. Retrieved

September 26, 2016, from http://www.fiercepharma.com/r-d/dna-origami-could-allow-

for-autonomous-delivery

Jiang, Q., Song, C., Nangreave, J., Liu, X., Lin, L., Qiu, D., . . . Ding, B. (2012). DNA Origami

as a Carrier for Circumvention of Drug Resistance. J. Am. Chem. Soc. Journal of the

American Chemical Society, 134(32), 13396-13403. doi:10.1021/ja304263n

Kean, S. (2016, September). Fun With DNA. Retrieved from

http://www.theatlantic.com/magazine/archive/2016/09/fun-with-dna/492743/

Lin, C., Liu, Y., Rinker, S., & Yan, H. (2006). DNA Tile Based Self-Assembly: Building

Complex Nanoarchitectures. ChemPhysChem, 7(8), 1641-1647.

doi:10.1002/cphc.200600260

Perrault, S. D., & Shih, W. M. (2014, April 2). System Maintenance: From Monday, September

26, 7pm to 11pm EDT. Retrieved September 26, 2016, from

http://pubs.acs.org/doi/full/10.1021/nn5011914

Rothemund, P. W. (2006). Folding DNA to create nanoscale shapes and patterns. Nature,

440(7082), 297-302. doi:10.1038/nature04586

Rothemund, P. (2016). DNA Origami: Folded DNA as a Building Material for Molecular

Devices - P. Rothemund - 5/25/16. Retrieved September 26, 2016, from

https://www.youtube.com/watch?v=yPkQsrQwpj8

Said, H., Schüller, V. J., Eber, F. J., Wege, C., Liedl, T., & Richert, C. (2013). M1.3 – a small

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scaffold for DNA origami . Nanoscale, 5(1), 284-290. doi:10.1039/c2nr32393a

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nanobots-deliver-drugs-in-living-cockroaches/

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https://www.newscientist.com/article/mg22229643-100-dna-

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