DNA Origami for Nanomanufacturing, Biosensing and Drug Delivery

What is DNA Origami?

DNA origami is a molecular self-folding technique in which long single-stranded DNA scaffolds, typically M13 phage genomic DNA, are folded into well-defined objects by hundreds of short-stranded DNA staples in a one-pot method. The intrinsic addressability of DNA origami is achieved by supplementing different parts of the long single-stranded DNA scaffold with a unique set of DNA staples driven by Watson-Crick base-pairing of double-stranded DNA (dsDNA) hybridization without the need for restriction enzymes or DNA ligases.The concept of DNA origami was pioneered in 2006 by Rothemund, who utilized the DNA Using DNA origami, he constructed a series of two-dimensional DNA nanostructures such as triangles, pentagrams and smiley faces. This assembled DNA origami (~100 nm) exhibits a programmable pattern with a spatial resolution of 6 nm, enabling the design of more complex or larger structures. First, with bottom-up DNA origami, DNA structures are no longer limited to some simple, regular geometric patterns. Large-scale, structurally stable and arbitrary DNA shapes can be constructed, which compensates for the poor size control problems faced by DNA tiles. Second, M13 phage genomic DNA is the most commonly used scaffold for DNA origami assembly, and the corresponding peg sequences can be automatically designed based on the routing path. Finally, DNA origami assembly does not have stringent requirements on the stoichiometric ratio and purity of the DNA backbone.

Figure 1. The construction of intricate DNA origamis. (Z, M, He.; et al, 2023)

Assembly of DNA Origami Structures

• Two-dimensional Assembly of DNA Origami

Since individual DNA origami is limited in size, two-dimensional assembly is generally used to scale up. A simple strategy for assembling single-stranded DNA into desired two-dimensional patterns was born. Staple strands in the edge portion are paired by complementary bases to produce an adjustable combination of shapes, avoiding high concentrations of aggregation by introducing a hairpin structure. This idea laid the foundation for subsequent approaches to DNA origami assembly. For example, based on the above background, the researchers proposed a method for designing large-scale 2D DNA origami using rectangular DNA tiles as staple tiles instead of using conventional staple strands. This strategy can be used to fabricate large DNA origami objects with a size range comparable to conventional lithography, thus combining bottom-up assembly with top-down lithography.

• Three-dimensional Assembly of DNA Origami

Expanding the size of DNA origami through 3D assembly can broaden the applications of DNA origami. Using reconfigurable DNA origami nanoarrays, the researchers propose a modular conversion method that enables controlled conversion from 2D structures to 3D architectures. 2D DNA structures can be modularized into multiple connected units for independent conversion, and this modular conversion also enables structural conversion between 2D and 3D nanostructures.

Applications of DNA Origami

DNA origami allows the construction of two- and three-dimensional nanostructures with specific shapes and functions by precisely designing and controlling DNA sequences. This technique not only provides new possibilities for precise assembly at the nanoscale, but also opens up new directions for the development of nanotechnology in different fields.

Figure 2. DNA origami in nanofabrication, biosensing, drug delivery, and computational storage. (Z, M, He.; et al, 2023)

• Nanofabrication: DNA origami structures are used as templates or frameworks for assembling or synthesizing a wide variety of materials with nanoprecision, and thus show great promise for nanofabrication of inorganic (metallic or non-metallic), polymeric, and biomolecular assemblies, etc., with enhanced structural stability and desired physicochemical properties.
• Nanophotonics and Electronics: Many optical and electronic properties arise from structural features with dimensions < 100 nm. However, it is challenging to precisely sculpt materials at this scale. DNA origami template nanostructures have high structural programmability at the nanoscale, enabling customizable optical or electronic properties including tunable conductivity, plasmonic coupling, Fano resonance, plasmonic chirality, and more.
• Catalysis: Biocatalytic transformations are central to the production of metabolites, biomolecules, and energy conversion in biological systems. DNA origami, provides an excellent platform for the spatial organization of enzymes. By encapsulating enzymes in closed nanostructures, the rate of enzyme cascades can be significantly increased.
• Biomedicine: Scientists will use DNA strands to DNA origami technology to form a substance called framework nucleic acids, just as in the real world to build a house when the first construction of the frame, this kind of framework nucleic acids is the nano-world of the house, so that the antibodies, proteins, enzymes and other substances to live in this kind of house, which can be used in cancer detection and treatment. DNA origami carriers can be used to deliver a variety of therapeutic molecules and materials, including doxorubicin, immunostimulatory nucleic acids, and small interfering RNAs, which can be loaded onto the carriers. In addition, DNA origami structures can be used as containers with docking sites inside or in specialized cavities to protect the payload from the environment.
• Computational Biology: DNA origami frameworks spatially organize DNA-based circuits to accelerate reaction rates, modulate reaction structures, limit or promote the output of specific pathways, segregate different functional modules (e.g., sensing, computation, actuation) to minimize computational errors due to crosstalk, and improve the recyclability of nucleic acids.

For the time being, the application of DNA origami is still in its infancy and faces some practical challenges as follows.

• In view of the high cost of DNA origami, especially DNA staples, it is necessary to develop easy and cost-effective synthesis and assembly strategies to reduce the cost and increase the yield in favor of practical applications.
• There is an urgent need to further develop convenient, automated and efficient design software to simplify the design process of DNA origami.
• The current size of DNA origami is limited by the lack of diverse DNA scaffolds.
• Considering that DNA origami is highly dependent on high concentrations of Mg 2+ and Na + to maintain its structural integrity, as well as its sensitivity to nuclease digestion, which is prevalent in living organisms, strategies need to be developed to enhance the stability of DNA origami.

Several approaches, such as covalent attachment of DNA bases, protein coatings, virus-inspired membrane encapsulation, polyethylene glycol grafting, and silica coatings, have been explored to enhance the stability of DNA origami.

Reference

1. Z, M, He.; et al. Self-Assembly of DNA Origami for Nanofabrication, Biosensing, Drug Delivery, and Computational Storage. Animal Biotechnology. 2023, 26(5): 106638.