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Nanoscale stepper motors such as kinesin and dynein play a key role in numerous natural processes such as mitotic spindle formation during cell division or intracellular organelle transport. Their high efficacy in terms of operational speed and processivity has inspired the investigation of biomimetic technologies based on the use of programmable molecules. In particular, several designs of molecular walkers have been explored using DNA nanotechnology. Here, we study the actuation of a DNA-origami walker on a DNA-origami track based on three principles: 1) octapedal instead of bipedal walking for greater redundancy; 2) three pairs of orthogonal sequences, each of which fuels one repeatable stepping phase for cyclically driven motion with controlled directionality based on strain-based step selection; 3) designed size of only 3.5 nm per step on an origami track. All three principles are innovative in the sense that earlier demonstrations of steppers relied on a maximum of four legs on at least four orthogonal sequences to drive cyclic stepping, and took steps much larger than 3.4 nm in size. Using gel electrophoresis and negative-stain electron microscopy, we demonstrate cyclic actuation of DNA-origami structures through states defined by three sets of specific sequences of anchor points. However, this mechanism was not able to provide the intended control over directionality of movement. DNA-origami-based stepper motors will offer a future platform for investigating how increasing numbers of legs can be exploited to achieve robust stepping with relatively small step sizes.

Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with angstrom-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics. DNA nanoswitch calipers can measure distances within single molecules with atomic resolution. Applied to single-molecule proteomics, they can enable the identification and quantification of molecules in trace samples via mechanical fingerprinting.

Cells often spatially organize biomolecules to regulate biological interactions. Synthetic mimicry of complex spatial organization may provide a route to similar levels of control for artificial systems. As a proof-of-principle, we constructed an RNA-extruding nanofactory using a DNA-origami barrel with an outer diameter of 60 nm as a chassis for integrated rolling-circle transcription and processing of RNA through spatial organization of DNA templates, RNA polymerases, and RNA endonucleases. The incorporation efficiency of molecular components was quantified to be roughly 50% on designed sites within the DNA-origami chassis. Each integrated nanofactory with RNA-producing units, composed of DNA templates and RNA polymerases, produced 100 copies of target RNA in 30 min on average. Further integration of RNA endonucleases that cleave rolling-circle transcripts from concatemers into monomers resulted in 30% processing efficiency. Disabling spatial organization of molecular components on DNA origami resulted in suppression of RNA production as well as processing.

Living systems achieve robust self-assembly across length scales. Meanwhile, nanofabrication strategies such as DNA origami have enabled robust self-assembly of submicron-scale shapes.However, erroneous and missing linkages restrict the number of unique origami that can be practically combined into a single supershape. We introduce crisscross polymerization of DNA-origami slats for strictly seed-dependent growth of custom multi-micron shapes with user-defined nanoscale surface patterning. Using a library of ~2000 strands that can be combinatorially assembled to yield any of ~1e48 distinct DNA origami slats, we realize five-gigadalton structures composed of >1000 uniquely addressable slats, and periodic structures incorporating >10,000 slats. Thus crisscross growth provides a generalizable route for prototyping and scalable production of devices integrating thousands of unique components that each are sophisticated and molecularly precise.One-sentence summaryCrisscross polymerization of DNA-origami slats can yield micron-scale structures with uniquely addressable nanoscale features.

Cells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in in vitro gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bioanalysis, and the design of adaptive materials.

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology., The design and optimisation of 3D DNA-origami can be a barrier to rapid application. Here the authors design barrel structure of stacked 2D double helical rings with complex surface patterns.

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved the data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets., Krieg et al. describe a methanol responsive polymer that can capture complementary DNA using grafted oligonucleotides. They successfully demonstrate its efficacy with simultaneous and sequence-specific isolation of three target genes (cDNA) from clinical NGS libraries with high efficiency. This method is fast, effective, scalable, modular, and versatile.

Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

Life operates at the intersection of chemistry and mechanics. Over the years, we have made remarkable progress in understanding life from a biochemical perspective and the mechanics of life at the single-molecule scale. Yet the full integration of physical and mechanical models into mainstream biology has been impeded by technical and conceptual barriers, including limitations in our ability to 1) easily measure and apply mechanical forces to biological systems, 2) scale these measurements from single-molecule characterization to more complex biomolecular systems, and 3) model and interpret biophysical data in a coherent way across length scales that span single molecules to cells to multicellular organisms. In this manuscript, through a look at historical and recent developments in force spectroscopy techniques and a discussion of a few exemplary open problems in cellular biomechanics, we aim to identify research opportunities that will help us reach our goal of a more complete and integrated understanding of the role of force and mechanics in biological systems.

