Binding Site Programmable Self-Assembly of 3D Hierarchical DNA Origami Nanostructures.

DNA nanotechnology has broad applications in biomedical drug delivery and programmable materials. Characterization of the self-assembly of DNA origami and quantum dots (QDs) is necessary for the development of new DNA-based nanostructures. We use computation and experiment to show that the self-assembly of 3D hierarchical nanostructures can be controlled by programming the binding site number and their positions on DNA origami. Using biotinylated pentagonal pyramid wireframe DNA origamis and streptavidin capped QDs, we demonstrate that DNA origami with 1 binding site at the outer vertex can assemble multimeric origamis with up to 6 DNA origamis on 1 QD, and DNA origami with 1 binding site at the inner center can only assemble monomeric and dimeric origamis. Meanwhile, the yield percentages of different multimeric origamis are controlled by the QD:DNA-origami stoichiometric mixing ratio. DNA origamis with 2 binding sites at the αγ positions (of the pentagon) make larger nanostructures than those with binding sites at the αß positions. In general, increasing the number of binding sites leads to increases in the nanostructure size. At high DNA origami concentration, the QD number in each cluster becomes the limiting factor for the growth of nanostructures. We find that reducing the QD size can also affect the self-assembly because of the reduced access to the binding sites from more densely packed origamis.

Sculpting photoproducts with DNA origami.

Natural light-harvesting systems spatially organize densely packed dyes in different configurations to either transport excitons or convert them into charge photoproducts, with high efficiency. In contrast, artificial photosystems like organic solar cells and light-emitting diodes lack this fine structural control, limiting their efficiency. Thus, biomimetic multi-dye systems are needed to organize dyes with the sub-nanometer spatial control required to sculpt resulting photoproducts. Here, we synthesize 11 distinct perylene diimide (PDI) dimers integrated into DNA origami nanostructures and identify dimer architectures that offer discrete control over exciton transport versus charge separation. The large structural-space and site-tunability of origami uniquely provides controlled PDI dimer packing to form distinct excimer photoproducts, which are sensitive to interdye configurations. In the future, this platform enables large-scale programmed assembly of dyes mimicking natural systems to sculpt distinct photophysical products needed for a broad range of optoelectronic devices, including solar energy converters and quantum information processors.

Discovery and Quantification of Long-Range RNA Base Pairs in Coronavirus Genomes with SEARCH-MaP and SEISMIC-RNA.

RNA molecules perform a diversity of essential functions for which their linear sequences must fold into higher-order structures. Techniques including crystallography and cryogenic electron microscopy have revealed 3D structures of ribosomal, transfer, and other well-structured RNAs; while chemical probing with sequencing facilitates secondary structure modeling of any RNAs of interest, even within cells. Ongoing efforts continue increasing the accuracy, resolution, and ability to distinguish coexisting alternative structures. However, no method can discover and quantify alternative structures with base pairs spanning arbitrarily long distances - an obstacle for studying viral, messenger, and long noncoding RNAs, which may form long-range base pairs. Here, we introduce the method of Structure Ensemble Ablation by Reverse Complement Hybridization with Mutational Profiling (SEARCH-MaP) and software for Structure Ensemble Inference by Sequencing, Mutation Identification, and Clustering of RNA (SEISMIC-RNA). We use SEARCH-MaP and SEISMIC-RNA to discover that the frameshift stimulating element of SARS coronavirus 2 base-pairs with another element 1 kilobase downstream in nearly half of RNA molecules, and that this structure competes with a pseudoknot that stimulates ribosomal frameshifting. Moreover, we identify long-range base pairs involving the frameshift stimulating element in other coronaviruses including SARS coronavirus 1 and transmissible gastroenteritis virus, and model the full genomic secondary structure of the latter. These findings suggest that long-range base pairs are common in coronaviruses and may regulate ribosomal frameshifting, which is essential for viral RNA synthesis. We anticipate that SEARCH-MaP will enable solving many RNA structure ensembles that have eluded characterization, thereby enhancing our general understanding of RNA structures and their functions. SEISMIC-RNA, software for analyzing mutational profiling data at any scale, could power future studies on RNA structure and is available on GitHub and the Python Package Index.

