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Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy number, spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clinical vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to ~25–30 nm monotonically increases B-cell receptor activation. Moreover, scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses.DNA origami allows the precise spatial patterning of antigens to investigate the impact of antigen spacing and arrangement on B-cell activation in vitro, which is important for the design of efficient vaccination strategies.

Many intricate nanostructures have been made with DNA origami. This process occurs when a long DNA scaffold develops a particular shape after hybridization with short staple strands. Most designs, however, require a difficult iterative procedure of refining the base pairing. Veneziano et al. now report algorithms that automate the design of arbitrary DNA wireframe structures. Synthesizing and structurally characterizing a variety of nanostructures allowed for verification of the algorithms' accuracy. Science , this issue p. 1534 A top-down algorithm can program the design of arbitrary three-dimensional DNA structures. Scaffolded DNA origami is a versatile means of synthesizing complex molecular architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, we report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asymmetric polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biological as well as nonbiological applications.

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxicity. However, ssDNA synthesis strategies are limited by the maximum length attainable, ranging from a few hundred nucleotides for chemical synthesis to a few thousand nucleotides for enzymatic synthesis, as well as limited control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kilobases (kb) using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and theranostic applications.

The intra-image identification of DNA structures is essential to rapid prototyping and quality control of self-assembled DNA origami scaffold systems. We postulate that the YOLO modern object detection platform commonly used for facial recognition can be applied to rapidly scour atomic force microscope (AFM) images for identifying correctly formed DNA nanostructures with high fidelity. To make this approach widely available, we use open-source software and provide a straightforward procedure for designing a tailored, intelligent identification platform which can easily be repurposed to fit arbitrary structural geometries beyond AFM images of DNA structures. Here, we describe methods to acquire and generate the necessary components to create this robust system. Beginning with DNA structure design, we detail AFM imaging, data point annotation, data augmentation, model training, and inference. To demonstrate the adaptability of this system, we assembled two distinct DNA origami architectures (triangles and breadboards) for detection in raw AFM images. Using the images acquired of each structure, we trained two separate single class object identification models unique to each architecture. By applying these models in sequence, we correctly identified 3470 structures from a total population of 3617 using images that sometimes included a third DNA origami structure as well as other impurities. Analysis was completed in under 20 s with results yielding an F1 score of 0.96 using our approach.

Natural light-harvesting systems spatially organize densely packed chromophore aggregates using rigid protein scaffolds to achieve highly efficient, directed energy transfer. Here, we report a synthetic strategy using rigid DNA scaffolds to similarly program the spatial organization of densely packed, discrete clusters of cyanine dye aggregates with tunable absorption spectra and strongly coupled exciton dynamics present in natural light-harvesting systems. We first characterize the range of dye-aggregate sizes that can be templated spatially by A-tracts of B-form DNA while retaining coherent energy transfer. We then use structure-based modelling and quantum dynamics to guide the rational design of higher-order synthetic circuits consisting of multiple discrete dye aggregates within a DX-tile. These programmed circuits exhibit excitonic transport properties with prominent circular dichroism, superradiance, and fast delocalized exciton transfer, consistent with our quantum dynamics predictions. This bottom-up strategy offers a versatile approach to the rational design of strongly coupled excitonic circuits using spatially organized dye aggregates for use in coherent nanoscale energy transport, artificial light-harvesting, and nanophotonics.

DNA hydrogels are self-assembled biomaterials that rely on Watson–Crick base pairing to form large-scale programmable three-dimensional networks of nanostructured DNA components. The unique mechanical and biochemical properties of DNA, along with its biocompatibility, make it a suitable material for the assembly of hydrogels with controllable mechanical properties and composition that could be used in several biomedical applications, including the design of novel multifunctional biomaterials. Numerous studies that have recently emerged, demonstrate the assembly of functional DNA hydrogels that are responsive to stimuli such as pH, light, temperature, biomolecules, and programmable strand-displacement reaction cascades. Recent studies have investigated the role of different factors such as linker flexibility, functionality, and chemical crosslinking on the macroscale mechanical properties of DNA hydrogels. In this review, we present the existing data and methods regarding the mechanical design of pure DNA hydrogels and hybrid DNA hydrogels, and their use as hydrogels for cell culture. The aim of this review is to facilitate further study and development of DNA hydrogels towards utilizing their full potential as multifeatured and highly programmable biomaterials with controlled mechanical properties.

DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage’s circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design—allowing for increasing structural complexity—and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applications. To this end, several techniques have been developed in recent years for the scalable synthesis of single stranded DNA scaffolds with custom lengths and sequences. This review focuses on these methods and the progress that has been made to address the challenges confronting custom scaffold production for large-scale DNA origami assembly.

Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones. Multiplexed imaging of synaptic proteins can provide useful information on the heterogeneity of synaptic architecture and plasticity. Here the authors use high affinity locked nucleic acid probes and low affinity DNA imaging probes to achieve multiplexed confocal and super-resolution imaging of synaptic and cytoskeletal proteins.

Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study's in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions.

Ensuring bacterial safety of blood transfusions remains a critical focus in medicine. We investigated a novel pathogen reduction technology utilizing nylon functionalized with synthetic dyes (nylon affinity networks) to capture and remove bacteria from plasma. In the initial screening process, we spiked phosphate buffer solution (PBS) and human plasma (1 mL each) with 10 or 100 colony forming units (cfu) of either Escherichia coli or Staphylococcus epidermidis, exposed the suspensions to affinity networks and assessed the extent of bacterial reduction using agar plate cultures as the assay output. Nineteen synthetic dyes were tested. Among these, Alcian Blue exhibited the best performance with both bacterial strains in both PBS and plasma. Next, bacterial suspensions of approximately 1 and 2 cfu/mL in 10 and 50 mL, respectively, were treated with Alcian Blue affinity networks in three sequential capture steps. This procedure resulted in complete bacterial depletion, as demonstrated by the lack of bacterial growth in the remaining fraction. The viability of the captured bacteria was confirmed by plating the post-treatment affinity networks on agar. Alcian Blue affinity networks captured and sequestered a few plasma proteins identified by liquid chromatography tandem mass spectrometry. These findings support the potential applicability of nylon affinity networks to enhance transfusion safety, although additional investigations are needed.