Here we present a modular method for manufacturing large-sized nanodiscs using DNA-origami barrels as scaffolding corrals. Large-sized nanodiscs can be produced by first decorating the inside of DNA barrels with small lipid-bilayer nanodiscs, which open up when adding extra lipid to form large nanodiscs of diameters ~45 or ~70 nm as prescribed by the enclosing barrel dimension. Densely packed membrane protein arrays are then reconstituted within these large nanodiscs for potential structure determination. Furthermore, we demonstrate the potential of these nanodiscs as model membranes to study poliovirus entry.

Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology—toehold exchange—through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA ‘acrobat’ for rapid movement, we measure a stepping rate constant approaching 1 s−1, which is 10- to 100-fold faster than prior DNA walkers. Finally, we use single-particle tracking to demonstrate movement of the walker over hundreds of nanometres within 10 min, in quantitative agreement with predictions from stepping kinetics. These results suggest that substantial improvements in the operating rates of broad classes of DNA nanomachines utilizing strand displacement are possible. Single-molecule optimization leads to a cartwheeling DNA walker with a more than tenfold improved stepping rate.

Scalable methods currently are lacking for isolation of long ssDNA, an important material for numerous biotechnological applications. Conventional biomolecule purification strategies achieve target capture using solid supports, which are limited in scale and susceptible to contamination owing to nonspecific adsorption and desorption on the substrate surface. We herein disclose selective nascent polymer catch and release (SNAPCAR), a method that utilizes the reactivity of growing poly(acrylamide-co-acrylate) chains to capture acrylamide-labeled molecules in free solution. The copolymer acts as a stimuli-responsive anchor that can be precipitated on demand to pull down the target from solution. SNAPCAR enabled scalable isolation of multi-kilobase ssDNA with high purity and 50-70 % yield. The ssDNA products were used to fold various DNA origami. SNAPCAR-produced ssDNA will expand the scope of applications in nanotechnology, gene editing, and DNA library construction.

Chemical clocks usually achieve well-defined temporal delays through concentration thresholding coupled to the production, degradation, activation, or inhibition of downstream effectors. In this way, the stochastic dynamics of many individual molecules yield essentially deterministic bulk behavior through ensemble averaging. As a result, their temporal evolution is governed by ensemble dynamics rather than by the behavior of an individual molecule or complex. Here, we present a general approach for the design of single-molecule clocks that permits quasi-deterministic control over the lifetime of single molecular interactions without any external synchronization. By coupling the dissociation of a bimolecular complex to a series of irreversible chemical steps, we interpose a well-defined time delay between binding and dissociation. The number and speed of irreversible steps can be varied to systematically tune both the lifetimes of complexes and the precision of the time delay, raising the prospect of localized timekeeping in nanoscale systems and devices.

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications., The instability of DNA nanostructures in physiological environments has hampered their use as therapeutics and diagnostic agents in in vivo applications. Here, the authors show that coating DNA origami with oligolysine-PEG moieties improves their pharmacokinetic properties in mouse models.

Natural biomolecular assemblies such as actin filaments or microtubules polymerize in a nucleation-limited fashion1,2. The barrier to nucleation arises in part from chelate cooperativity, where stable capture of incoming monomers requires straddling multiple subunits on a filament end3. For programmable self-assembly from building blocks such as synthetic DNA4–23, it is likewise desirable to be able to suppress spontaneous nucleation24–31. However, existing approaches that exploit just a low level of cooperativity can limit spontaneous nucleation only for slow growth, near-equilibrium conditions32. Here we introduce ultracooperative assembly of ribbons densely woven from single-stranded DNA slats. An inbound “crisscross” slat snakes over and under six or more previously captured slats on a growing ribbon end, forming weak but specific half-duplex interactions with each. We demonstrate growth of crisscross ribbons with distinct widths and twists to lengths representing many thousands of slat additions. Strictly seed-initiated extension is attainable over a broad range of temperatures, divalent-cation concentrations, and free-slat concentrations, without unseeded ribbons arising even after a hundred hours to the limit of agarose-gel detection. We envision that crisscross assembly will be broadly enabling for all-or-nothing formation of microstructures with nanoscale features, algorithmic self-assembly, and signal amplification in diagnostic applications requiring extreme sensitivity.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40–50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

DNA bricks build nanotube transistors Semiconducting carbon nanotubes (CNTs) are an attractive platform for field-effect transistors (FETs) because they potentially can outperform silicon as dimensions shrink. Challenges to achieving superior performance include creating highly aligned and dense arrays of nanotubes as well as removing coatings that increase contact resistance. Sun et al. aligned CNTs by wrapping them with single-stranded DNA handles and binding them into DNA origami bricks that formed an array of channels with precise intertube pitches as small as 10.4 nanometers. Zhao et al. then constructed single and multichannel FETs by attaching the arrays to a polymer-templated silicon wafer. After adding metal contacts across the CNTs to fix them to the substrate, they washed away all of the DNA and then deposited electrodes and gate dielectrics. The FETs showed high on-state performance and fast on-off switching. Science , this issue p. 874 , p. 878

DNA-based devices often operate through a series of toehold-mediated strand-displacement reactions. To achieve cycling, fluidic mixing can be used to introduce ‘recovery’ strands to reset the system. However, such mixing can be cumbersome, non-robust, and wasteful of materials. Here we demonstrate mixing-free thermal cycling of DNA devices that operate through associative strand-displacement cascades. These cascades are favored at low temperatures due to the primacy of a net increase in base pairing, whereas rebinding of ‘recovery’ strands is favored at higher temperatures due to the primacy of a net release of strands. The temperature responses of the devices could be modulated by adjustment of design parameters such as the net increase of base pairs and the concentrations of strands. Degradation of function was not observable even after 500 thermal cycles. We experimentally demonstrated simple digital-logic circuits that evaluate at 35°C and reset after transient heating to 65°C. Thus associative strand displacement enables robust thermal cycling of DNA-based devices in a closed system.

Cells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in cell-free gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic-acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic-acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic-acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic-acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bio-analysis, and the design of adaptive materials.

We present a rapid, scalable, user-friendly method for in vitro production of high-purity single-stranded DNA (ssDNA) ranging from 89–3315 nucleotides in length. PCR with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology-directed repair in human cells, DNA-origami folding, and fluorescent in situ hybridization.

Scaffolded DNA origami has proven to be a versatile method for generating functional nanostructures with prescribed sub-100 nm shapes. Programming DNA-origami tiles to form large-scale 2D lattices that span hundreds of nanometers to the micrometer scale could provide an enabling platform for diverse applications ranging from metamaterials to surface-based biophysical assays. Toward this end, here we design a family of hexagonal DNA-origami tiles using computer-aided design and demonstrate successful self-assembly of micrometer-scale 2D honeycomb lattices and tubes by controlling their geometric and mechanical properties including their interconnecting strands. Our results offer insight into programmed self-assembly of low-defect supra-molecular DNA-origami 2D lattices and tubes. In addition, we demonstrate that these DNA-origami hexagon tiles and honeycomb lattices are versatile platforms for assembling optical metamaterials via programmable spatial arrangement of gold nanoparticles (AuNPs) into cluster and superlattice geometries.

The creation of nanometre-sized structures that exhibit controllable motions and functions is a critical step towards building nanomachines. Recent developments in the field of DNA nanotechnology have begun to address these goals, demonstrating complex static or dynamic nanostructures made of DNA. Here we have designed and constructed a rhombus-shaped DNA origami ‘nanoactuator' that uses mechanical linkages to copy distance changes induced on one half (‘the driver') to be propagated to the other half (‘the mirror'). By combining this nanoactuator with split enhanced green fluorescent protein (eGFP), we have constructed a DNA–protein hybrid nanostructure that demonstrates tunable fluorescent behaviours via long-range allosteric regulation. In addition, the nanoactuator can be used as a sensor that responds to specific stimuli, including changes in buffer composition and the presence of restriction enzymes or specific nucleic acids., The construction of nano-machines requires building nano-scale structures with controllable functions. Here the authors use DNA origami to construct an allosteric actuator which can act as signal propagator and an environmental sensor.

Designer nanoparticles with controlled shapes and sizes are increasingly popular vehicles for therapeutic delivery due to their enhanced cell-delivery performance. However, our ability to fashion nanoparticles has offered only limited control over these parameters. Structural DNA nanotechnology has an unparalleled ability to self-assemble three-dimensional nanostructures with near-atomic resolution features, and thus, it offers an attractive platform for the systematic exploration of the parameter space relevant to nanoparticle uptake by living cells. In this study, we examined the cell uptake of a panel of 11 distinct DNA-origami shapes, with the largest dimension ranging from 50-400 nm, in 3 different cell lines. We found that larger particles with a greater compactness were preferentially internalized compared with elongated, high-aspect-ratio particles. Uptake kinetics were also found to be more cell-type-dependent than shape-dependent, with specialized endocytosing dendritic cells failing to saturate over 12 h of study. The knowledge gained in the current study furthers our understanding of how particle shape affects cellular uptake and heralds the development of DNA nanotechnologies toward the improvement of current state-of-the-art cell-delivery vehicles.

Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology-toehold exchange-through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA 'acrobat' for rapid movement, we measure a stepping rate constant approaching 1 s

The ability to assemble functional materials with precise spatial arrangements is important for applications ranging from protein crystallography to photovoltaics. Here, we describe a general framework for constructing two-dimensional crystals with prescribed depths and sophisticated three-dimensional features. The crystals are self-assembled from single-stranded DNA components called DNA bricks. We demonstrate the experimental construction of DNA brick crystals that can grow to micrometre size in their lateral dimensions with precisely controlled depths up to 80 nm. They can be designed to pack DNA helices at angles parallel or perpendicular to the plane of the crystal and to display user-specified sophisticated three-dimensional nanoscale features, such as continuous or discontinuous cavities and channels.

DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental data. Our study thus describes considerations that are vital for researchers undertaking in vitro tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments.

Furthermore, as shown by Zhang and co-workers, where longrange PCR was used for the production of single-stranded scaffolds of up to 26 kb, [ 17 ] alternative scaffold sources enable the assembly of larger single structures while avoiding often low-yield hierarchical assembly procedures. [ 18–20 ] All these methods extend the DNA origami technique considerably but also rely on various time consuming and tedious modifi cation steps of either naturally occurring or synthetic template DNA such as several purifi cation steps, PCR, strand separation, enzymatic digestion and/or modifi cation. In addition, for many promising applications of the DNA origami technique – such as nanomedicine or drug delivery – a large quantity of the material is needed. Scaling up the assembly process and maximizing the assembly yield while reducing both labor and cost remains a major challenge for the future development of the fi eld. [ 21 ]

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.

Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes., Characterizing the mechanical response of molecular motors involves the use of methods such as optical trapping to apply force. Here the authors develop a DNA origami nanospring to apply progressive force to human myosin VI, and discover that it adopts different stepping modes when subjected to low load or high load.

Thirty percent of the human proteome is composed of membrane proteins that can perform a wide range of cellular functions and communications. They represent the core of modern medicine as the targets of about 50 % of all prescription pharmaceuticals. However, elucidating the structure of membrane proteins has represented a constant challenge, even in the modern era. To date, only a few hundred high-resolution structural models of membrane proteins are available. This chapter describes the emergence of DNA nanotechnology as a powerful tool for the structural characterization of membrane protein using solution-state nuclear magnetic resonance (NMR) spectroscopy. Here, we detail the large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into a dilute liquid crystal to be used as a weak-alignment media in solution NMR structure determination of membrane proteins.

Structural DNA nanotechnology methods such as DNA origami allow for the synthesis of highly precise nanometer-scale materials (Rothemund, Nature 440:297-302, 2006; Douglas et al., Nature 459:414-418, 2009). These offer compelling advantages for biomedical applications. Such materials can suffer from structural instability in biological environments due to denaturation and nuclease digestion (Hahn et al., ACS Nano 2014; Perrault and Shih, ACS Nano 8:5132-5140, 2014). Encapsulation of DNA nanostructures in a lipid membrane compartmentalizes them from their environment and prevents denaturation and nuclease digestion (Perrault and Shih, ACS Nano 8:5132-5140, 2014). Here, we describe the encapsulation of a 50 nm DNA nanostructure having the geometry of a wireframe octahedron in a phospholipid membrane containing poly-(ethylene glycol), resulting in biocompatible DNA nanostructures.