Scalable search of massively pooled nucleic acid samples enabled by a molecular database query language.

The surge in nucleic acid analytics requires scalable storage and retrieval systems akin to electronic databases used to organize digital data. Such a system could transform disease diagnosis, ecological preservation, and molecular surveillance of biothreats. Current storage systems use individual containers for nucleic acid samples, requiring single-sample retrieval that falls short compared with digital databases that allow complex and combinatorial data retrieval on aggregated data. Here, we leverage protective microcapsules with combinatorial DNA labeling that enables arbitrary retrieval on pooled biosamples analogous to Structured Query Languages. Ninety-six encapsulated pooled mock SARS-CoV-2 genomic samples barcoded with patient metadata are used to demonstrate queries with simultaneous matches to sample collection date ranges, locations, and patient health statuses, illustrating how such flexible queries can be used to yield immunological or epidemiological insights. The approach applies to any biosample database labeled with orthogonal barcodes, enabling complex post-hoc analysis, for example, to study global biothreat epidemiology.

Force-free activation of Notch with DNA origami.

The Notch signaling pathway is a highly conserved, fundamental process to embryogenesis and neurogenesis. While force-induced conformational change is known to activate Notch receptors, Smyrlaki et al. recently used DNA origami to reveal an additional, force-independent mode of Notch activation via soluble presentation of spatially controlled ligand nanopatterns.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

Isometric Hamming embeddings of weighted graphs.

A mapping α:V(G)→V(H) from the vertex set of one graph G to another graph H is an isometric embedding if the shortest path distance between any two vertices in G equals the distance between their images in H. Here, we consider isometric embeddings of a weighted graph G into unweighted Hamming graphs, called Hamming embeddings, when G satisfies the property that every edge is a shortest path between its endpoints. Using a Cartesian product decomposition of G called its canonical isometric representation, we show that every Hamming embedding of G may be partitioned into a canonical partition, whose parts provide Hamming embeddings for each factor of the canonical isometric representation of G. This implies that G permits a Hamming embedding if and only if each factor of its canonical isometric representation is Hamming embeddable. This result extends prior work on unweighted graphs that showed that an unweighted graph permits a Hamming embedding if and only if each factor is a complete graph. When a graph G has nontrivial isometric representation, determining whether G has a Hamming embedding can be simplified to checking embeddability of two or more smaller graphs.

Ultrafast dense DNA functionalization of quantum dots and rods for scalable 2D array fabrication with nanoscale precision.

Scalable fabrication of two-dimensional (2D) arrays of quantum dots (QDs) and quantum rods (QRs) with nanoscale precision is required for numerous device applications. However, self-assembly-based fabrication of such arrays using DNA origami typically suffers from low yield due to inefficient QD and QR DNA functionalization. In addition, it is challenging to organize solution-assembled DNA origami arrays on 2D device substrates while maintaining their structural fidelity. Here, we reduced manufacturing time from a few days to a few minutes by preparing high-density DNA-conjugated QDs/QRs from organic solution using a dehydration and rehydration process. We used a surface-assisted large-scale assembly (SALSA) method to construct 2D origami lattices directly on solid substrates to template QD and QR 2D arrays with orientational control, with overall loading yields exceeding 90%. Our fabrication approach enables the scalable, high fidelity manufacturing of 2D addressable QDs and QRs with nanoscale orientational and spacing control for functional 2D photonic devices.

Engineering Exciton Dynamics with Synthetic DNA Scaffolds.

