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DNA origami is the nanoscale folding of DNA to create arbitrary two- and three-dimensional shapes at the nanoscale. The specificity of the interactions between complementary base pairs make DNA a useful construction material, through design of its base sequences. DNA is a well-understood material that is suitable for creating scaffolds that hold other molecules in place or to create structures all on its own.

DNA origami was the cover story of Nature on March 16, 2006. Since then, DNA origami has progressed past an art form and has found a number of applications from drug delivery systems to uses as circuitry in plasmonic devices; however, most commercial applications remain in a concept or testing phase.

Overview
The idea of using DNA as a construction material was first introduced in the early 1980s by Nadrian Seeman. The current method of DNA origami was developed by Paul Rothemund at the California Institute of Technology. The process involves the folding of a long single strand of viral DNA (typically the 7,249 bp genomic DNA of M13 bacteriophage) aided by multiple smaller "staple" strands. These shorter strands bind the longer in various places, resulting in the formation of a pre-defined two- or three-dimensional shape. Examples include a smiley face and a coarse map of China and the Americas, along with many three-dimensional structures such as cubes.

There are several DNA properties that make the molecule an ideal building material for DNA origami. DNA strands have a natural tendency to bind to their complementary sequences through Watson-Crick base pairing. This allows staple strands to locate the position on the scaffold strand without any external manipulation, leading to self-assembly of the desired structure.

The specific sequence of bases in DNA gives the material an element of programmability by determining its binding behavior. Carefully designing the sequences of the staple strands enables scientists to precisely direct the scaffold strand’s folding into a predetermined shape with high precision.

On a chemical level, the hydrogen bonds that exist between the complementary base pairs provide strength and stability to the folded DNA origami structures. Additionally, DNA is a relatively stable molecule, offering resilience in physiological conditions.

One of the advantages of using a DNA Origami nanostructure over an otherwise classified DNA nanostructure is the ease of defining finite structures. In the design of some other DNA nanostructures, it can be impractical to design the extremely large number of individualized strands if the entire structure is composed of smaller strands. One method of bypassing the need for a huge number of different strands is to use repeating units, which comes with the disadvantage of a distribution of sizes and sometimes shapes. DNA Origami, however, forms discrete structures.

Applications for DNA Origami are primarily focused around the ability to exert fine control on systems, especially by constraining positions of molecules, typically by attachment to the DNA Origami nanostructures. Current applications are primarily focused around sensing and drug delivery, but many additional applications have been investigated.

Fabrication
Fabrication of DNA origami objects requires an initial intuition of 3-dimensional DNA structural design. This can be difficult to grasp due to the complexity of exclusively using adenine-thymine pairings and guanine-cytosine pairings to both fold and unravel double helical DNA molecules such that the output strands produce uniquely desired shapes.

Indeed the design software and the choice of base-pair sequences become crucial for creating intricate 2D or even 3D shapes as the key to DNA origami lies in the precise base-pairing between the technique’s two building blocks: staple strands and the scaffold. This ensures specific binding and accurate folding. A scaffold strand is a long, single-stranded DNA molecule, often sourced from a virus like M13 bacteriophage. Staple strands are shorter DNA strands designed to bind to specific sequences on the scaffold strand, dictating its folding.

To produce a desired shape, images are drawn with a raster fill of a single long DNA molecule. This design is then fed into a computer program that calculates the placement of individual staple strands. Each staple binds to a specific region of the DNA template, and thus due to Watson-Crick base pairing, the necessary sequences of all staple strands are known and displayed. The DNA is mixed, then heated and cooled. As the DNA cools, the various staples pull the long strand into the desired shape. Designs are directly observable via several methods, including electron microscopy, atomic force microscopy, or fluorescence microscopy when DNA is coupled to fluorescent materials. Since the creation of this method, software was developed to assist the process using CAD software. This allows researchers to use a computer to determine the way to create the correct staples needed to form a certain shape. One such software called caDNAno is an open source software for creating such structures from DNA. The use of software has not only increased the ease of the process but has also drastically reduced the errors made by manual calculations.