International audience; Thirty percent of the human proteome is composed of membrane proteins that can perform a wide range of cellular functions and communications. They represent the core of modern medicine as the targets of about 50 % of all prescription pharmaceuticals. However, elucidating the structure of membrane proteins has represented a constant challenge, even in the modern era. To date, only a few hundred high-resolution structural models of membrane proteins are available. This chapter describes the emergence of DNA nanotechnology as a powerful tool for the structural characterization of membrane protein using solution-state nuclear magnetic resonance (NMR) spectroscopy. Here, we detail the large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into a dilute liquid crystal to be used as a weak-alignment media in solution NMR structure determination of membrane proteins.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.

DNA is not just the stuff of our genetic code; it is also a means to design self-assembling materials. Grafting DNA onto nano- and microparticles can, in principle, ‘program’ them with information that tells them exactly how to self-assemble. Although fully programmable assembly has not yet been realized, the groundwork has been laid: with an understanding of how specific interparticle attractions arise from DNA hybridization, we can now make systems that reliably assemble in and out of equilibrium. We discuss these advances, and the design rules that will allow us to control — and ultimately program — the assembly of new materials. Grafting DNA strands onto colloidal nano- and microparticles endows them with sequence-specific interactions. This Review explains how these interactions emerge from reactions between the strands and how the DNA sequences can add information that tells the particles how to self-assemble.

Studies in mechanochemistry have greatly benefited from advances in single molecule force spectroscopy (SMFS) techniques. Despite success, conventional SMFS techniques are inherently low throughput. For example, Atomic Force Microscopy (AFM) and optical tweezers are serial techniques. In recent years, considerable effort has been made to develop massively parallel force spectroscopy methods such as the centrifugal force microscope (CFM), which applies centrifugal force to tethered beads. The capability to apply centrifugal force on a large number of beads opens new multiplexing possibilities.Here, we report the development of a novel technique — Multiplexed Mechanochemistry Assay (MMA) — enabling large-scale study of single molecule chemical bond rupture events. Briefly, in a bead surface assay configuration, we designed a centrifugal system for the application of pre-calibrated forces on up to 320 samples simultaneously. Our system enables two-dimensional multiplexing, (1) number of beads being studied simultaneously, and (2) number of experimental conditions being probed. The force range afforded by our equipment ranges from a fraction of a pN to ∼30 nN. This unprecedented level of ultraplexing afforded by our approach, hundreds of experimental conditions tested against orders of magnitude variation in force, provides a dramatic improvement in throughput of individual experiments over conventional SMFS techniques. As system validation, we report a case study on studying bond rupture of covalent bonds. We believe this simple tool will greatly increase the rate of progress in probing the relation between force-lifetime and chemistry in single molecular bonds.

Our group previously solved a key challenge for nanotechnology: programmable self-assembly of complex, three-dimensional nanostructures. Our solution was to build custom three-dimensional structures that can be conceived as stacks of nearly flat layers of DNA. I will discuss applications of this technology for molecular biophysics and therapeutics, including our efforts to build nanoscale capsules as delivery vehicles in vivo.

Push Me, Release, Pull You In eukaryotic cells, nearly all long-distance transport of cargos is carried out by the microtubule-based motors kinesin and dynein. These opposite-polarity motors move cargos bidirectionally so that they reach their cellular destinations with spatial and temporal specificity. To understand transport by motor ensembles, Derr et al. (p. 662 , published online 11 October; see the Persective by Diehl ) used a DNA scaffold for building an artificial cargo that could be programmed to bind different numbers and types of molecular motors with defined geometry. A cargo with multiple copies of the same motor was transported with minimal interference, suggesting that similar-polarity motors can coordinate without the need for additional cellular factors. However, ensembles of opposite-polarity motors frequently engaged in a sort of “tug of war,” which could only be resolved by releasing one motor from the microtubule track. Thus, within the cell, it is likely that regulation is required for bidirectional transport.

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ∼40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

"Scaffolded DNA origami" has been proven to be a powerful and efficient approach to construct two-dimensional or three-dimensional objects with great complexity. Multilayer DNA origami has been demonstrated with helices packing along either honeycomb-lattice geometry or square-lattice geometry. Here we report successful folding of multilayer DNA origami with helices arranged on a close-packed hexagonal lattice. This arrangement yields a higher density of helical packing and therefore higher resolution of spatial addressing than has been shown previously. We also demonstrate hybrid multilayer DNA origami with honeycomb-lattice, square-lattice, and hexagonal-lattice packing of helices all in one design. The availability of hexagonal close-packing of helices extends our ability to build complex structures using DNA nanotechnology.