Excitons are the molecular-scale currency of electronic energy. Control over excitons enables energy to be directed and harnessed for light harvesting, electronics, and sensing. Excitonic circuits achieve such control by arranging electronically active molecules to prescribe desired spatiotemporal dynamics. Photosynthetic solar energy conversion is a canonical example of the power of excitonic circuits, where chromophores are positioned in a protein scaffold to perform efficient light capture, energy transport, and charge separation. Synthetic systems that aim to emulate this functionality include self-assembled aggregates, molecular crystals, and chromophore-modified proteins. While the potential of this approach is clear, these systems lack the structural precision to control excitons or even test the limits of their power. In recent years, DNA origami has emerged as a designer material that exploits biological building blocks to construct nanoscale architectures. The structural precision afforded by DNA origami has enabled the pursuit of naturally inspired organizational principles in a highly precise and scalable manner. In this Account, we describe recent developments in DNA-based platforms that spatially organize chromophores to construct tunable excitonic systems. The high fidelity of DNA base pairing enables the formation of programmable nanoscale architectures, and sequence-specific placement allows for the precise positioning of chromophores within the DNA structure. The integration of a wide range of chromophores across the visible spectrum introduces spectral tunability. These excitonic DNA-chromophore assemblies not only serve as model systems for light harvesting, solar conversion, and sensing but also lay the groundwork for the integration of coupled chromophores into larger-scale nucleic acid architectures.We have used this approach to generate DNA-chromophore assemblies of strongly coupled delocalized excited states through both sequence-specific self-assembly and the covalent attachment of chromophores. These strategies have been leveraged to independently control excitonic coupling and system-bath interaction, which together control energy transfer. We then extended this framework to identify how scaffold configurations can steer the formation of symmetry-breaking charge transfer states, paving the way toward the design of dual light-harvesting and charge separation DNA machinery. In an orthogonal application, we used the programmability of DNA chromophore assemblies to change the optical emission properties of strongly coupled dimers, generating a series of fluorophore-modified constructs with separable emission properties for fluorescence assays. Upcoming advances in the chemical modification of nucleotides, design of large-scale DNA origami, and predictive computational methods will aid in constructing excitonic assemblies for optical and computing applications. Collectively, the development of DNA-chromophore assemblies as a platform for excitonic circuitry offers a pathway to identifying and applying design principles for light harvesting and molecular electronics.

Functionalizing DNA origami to investigate and interact with biological systems.

DNA origami has emerged as a powerful method to generate DNA nanostructures with dynamic properties and nanoscale control. These nanostructures enable complex biophysical studies and the fabrication of next-generation therapeutic devices. For these applications, DNA origami typically needs to be functionalized with bioactive ligands and biomacromolecular cargos. Here, we review methods developed to functionalize, purify, and characterize DNA origami nanostructures. We identify remaining challenges, such as limitations in functionalization efficiency and characterization. We then discuss where researchers can contribute to further advance the fabrication of functionalized DNA origami.

Factorization and pseudofactorization of weighted graphs.

For unweighted graphs, finding isometric embeddings of a graph G is closely related to decompositions of G into Cartesian products of smaller graphs. When G is isomorphic to a Cartesian graph product, we call the factors of this product a factorization of G. When G is isomorphic to an isometric subgraph of a Cartesian graph product, we call those factors a pseudofactorization of G. Prior work has shown that an unweighted graph's pseudofactorization can be used to generate a canonical isometric embedding into a product of the smallest possible pseudofactors. However, for arbitrary weighted graphs, which represent a richer variety of metric spaces, methods for finding isometric embeddings or determining their existence remain elusive, and indeed pseudofactorization and factorization have not previously been extended to this context. In this work, we address the problem of finding the factorization and pseudofactorization of a weighted graph G, where G satisfies the property that every edge constitutes a shortest path between its endpoints. We term such graphs minimal graphs, noting that every graph can be made minimal by removing edges not affecting its path metric. We generalize pseudofactorization and factorization to minimal graphs and develop new proof techniques that extend the previously proposed algorithms due to Graham and Winkler [Graham and Winkler, '85] and Feder [Feder, '92] for pseudofactorization and factorization of unweighted graphs. We show that any n-vertex, m-edge graph with positive integer edge weights can be factored in O(m2) time, plus the time to find all pairs shortest paths (APSP) distances in a weighted graph, resulting in an overall running time of O(m2+n2 log log n) time. We also show that a pseudofactorization for such a graph can be computed in O(mn) time, plus the time to solve APSP, resulting in an O(mn+n2 log log n) running time.

Evaluation of Nonmodified Wireframe DNA Origami for Acute Toxicity and Biodistribution in Mice.

Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not been previously characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following a therapeutically relevant dosage of nonmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver and kidney biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.

A synaptic molecular dependency network in knockdown of autism- and schizophrenia-associated genes revealed by multiplexed imaging.

The complex functions of neuronal synapses depend on their tightly interconnected protein network, and their dysregulation is implicated in the pathogenesis of autism spectrum disorders and schizophrenia. However, it remains unclear how synaptic molecular networks are altered biochemically in these disorders. Here, we apply multiplexed imaging to probe the effects of RNAi knockdown of 16 autism- and schizophrenia-associated genes on the simultaneous joint distribution of 10 synaptic proteins, observing several protein composition phenotypes associated with these risk genes. We apply Bayesian network analysis to infer hierarchical dependencies among eight excitatory synaptic proteins, yielding predictive relationships that can only be accessed with single-synapse, multiprotein measurements performed simultaneously in situ. Finally, we find that central features of the network are affected similarly across several distinct gene knockdowns. These results offer insight into the convergent molecular etiology of these widespread disorders and provide a general framework to probe subcellular molecular networks.

Evaluation of non-modified wireframe DNA origami for acute toxicity and biodistribution in mice.

Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not previously been characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following therapeutically relevant dosage of unmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.

3D RNA-scaffolded wireframe origami.

Hybrid RNA:DNA origami, in which a long RNA scaffold strand folds into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of three-dimensional wireframe RNA-scaffolded origami rendered as polyhedra composed of dual-duplex edges. We computationally design, fabricate, and characterize tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds. We characterize secondary and tertiary structures using dimethyl sulfate mutational profiling and cryo-electron microscopy, revealing insight into both global and local, base-level structures of origami. Our top-down sequence design strategy enables the use of long RNAs as functional scaffolds for complex wireframe origami.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds.

Multivalent antigen display is a well-established principle to enhance humoral immunity. Protein-based virus-like particles (VLPs) are commonly used to spatially organize antigens. However, protein-based VLPs are limited in their ability to control valency on fixed scaffold geometries and are thymus-dependent antigens that elicit neutralizing B cell memory themselves, which can distract immune responses. Here, we investigated DNA origami as an alternative material for multivalent antigen display in vivo, applied to the receptor binding domain (RBD) of SARS-CoV2 that is the primary antigenic target of neutralizing antibody responses. Icosahedral DNA-VLPs elicited neutralizing antibodies to SARS-CoV-2 in a valency-dependent manner following sequential immunization in mice, quantified by pseudo- and live-virus neutralization assays. Further, induction of B cell memory against the RBD required T cell help, but the immune sera did not contain boosted, class-switched antibodies against the DNA scaffold. This contrasted with protein-based VLP display of the RBD that elicited B cell memory against both the target antigen and the scaffold. Thus, DNA-based VLPs enhance target antigen immunogenicity without generating off-target, scaffold-directed immune memory, thereby offering a potentially important alternative material for particulate vaccine design.

Innate Immune Stimulation Using 3D Wireframe DNA Origami.

Three-dimensional wireframe DNA origami have programmable structural and sequence features that render them potentially suitable for prophylactic and therapeutic applications. However, their innate immunological properties, which stem from parameters including geometric shape and cytosine-phosphate-guanine dinucleotide (CpG) content, remain largely unknown. Here, we investigate the immunostimulatory properties of 3D wireframe DNA origami on the TLR9 pathway using both reporter cell lines and primary immune cells. Our results suggest that bare 3D polyhedral wireframe DNA origami induce minimal TLR9 activation despite the presence of numerous internal CpG dinucleotides. However, when displaying multivalent CpG-containing ssDNA oligos, wireframe DNA origami induce robust TLR9 pathway activation, along with enhancement of downstream immune response as evidenced by increases in Type I and Type III interferon (IFN) production in peripheral blood mononuclear cells. Further, we find that CpG copy number and spatial organization each contribute to the magnitude of TLR9 signaling and that NANP-attached CpGs do not require phosphorothioate stabilization to elicit signaling. These results suggest key design parameters for wireframe DNA origami that can be programmed to modulate immune pathway activation controllably for prophylactic and therapeutic applications.

