Shawn M. Douglas

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.

A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads

Nanorobots Deliver DNA aptamers are short strands that have high binding affinity for a target protein that can be used as triggers for releasing cargo from delivery vehicles. Douglas et al. (p. 831) used this strategy to design DNA origami “nanorobots”—complex shaped structures created by manipulating a long DNA strand through binding with shorter “staple” strands—that could deliver payloads such as gold nanoparticles or fluorescently labeled antibody fragments. These nanorobots were designed to open in response to specific cell-surface proteins, releasing molecules that triggered cell signaling. Cargoes stored in folded DNA origami are released when aptamers in the structure bind target protein molecules. We describe an autonomous DNA nanorobot capable of transporting molecular payloads to cells, sensing cell surface inputs for conditional, triggered activation, and reconfiguring its structure for payload delivery. The device can be loaded with a variety of materials in a highly organized fashion and is controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues. We implemented several different logical AND gates and demonstrate their efficacy in selective regulation of nanorobot function. As a proof of principle, nanorobots loaded with combinations of antibody fragments were used in two different types of cell-signaling stimulation in tissue culture. Our prototype could inspire new designs with different selectivities and biologically active payloads for cell-targeting tasks.

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.

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.

A public resource facilitating clinical use of genomes

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved “open consent” process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain—we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.

Robotic Cloning and Protein Production Platform of the Northeast Structural Genomics Consortium

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

Folding complex DNA nanostructures from limited sets of reusable sequences

Abstract Scalable production of DNA nanostructures remains a substantial obstacle to realizing new applications of DNA nanotechnology. Typical DNA nanostructures comprise hundreds of DNA oligonucleotide strands, where each unique strand requires a separate synthesis step. New design methods that reduce the strand count for a given shape while maintaining overall size and complexity would be highly beneficial for efficiently producing DNA nanostructures. Here, we report a method for folding a custom template strand by binding individual staple sequences to multiple locations on the template. We built several nanostructures for well-controlled testing of various design rules, and demonstrate folding of a 6-kb template by as few as 10 unique strand sequences binding to 10 ± 2 locations on the template strand.

Gelbox - An Interactive Simulation Tool for Gel Electrophoresis

Gel electrophoresis enables separation and visualization of biomolecules such as DNA, RNA, or proteins. Like many powerful tools, mastering the use of gels and making the information gained from them productive, can be dificult. Gelbox is a simulation tool that helps users understand how changing experimental input parameters can affect the data output from gel electrophoresis. Our simulation model handles a wide range of settings, including many “suboptimal” values that may be useful for troubleshooting common mistakes made by novices.

Construction of a novel phagemid to produce custom DNA origami scaffolds

Abstract DNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the ‘scaffold’ to template assembly of numerous short DNA oligonucleotide ‘staples’. The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and those sequences cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here, we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 to 10 080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications.