Challenges and opportunities for structural DNA nanotechnology

Abstract

DNA molecules have been used to build a variety of nanoscale structures and devices over the past 30 years, and potential applications have begun to emerge. But the development of more advanced structures and applications will require a number of issues to be addressed, the most significant of which are the high cost of DNA and the high error rate of self-assembly. Here we examine the technical challenges in the field of structural DNA nanotechnology and outline some of the promising applications that could be developed if these hurdles can be overcome. In particular, we highlight the potential use of DNA nanostructures in molecular and cellular biophysics, as biomimetic systems, in energy transfer and photonics, and in diagnostics and therapeutics for human health.

Figure 1. Examples of structural DNA nanotechnology

a, Seeman's original proposal consisted of using immobile DNA junctions (left) to build 3D scaffolds that could be used to organize proteins (right). b, Important milestones in structural DNA nanotechnology: the first wireframe 3D cube (left), DNA origami (centre) and a 3D periodic structure composed of tensegrity triangles (right). c, DNA periodic arrays composed of double-crossover tiles (left), 4 × 4 tiles (centre left), three-point star tiles (centre right) and double-crossover-tile-based algorithmic assembly of Sierpinski triangles (right). d, Three-dimensional DNA origami: a hollow box (left pair of images), a multi-layer square nut (centre left pair), a square-toothed gear (centre right pair) and a nanoflask (right pair). e, DNA nanostructure-directed patterning of heteroelements: double-crossover tiles for the organization of gold nanoparticle arrays (left), DNA origami for the assembly of carbon nanotubes (centre left), biotin-streptavidin protein patterning of 4 × 4 tiles (centre), aptamer-directed assembly of thrombin arrays on triple crossover tiles (centre right), and Snap-tag and His-tag mediated orthogonal decoration of DNA origami (right). Figures reproduced with permission from: b, Nadrian C. Seeman (left), ref. 42, © 2006 NPG (centre), ref. 38, © 2009 NPG (right); c, ref. 19, © 1998 NPG (left), ref. 22, © 2003 AAAS (centre left), ref. 24, © 2005 ACS (centre right), ref. 30, courtesy of P. Rothemund (right); d, ref. 43, © 2009 NPG (left), ref. 47, © 2009 NPG (centre left), ref. 49, © 2009 AAAS (centre right), ref. 50, © 2011 AAAS (right); e, ref. 62,© 2004 ACS (left), ref. 75, © 2010 NPG (centre left), ref. 22, © 2003 NPG (centre), ref. 53, © 2005 Wiley (centre right), ref. 57, © 2010 Wiley (right).

Figure 2. Challenges for DNA nanostructures

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.

Figure 3. DNA nanotechnology for biophysical studies

a, DNA origami can act as fully addressable molecular pegboards that can be used as molecular rulers for the organization of heteroelements (blue and red spheres). The purple and green blocks can be any DNA structure that directs the sphere position along a platform. A particularly interesting application is the spatial arrangement of enzyme components of cascade reactions. The relative positions of components can be designed with nanometre accuracy, possibly allowing biochemists to suppress diffusion-dependent effects in cascade reactions. This would open classic biochemical systems to new functional properties, and potential improved performances, distinct from bulk reaction measurements. Moreover, such assemblies could be used as models of intracellular compartmentalization or in vivo clustering. b, When current real-time measurement tools are employed, many in vivo interactions elude detection. Fluorescence, and in particular Förster resonance energy transfer, or single-dye fluorescent markers, yield narrow snapshots of in vivo reality. DNA scissors, tweezers or tensegrity structures (shown as cross-like structures within translucent pink oval, which represents a cell) may be used for real-time and dynamic measurement of target protein activity, or the specific detection and size estimation of protein complexes required for cellular functions. The DNA nanostructure switches conformation to accompany changes in the shape and size of target structures in their native medium: this allows them to serve as relays between the length scales associated with interactions between protein constructs such as DNA-promoter complexes ~10s of nanometres) and those associated with fluorescence reporting (a few nanometres or less). Two such structures are shown here.

Figure 4. DNA nanostructures as biomimetic and in vivo active systems

Aldaye and co-workers recently reported the assembly of two enzymes of a hydrogen-production cascade reaction using RNA arrays, which led to improved yields. In vivo replication of complex DNA structures allows intracellular components (blue, pink and yellow objects) to be organized with tighter and more complex spatial control for the study of cellular properties or new capabilities due to the cytosol clustering effect. Conversely, DNA structures can be designed and `expressed' that fold into biomimetic structures, such as DNA-based nanopores, channels or pumps, introducing artificial layers of cell communication and interaction with its external medium. Also, DNA nanostructures can induce immune responses and actively modulate cell-cell communication on clustering and spatial organization of membrane protein markers, or, in a more abstract concept, acting as specific cell-cell glue (here shown as light blue and red rods connecting the dark blue and pink cells).

Figure 5. DNA nanotechnology for energy transfer and photonics

DNA nanostructures provide a useful tool for the organization of photonic components in a linear fashion or in branched networks. The modularity of assembly, along with the plethora of DNA functionalization of photonic components, allows for the construction of photonic molecular circuits. Light-harvesting complexes can be spatially clustered and aligned, where sequential energy or charge-transfer processes lead to optimized channelling efficiency, to create a new generation of photonic wires, plasmonic or conducting devices (blue, green and red spheres and orange rods represent photonic components that can serve as light-harvesting and energy-transfer materials). Enzymes or membrane complexes (uneven green spheres) can be used as final energy or electron acceptors, acting as molecular transducer units, where light is transformed into chemical potential (represented by the transformation of substrate (triangles) into a higher-energy product (stars)). Physical separation of photonic components creates a new layer of spectral separation, allowing the construction of larger and more complex photonic circuitry.

Figure 6. Structural DNA nanotheranostics

DNA structures can be used to build disease-targeting units for diagnostics and therapeutics (or `theranostics'). Hollow structures are designed in a modular fashion, where multiple pharmacologically active species can be caged into different compartments. Advances in DNA computing may allow the detection of several disease markers (such as interaction between aptamers and membrane receptors, or hormone-activated switches) that are input into a programmed response. The use of multiple input stimuli for the controlled release of drugs may increase drug delivery specificity. This way, the presence of pathogens or multiple cancer markers, for example, can be simultaneously analysed, triggering suitable therapeutics. The magnitude and duration of the response can also be programmed, from continuous cargo release to threshold-controlled dumping. Such a system might be regarded as a platform model of an artificial immune system.