Activating charge-transfer state formation in strongly-coupled dimers using DNA scaffolds.

Strongly-coupled multichromophoric assemblies orchestrate the absorption, transport, and conversion of photonic energy in natural and synthetic systems. Programming these functionalities involves the production of materials in which chromophore placement is precisely controlled. DNA nanomaterials have emerged as a programmable scaffold that introduces the control necessary to select desired excitonic properties. While the ability to control photophysical processes, such as energy transport, has been established, similar control over photochemical processes, such as interchromophore charge transfer, has not been demonstrated in DNA. In particular, charge transfer requires the presence of close-range interchromophoric interactions, which have a particularly steep distance dependence, but are required for eventual energy conversion. Here, we report a DNA-chromophore platform in which long-range excitonic couplings and short-range charge-transfer couplings can be tailored. Using combinatorial screening, we discovered chromophore geometries that enhance or suppress photochemistry. We combined spectroscopic and computational results to establish the presence of symmetry-breaking charge transfer in DNA-scaffolded squaraines, which had not been previously achieved in these chromophores. Our results demonstrate that the geometric control introduced through the DNA can access otherwise inaccessible processes and program the evolution of excitonic states of molecular chromophores, opening up opportunities for designer photoactive materials for light harvesting and computation.

Nanoscale 3D spatial addressing and valence control of quantum dots using wireframe DNA origami.

Control over the copy number and nanoscale positioning of quantum dots (QDs) is critical to their application to functional nanomaterials design. However, the multiple non-specific binding sites intrinsic to the surface of QDs have prevented their fabrication into multi-QD assemblies with programmed spatial positions. To overcome this challenge, we developed a general synthetic framework to selectively attach spatially addressable QDs on 3D wireframe DNA origami scaffolds using interfacial control of the QD surface. Using optical spectroscopy and molecular dynamics simulation, we investigated the fabrication of monovalent QDs of different sizes using chimeric single-stranded DNA to control QD surface chemistry. By understanding the relationship between chimeric single-stranded DNA length and QD size, we integrated single QDs into wireframe DNA origami objects and visualized the resulting QD-DNA assemblies using electron microscopy. Using these advances, we demonstrated the ability to program arbitrary 3D spatial relationships between QDs and dyes on DNA origami objects by fabricating energy-transfer circuits and colloidal molecules. Our design and fabrication approach enables the geometric control and spatial addressing of QDs together with the integration of other materials including dyes to fabricate hybrid materials for functional nanoscale photonic devices.

Controlling Nuclease Degradation of Wireframe DNA Origami with Minor Groove Binders.

Viruslike particles (VLPs) fabricated using wireframe DNA origami are emerging as promising vaccine and gene therapeutic delivery platforms due to their programmable nature that offers independent control over their size and shape, as well as their site-specific functionalization. As materials that biodegrade in the presence of endonucleases, specifically DNase I and II, their utility for the targeting of cells, tissues, and organs depends on their stability in vivo. Here, we explore minor groove binders (MGBs) as specific endonuclease inhibitors to control the degradation half-life of wireframe DNA origami. Bare, unprotected DNA-VLPs composed of two-helix edges were found to be stable in fetal bovine serum under typical cell culture conditions and in human serum for 24 h but degraded within 3 h in mouse serum, suggesting species-specific endonuclease activity. Inhibiting endonucleases by incubating DNA-VLPs with diamidine-class MGBs increased their half-lives in mouse serum by more than 12 h, corroborated by protection against isolated DNase I and II. Our stabilization strategy was compatible with the functionalization of DNA-VLPs with HIV antigens, did not interfere with B-cell signaling activity of DNA-VLPs in vitro, and was nontoxic to B-cell lines. It was further found to be compatible with multiple wireframe DNA origami geometries and edge architectures. MGB protection is complementary to existing methods such as PEGylation and chemical cross-linking, offering a facile protocol to control DNase-mediated degradation rates for in vitro and possibly in vivo therapeutic and vaccine applications.