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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.

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.

Macrophages destroy pathogens and diseased cells through Fcγ receptor (FcγR)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple FcγRs, but the mechanism controlling the threshold for response is unclear. We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands. When the number of ligands remains constant, reducing ligand spacing from 17.5 nm to 7 nm potently enhances engulfment, primarily by increasing efficiency of the engulfment-initiation process. Tighter ligand clustering increases receptor phosphorylation, as well as proximal downstream signals. Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect, indicating that clustering of multiple receptors is required. Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions, which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies. The word ‘phagocytosis’ means cellular eating. It is the process by which cells extend their membranes around foreign particles and engulf them. Macrophages, a type of immune cell found in every tissue of the body, perform phagocytosis to eat pathogens and diseased cells. To avoid eating healthy cells, macrophages focus on targets marked by proteins called antibodies. They look for cells coated with high levels of a type of antibody called immunoglobulin G, or IgG for short, but only eat cells coated with enough IgG, raising the question, can macrophages count? Macrophages recognize IgG antibodies using cell surface receptors called Fc-gamma Receptors. When these receptors bind to IgG, they cluster together. Researchers do not yet know how the number of IgG antibodies per cluster, or the spacing between them, affects phagocytosis. To find this out, researchers need to be able to manipulate the clustering experimentally. One way to do this is using a technique called DNA origami. This technique creates nanoscale patterns of DNA strands on a target surface. If the part of a receptor that interacts with its target is then replaced with a complementary DNA strand to the strands on the target surface, the receptor will bind the surface following the nanoscale pattern. This allows researchers to generate synthetic targets with specific patterns of receptor-target interaction. Kern et al. replaced the part of the macrophage Fc-gamma Receptor that interacts with IgG with a strand of DNA. They then used DNA origami to arrange complementary DNA strands on pegboards and attached these pegboards to silica beads. The different arrangements of DNA on these pegboards mimicked the types of antibody clusters macrophages might encounter on the surfaces of the cells and particles they have to engulf in the body. Kern et al. found that tight clusters of the DNA targets on the pegboards made the macrophages most likely to begin phagocytosis, particularly clusters of eight or more DNA strands spaced less than seven nanometers apart. Macrophages encountering these tight clusters showed an increase in Fc-gamma receptor activation, which is crucial for macrophage attack. Whether or not macrophages can count, they can at least sense the level of clustering of IgG antibodies to determine if a target should be engulfed. Doctors use antibody therapies that rely on Fc-gamma receptor engagement to treat cancer, autoimmune and neurodegenerative diseases. Understanding how clustering affects phagocytosis could aid in the design of new antibody treatments. It could also help improve the design of synthetic receptors to create designer immune cells that can attack specific targets. The next step will be to recreate the results from the synthetic system used by Kern et al. with natural receptors and antibodies.

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.

Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. We apply DNA origami to construct molecular goniometers—instruments that precisely orient objects—and use them to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. We construct goniometers with 14 different stage configurations to orient and visualize the protein just above the cryo-EM grid surface. Each goniometer has a distinct barcode pattern that we use during particle classification to assign angle priors to the bound protein. We use goniometers to obtain a 6.5-Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. Our approach should be adaptable to other DNA-binding proteins as well as small proteins fused to DNA-binding domains. DNA origami orients single proteins on a surface to aid structure determination by cryo-electron microscopy.

Nanomaterials get complicated An important goal in nanotechnology is the programmable self-assembly of complex, three-dimensional nanostructures. With DNA as the building block, synthesis techniques have developed to the stage where two-dimensional designer structures and certain three-dimensional structures can be produced. Douglas et al . describe a refinement of the scaffolded DNA origami technique capable of producing three-dimensional objects of more or less any desired form, to a scale of ten to a hundred nanometres, and with an impressive degree of control over the positions of the various DNA helices. The synthesis involves DNA helices arranged on pleated strands and assembled into honeycomb-like three-dimensional structures. The various strands link together via phosphate groups. The method produces complex objects that are slow to assemble. But it also provides a route towards assembling custom devices with nanometre-scale features, as demonstrated by the construction of objects with shapes resembling a square nut, slotted cross and wire-frame icosahedron. DNA has proved to be a versatile building block in the creation of complex structures through self-assembly, exploiting the intermolecular forces between the components. Here, the arrangement of DNA helices on pleated strands which are then assembled into honeycomb-like three-dimensional structures, produces objects of unprecedented complexity. Molecular self-assembly offers a ‘bottom-up’ route to fabrication with subnanometre precision of complex structures from simple components 1 . DNA has proved to be a versatile building block 2 , 3 , 4 , 5 for programmable construction of such objects, including two-dimensional crystals 6 , nanotubes 7 , 8 , 9 , 10 , 11 , and three-dimensional wireframe nanopolyhedra 12 , 13 , 14 , 15 , 16 , 17 . Templated self-assembly of DNA 18 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’ 19 , 20 . 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.

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities.We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.

A molecular origami method uses double-stranded DNA scaffolds and protein staples to create hybrid nanostructures Molecular engineers have become increasingly adept at repurposing life's building blocks to make custom self-assembled shapes. Because a single drop of solution contains billions of such shapes, DNA origami smiley faces ( 1 ), RNA stars ( 2 ), and designer protein polyhedra ( 3 ) may vastly outnumber most other human-made objects on Earth. These shapes lack immediate practical utility, but they transmit a powerful message: Researchers are beginning to understand how molecules self-assemble. On page 1283 of this issue, Praetorius and Dietz make another leap forward by demonstrating a novel class of nanostructures, namely DNA-protein hybrid shapes ( 4 ). This is an important advance because it provides a method to create human-designed shapes out of ingredients that are generally compatible with living systems.

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.

The Nobel Prize in Physiology or Medicine in 2006 was awarded to Craig Mello and Andrew Fire for the development of essentially a new field, RNA Interference or RNAi. Because this field sprung from a singular discovery made very recently, we can track its growth in precise detail in the biological and literature databases.