DNA synthesis and sequence design. At current prices of about US$0.10 per base for oligonucleotide synthesis on the 25-nmol scale, the overall material cost for constructing a new M13-based origami is around US$700. A key technological opportunity is the emerging commercial availability of affordable arrays on which small amounts of each of the tens of thousands of unique oligonucleotide sequences are printed at a current price of less than US$0.001 per base. If reliable low-cost methods for enzymatic amplification of subsets of strands from these arrays could be further developed this would raise the possibility of custom-designed DNA nanostructures that are 1 gigadalton in mass (that is, around 100 times as complex as current M13-based origami) for a material cost of ~US$1,000. Large reductions in cost of enzymatic amplification would also enable production of gram to kilogram quantities of complex DNA nanostructures, which will be important for many but not all applications.
For DNA origami, a current constraint has been the reliance on the 7 kb genome of M13 as the primary source of scaffold. To create larger structures, ideally one could fold either a longer unique scaffold, or as an alternative, multiple scaffolds with distinct sequences. Furthermore, it seems unlikely that M13 encodes the optimal sequence for high-yield folding of all possible DNA nanostructures. Thus, one would want to generate many unique scaffold sequences, each tailored for optimized folding into a particular origami shape, or at least a large number of distinct generic scaffold sequences that can support independent foldings in a single pot. Affordability is a concern, but gene synthesis from array-printed DNA again may provide a solution. Consideration of these issues naturally leads to the question of what the rules are for effective sequence design, and our current ignorance in this area warrants much future work in this direction, involving an interplay between theory and experiment.
Hierarchical and templated assembly.
Conventional DNA origami uses a single long scaffold molecule as half the material. Using this approach to build a gigadalton DNA nanostructure, one would need a scaffold over 1 megabase long, approaching the length of the Escherichia coli genome. Such large DNA molecules are mechanically fragile and difficult to synthesize. Instead, we can imagine current origami as 'super-tiles' that can be linked together hierarchically to form larger superstructures. Each super-tile can be made a larger size by changing the design to enable use of a higher ratio of non-scaffold to scaffold-strand mass. The design of super-tile interfaces will need to be optimized to improve yield. Higher-order superstructure can be further enforced by use of a super-scaffold that organizes super-tiles. Both a super-scaffold and algorithmic assembly could be used to organize multiple orthogonal super-tiles in specific patterns within a given larger structure. Also, lithographically etched surfaces could be used to template long-range order on a collection of super-tiles merging top-down with bottom-up approaches in this way will attract the attention of the semiconductor industry for microfabrication applications.
a, Expanding size and complexity. Two main approaches are being explored to overcome the current dependence of the structural DNA nanotechnology community on the viral M13 genome: the use of longer DNA scaffold strands (top left) to fold larger structures (top right), or the assembly of pre-formed structures for the constructions of supramolecular assemblies (bottom). b, New functional nanostructures. The functionalization of specific protein surface residues (dark blue circles on the light blue proteins) with oligonucleotides, and subsequent purification, would allow for an extra dimension of positioning control of the protein into a DNA template. c, New generation of DNA walkers (green spheres with purple legs) with programmable routines and/or sensitive to state changes, such as light, for the selection of routes in multi-path systems. d, In vivo selection and amplification of DNA nanostructures. Creating procedures for the selection and evolution of biocompatible/bioactive shapes through environmental conditioning, or using cellular replication machinery for the high-throughput production of DNA structures, should lead to new applications of DNA nanotechnology.
Energy transfer and photonics
Supramolecular chemistry has contributed greatly to the design of artificial light harvesting, energy transfer and charge separation complexes. The main drawback to traditional approaches is the need for extensive organic synthesis efforts, leading to two extreme situations: small constructs with two to five functional units and ångström-level spatial control, or longer constructs with many repeating units, but reduced control over the overall shape and size.
The bottom-up assembly of organic suprastructures affords spatial control at the ångström level; DNA nanostructures can be used as the interface between molecular entities to provide nanometre-scale precise junctions to attach different molecular entities. For example, light-harvesting complexes can be put in close contact with charge transfer units in a modular fashion, using DNA as a molecular pegboard. This might constitute a fresh approach for the construction of 'artificial leaf' systems
Diagnostics and therapeutics for human health.
Probably the most seductive prospect for DNA nanotechnology is as effective drug-delivery nanovehicles for fighting disease. How can DNA-based nanoparticles help to overcome these myriad barriers? DNA nanostructures offer unprecedented control over shape, size, mechanical flexibility and anisotropic surface modification. Clearly, proper control over these aspects can increase circulation times by orders of magnitude, as can be seen for long-circulating particles such as erythrocytes and various pathogenic particles evolved to overcome this issue. But our current knowledge of the proper recipe for long circulation times is limited, so investigation in this area will be important. Surface display of the appropriate ligands can also mediate targeting to and passage through endothelial barriers to diseased tissues, as well as accelerating cellular uptake at the desired target. Mimesis of viral strategies for escaping endosomal compartments into the cytoplasm can in principle be addressed with the additional control over surface functionalization afforded by DNA nanostructures.
The surfaces of DNA nanocontainers are fully addressable, allowing for the incorporation of multiple ligands, labels for bioimaging, antibodies, hormones and so forth that might be used for efficient and site-specific drug delivery and release
Bright future of structural DNA nanotechnology
We have outlined several key challenges for advancing the field of structural DNA nanotechnology, and have suggested a few potential routes to meeting these milestones. Nature has developed sophisticated and complex behaviour at the nanoscale through millions of years of cellular evolution; we will need an aggressive pursuit of bold and forward-looking ideas if we are to catch up over a mere few decades. Along this trajectory of development, let us hope that the advances of structural DNA nanotechnology can be used by researchers in other fields, who will contribute with new approaches, techniques and expertise. Indeed, structural DNA nanotechnology has already become an interdisciplinary research field, with researchers from chemistry, materials science, computer science, biology and physics coming together to tackle important problems. As the field is progressing rapidly, we believe that exciting new directions will emerge well beyond the limited set described here.
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