William M. Shih

Self-assembly of DNA into nanoscale three-dimensional shapes

Molecular self-assembly offers a ‘bottom-up’ route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase ‘scaffold strand’ that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide ‘staple strands’. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes—monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross—with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometre scale.

Challenges and opportunities for structural DNA nanotechnology

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.

Folding DNA into Twisted and Curved Nanoscale Shapes

Stressful Self-Assembly One way to control shape during the assembly of an object is to design in stresses that cause a planned amount of deformation. Dietz et al. (p. 725; see the Perspective by Liu and Yan) designed DNA helix bundles, arranged in honeycomb lattices, in which some of the helices have insertions or deletions relative to the other helices in the bundles. The stresses help the bundles assemble into objects on the scale of tens of nanometers. Both the direction and degree of bending could be controlled, and curvatures as tight as 6 nanometers achieved. Complex shapes, such as square-toothed gears, could be created by combining multiple curved elements. Site-directed insertions and deletions of base pairs direct twist and curvature in crystal-like DNA arrays. We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly cross-linked double helices, arrayed in parallel to their helical axes. Targeted insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quantitatively controlled, and a radius of curvature as tight as 6 nanometers was achieved. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears.

A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron

Molecular self-assembly offers a means of spontaneously forming complex and well-defined structures from simple components. The specific bonding between DNA base pairs has been used in this way to create DNA-based nanostructures and to direct the assembly of material on the subnanometre to micrometre scale. In principle, large-scale clonal production of suitable DNA sequences and the directed evolution of sequence lineages towards optimized behaviour can be realized through exponential DNA amplification by polymerases. But known examples of three-dimensional geometric DNA objects are not amenable to cloning because they contain topologies that prevent copying by polymerases. Here we report the design and synthesis of a 1,669-nucleotide, single-stranded DNA molecule that is readily amplified by polymerases and that, in the presence of five 40-mer synthetic oligodeoxynucleotides, folds into an octahedron structure by a simple denaturation–renaturation procedure. We use cryo-electron microscopy to show that the DNA strands fold successfully, with 12 struts or edges joined at six four-way junctions to form hollow octahedra approximately 22 nanometres in diameter. Because the base-pair sequence of individual struts is not repeated in a given octahedron, each strut is uniquely addressable by the appropriate sequence-specific DNA binder.

Three-Dimensional Structures Self-Assembled from DNA Bricks

Building with DNA One route for assembling three-dimensional (3D) DNA nanostructures is to start with a long natural DNA single strand and attach short strands, or “staples,” that cause the entire “origami” structure to fold into a desired shape. Ke et al. (p. 1177, see the cover; see the Perspective by Gothelf) present an alternative approach to 3D assembly that builds upon modular assembly of 2D DNA tiles. One hundred and two distinct shapes were created from four-domain, 32-nucleotide single-stranded DNAs that assembled like childrens blocks; each block could bind to four neighboring blocks through specific pairing interactions. Computer design and stepwise assembly allowed assembly of hollow shapes with a variety of internal cavities. Stepwise assembly of 32-nucleotide DNA “bricks” can create a wide variety of nanoscale objects. We describe a simple and robust method to construct complex three-dimensional (3D) structures by using short synthetic DNA strands that we call “DNA bricks.” In one-step annealing reactions, bricks with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide brick is a modular component; it binds to four local neighbors and can be removed or added independently. Each 8–base pair interaction between bricks defines a voxel with dimensions of 2.5 by 2.5 by 2.7 nanometers, and a master brick collection defines a “molecular canvas” with dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we constructed a panel of 102 distinct shapes exhibiting sophisticated surface features, as well as intricate interior cavities and tunnels.

Rapid prototyping of 3D DNA-origami shapes with caDNAno

DNA nanotechnology exploits the programmable specificity afforded by base-pairing to produce self-assembling macromolecular objects of custom shape. For building megadalton-scale DNA nanostructures, a long ‘scaffold’ strand can be employed to template the assembly of hundreds of oligonucleotide ‘staple’ strands into a planar antiparallel array of cross-linked helices. We recently adapted this ‘scaffolded DNA origami’ method to producing 3D shapes formed as pleated layers of double helices constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here we present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3D honeycomb-pleated shapes A series of rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3D DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.

DNA-nanotube-induced alignment of membrane proteins for NMR structure determination.

Membrane proteins are encoded by 20–35% of genes but represent <1% of known protein structures to date. Thus, improved methods for membrane-protein structure determination are of critical importance. Residual dipolar couplings (RDCs), commonly measured for biological macromolecules weakly aligned by liquid-crystalline media, are important global angular restraints for NMR structure determination. For α-helical membrane proteins >15 kDa in size, Nuclear-Overhauser effect-derived distance restraints are difficult to obtain, and RDCs could serve as the main reliable source of NMR structural information. In many of these cases, RDCs would enable full structure determination that otherwise would be impossible. However, none of the existing liquid-crystalline media used to align water-soluble proteins are compatible with the detergents required to solubilize membrane proteins. We report the design and construction of a detergent-resistant liquid crystal of 0.8-μm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4-μm-long six-helix bundles each self-assembled from a 7.3-kb scaffold strand and >170 short oligonucleotide staple strands. We show that the DNA-nanotube liquid crystal enables the accurate measurement of backbone NH and CαHα RDCs for the detergent-reconstituted ζ-ζ transmembrane domain of the T cell receptor. The measured RDCs validate the high-resolution structure of this transmembrane dimer. We anticipate that this medium will extend the advantages of weak alignment to NMR structure determination of a broad range of detergent-solubilized membrane proteins.

Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT

Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or ‘uncoupling’ of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1–4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

Self-assembly of three-dimensional prestressed tensegrity structures from DNA.

Tensegrity or tensional integrity is a property of a structure that relies on a balance between components that are either in pure compression or in pure tension for its stability [1,2]. Tensegrity structures exhibit extremely high strength-to-weight ratios and great resilience, and are therefore widely used in engineering, robotics and architecture [3,4]. Here we report nanoscale, prestressed, three-dimensional tensegrity structures in which rigid bundles of DNA double helices resist compressive forces exerted by segments of single-stranded DNA that act as tension-bearing cables. Our DNA tensegrity structures can self-assemble against forces up to 14 pN, which is twice the stall force of powerful molecular motors such as kinesin or myosin [5,6]. The forces generated by this molecular prestressing mechanism can be employed to bend the DNA bundles or to actuate the entire structure through enzymatic cleavage at specific sites. In addition to being building blocks for nanostructures, tensile structural elements made of single-stranded DNA could be used to study molecular forces, cellular mechanotransduction, and other fundamental biological processes.