After meticulously planning the sequence of the staple strands with software to ensure they bind the scaffold strand at the right points, the designed staple strand sequences are synthesized in a lab using techniques like automated DNA synthesis. Finally, the scaffold strand and staple strands are mixed in a buffer solution and subjected to a specific temperature cycle. This cycle allows the staple strands to find their complementary sequences on the scaffold strand and bind through hydrogen bonding, causing the scaffold to fold into the desired shape.

Dynamic Structures and Modifications
As in the broader field of DNA nanotechnology, DNA Origami may be made dynamic in nature through the use of a variety of methods. The three primary methods of creating a dynamic DNA Origami machine are toehold mediated strand displacement, enzymatic reactions, and base stacking. While these methods are most commonly used, additional methods for creating dynamic DNA Origami machines exist, such as designing a directional component and using brownian motion to drive rotational movement of structures or leveraging less commonly used DNA self-assembly phenomena like G-quadruplexes or i-motifs which can be pH sensitive. .



Modifications can be otherwise used to affect structural properties, to impart unique chemistry to the nanostructures, or to add stimuli responses to the nanostructures. Modifications to structures can be made through conjugation of molecules such as proteins, or through chemical modification of the DNA bases themselves. pH dependent responses, light dependent responses, and more have been shown through modified systems.

One example application of creating dynamic structures is the ability to have a stimuli response resulting in drug release, which is presented by several groups. Other, less common applications comes in sensing moving mechanisms in vivo such as the unwinding of helicase.

Biomedical Applications
DNA Origami, being made of a natural biological polymer, is well suited to the biological environment when salt concentrations allow, and offers fine control over the positioning of molecules and structures in the system. This allows DNA Origami to be applicable to a number of scenarios in biomedical engineering. Current biomedical applications include drug release with 0 order mechanisms, vaccines , cell signaling , and sensing applications.

Researchers at the Harvard University Wyss Institute reported the self-assembling and self-destructing drug delivery vessels using the DNA origami in the lab tests. The DNA nanorobot they created is an open DNA tube with a hinge on one side which can be clasped shut. The drug-filled DNA tube is held shut by a DNA aptamer, configured to identify and seek certain diseased related proteins. Once the origami nanobots get to the infected cells, the aptamers break apart and release the drug. The first disease model the researchers used was leukemia and lymphoma.

Researchers in the National Center for Nanoscience and Technology in Beijing and Arizona State University reported a DNA origami delivery vehicle for Doxorubicin, a well-known anti-cancer drug. The drug was non-covalently attached to DNA origami nanostructures through intercalation and a high drug load was achieved. The DNA-Doxorubicin complex was taken up by human breast adenocarcinoma cancer cells (MCF-7) via cellular internalization with much higher efficiency than doxorubicin in free form. The enhancement of cell-killing activity was observed not only in regular MCF-7, more importantly, also in doxorubicin-resistant cells. The scientists theorized that the doxorubicin-loaded DNA origami inhibits lysosomal acidification, resulting in cellular redistribution of the drug to action sites, thus increasing the cytotoxicity against the tumor cells. Further testing on in vivo on mice suggests that over a 12 day period, Doxorubicin was more effective at reducing tumor sizes in mice when it was contained in DNA Origami Nanostructures or DONs.

Researchers from the Massachusetts Institute of Technology are developing a method to attach various viral antigens to Virus-shaped DNA particles to mimic the virus to be used to develop new vaccines. This was started in 2016 when Bathe’s lab created an algorithm known as DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined Structures) to generate precision-controlled three-dimensional shapes of DNA. Using the tool they designed virus-shaped scaffolding that can modularly attach different antigens to the surface of the DNA scaffold. Currently, MIT is working to develop optimal geometries for B cells to recognize HIV antigens. Further research has attempted to replace HIV antigens with SARS-CoV-2 and are testing whether vaccines show proper immune response from isolated B cells and in mice. Similarly, researchers from the Technical University of Munich have developed a method to have T-cells target tumor cells by using antigen coated DNA origami. The researchers developed a method to create chassis known as programable T-cell Engagers or (PTEs) which are DNA Origami structures that can be configured to bind to user-defined target cells and T-cells based on which antigens are coated on the surfaces of the nanostructure. The in vitro results show that after 24 hours of exposure 90% of the tumor cells were destroyed. Meanwhile in vivo testing showed that their PTEs were capable of binding to the target proteins for several hours which validates the mechanism they designed.

