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 makes DNA a useful construction material, through the 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 method of DNA origami was developed by Paul Rothemund at the California Institute of Technology. In contrast to common top-down fabrication methods such as 3D printing or lithography which involve depositing or removing material through a tool, DNA Nanotechnology, as well as DNA origami as a subset, is a bottom-up fabrication method. By rationally designing the constituent subunits of the DNA polymer, DNA can self-assemble into a variety of shapes. The process of constructing DNA origami 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 an 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 on sensing and drug delivery, but many additional applications have been investigated.
Fabrication Fabrication of DNA origami objects requires a preliminary 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.
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. 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.
Bottom-up self-assembly methods are considered promising alternatives that offer cheap, parallel synthesis of nanostructures under relatively mild conditions.
Since the creation of this method, software has been developed to assist the process using computer-aided design 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 intended 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 stimulus 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.
DNA Legos
Introduction and History of DNA Legos DNA origami has been used to assemble a range of two-dimensional and three-dimensional nanostructures; however, each different structure requires a new design and a new set of scaffold strand and staple strands. This limits scalability and structure yield efficiency. Researchers have developed “DNA brick” or “DNA Lego” technologies by making single stranded tiles (SSTs). SSTs allow for the production of larger structures without a scaffold strand and with the ease of arranging the predesigned blocks into bigger structures. Much like how actual Lego bricks allow specific assembly configurations, DNA Lego bricks bind at specific sites to form a range of shapes. Appropriate selection of blocks to include in an assembly mixture allows production of a variety of complex 3D shapes without a scaffold strand and without having to design an entirely different structure.
With the DNA Lego brick approach, the DNA strand binds to neighboring strands to form a “brick”. By selecting subsets of strands to form a larger shape, researchers demonstrated that many 3D structures could be assembled from the same general set of components or bricks. The authors called this a “voxel-based” design strategy meaning that each 8-base pair interaction between bricks defines a voxel with dimensions 2.5 by 2.5 nm. Since each DNA interaction can be analogized to Lego construction, we can also claim one voxel corresponds to one stud Lego brick .
This scaffold-free strategy has also been extended to larger multicomponent structures. For example, the construction of multi-micron structures into larger architectures was accomplished using “criss-cross” polymerization. In this case, the additive components are “slats” instead of bricks. These slats have binding domains that allow them to attach perpendicularly to form a cross-linked structure. This approach stabilizes the structure, allowing for larger, highly ordered structures to form. This concept further reinforces the concept of smaller DNA components used as Legos to create larger structures without a bottom-up design. This approach also contrasts with earlier DNA origami techniques which relied on intricate computer software to design the scaffold and staple strands to enable creation of larger DNA nanostructures.
Brick Structure Synthesis A canonical DNA brick is a “32-nucleotide single strand with four 8-base binding domains (sticky ends)”. While each DNA brick has a distinct nucleotide sequence, they are all able to adopt the brick structure consisting of two 16-nucleotide antiparallel helixes joined by a single phosphate linkage. The two domains attached to the phosphate linkage are designated as “head” and “tail” domains. To bind two DNA bricks, a “head” domain will bind to a neighboring brick if their “tail” domain is the complementary strand of the earlier, and vice versa, producing a 90° dihedral angle. The concept of a Lego-like model is meant to depict the design in a simpler manner. Looking at a singular model in this manner, the two protruding round holes represent the two “tail” domains while recessed round holes represent the two “head” domains. Also, the brick can adopt a horizontal or vertical orientation. Using 3D modeling software such as LegoGen and Cadnano, it is possible to design DNA bricks by simply defining the target shape and subsequently the computer program can analyze it and automatically select the correct subset of bricks for self-assembly of the shape without having to manually design every DNA interaction.
Structure Stabilization Improvements