DNA is folded into an octahedron and coated with a single bilayer of phospholipid, mimicking the envelope of a virus particle. The DNA nanoparticles, each at about the size of a virion, are able to remain in circulation for hours after being injected into mice. It also elicits a much lower immune response than the uncoated particles. It presents a potential use in drug delivery, reported by researchers at Wyss Institute at Harvard University.

Nanotechnology Applications
The field of nanotechnology has shown great promise in the field of biomedical sciences, with several potential uses such as medication delivery systems, enzyme immobilization, and materials self-assembly using nanotechnology. Researchers have investigated DNA's potential in nanorobotic applications, especially in the areas of drug delivery, enzyme immobilization, and algorithmic computing, despite the material's limits in structural and catalytic adaptability. The following noteworthy biological uses have been documented in published works

Self-Assembling Drug Delivery Vessels
Using DNA origami, researchers at the Harvard University Wyss Institute have developed drug-delivery vessels that can both self-assemble and self-destruct. The DNA nanorobots have an adjustable hinge on an open DNA tube that may be clasped shut. Targeting particular disease-related proteins, a DNA aptamer keeps the drug-filled DNA tube closed. The medication is released when the aptamers break apart when the nanorobots get to the contaminated cells. Models of leukemia and lymphoma have demonstrated potential in preliminary experiments.

In a study conducted by a group of scientists from iNANO center and CDNA Center at Aarhus university, researchers were able to construct a small multi-switchable 3D DNA Box Origami. The proposed nanoparticle was characterized by AFM, TEM and FRET. The constructed box was shown to have a unique reclosing mechanism, which enabled it to repeatedly open and close in response to a unique set of DNA or RNA keys. The authors proposed that this "DNA device can potentially be used for a broad range of applications such as controlling the function of single molecules, controlled drug delivery, and molecular computing.".

DNA is folded into an octahedron and coated with a single bilayer of phospholipid, mimicking the envelope of a virus particle. The DNA nanoparticles, each at about the size of a virion, are able to remain in circulation for hours after injected into mice. It also elicits much lower immune response than the uncoated particles. It presents a potential use in drug delivery, reported by researchers in Wyss Institute at Harvard University.

Drug Delivery Systems

Origami DNA nanostructures can be used as flexible platforms to deliver drugs to specific locations. Through the functionalization of DNA, origami surfaces with particular ligands, or targeting moieties, scientists can create drug carriers that minimize off-target effects while delivering therapeutic molecules to diseased cells with selectivity. Furthermore, because DNA origami is programmable, stimuli-responsive components can be added, allowing for the regulated release of medications in response to environmental cues like pH, temperature, or enzyme activity.

Molecular Imaging

For usage in molecular imaging applications, DNA origami nanostructures can be designed to exhibit exact spatial configurations of molecular probes. To observe biological structures and processes at the nanoscale, researchers can develop highly sensitive and selective imaging probes by adhering fluorescent dyes, nanoparticles, or contrast agents to specific spots on the DNA origami scaffold. Applications including intracellular imaging, tumor targeting, and biomolecule monitoring in living cells and organisms are made possible by this.

Nano Computing

Nanorobots made of DNA origami demonstrated computing capacities and completed pre-programmed task inside the living organism was reported by a team of bioengineers at Wyss Institute at Harvard University and Institute of Nanotechnology and Advanced Materials at Bar-Ilan University. As a proof of concept, the team injected various kinds of nanobots (the curled DNA encasing molecules with fluorescent markers) into live cockroaches. By tracking the markers inside the cockroaches, the team found the accuracy of delivery of the molecules (released by the uncurled DNA) in target cells, the interactions among the nanobots and the control are equivalent to a computer system. The complexity of the logic operations, the decisions and actions, increases with the increased number of nanobots. The team estimated that the computing power in the cockroach can be scaled up to that of an 8-bit computer.

Similar approaches
The idea of using protein design to accomplish the same goals as DNA origami has surfaced as well. Researchers at the National Institute of Chemistry in Slovenia are working on using rational design of protein folding to create structures much like those seen with DNA origami. The main focus of current research in protein folding design is in the drug delivery field, using antibodies attached to proteins as a way to create a targeted vehicle.