DNA Origami Scaffolds Research Articles

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.

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.

Nanoscale organization of two-dimensional multimeric pMHC reagents with DNA origami for CD8+ T cell detection

Peptide-MHC (pMHC) multimers have excelled in the detection of antigen-specific T cells and have allowed phenotypic analysis using other reagents, but their use for detection of low-affinity T cells remains a challenge. Here we develop a multimeric T cell identifying reagent platform using two-dimensional DNA origami scaffolds to spatially organize pMHCs (termed as dorimers) with nanoscale control. We show that these dorimers enhance the binding avidity for low-affinity antigen-specific T cell receptors (TCRs). The dorimers are able to detect more antigen-specific T cells in mouse CD8+ T cells and early-stage CD4+CD8+ double-positive thymocytes that express less dense TCRs, compared with the equivalent tetramers and dextramers. Moreover, we demonstrate dorimer function in the analysis of autoimmune CD8+ T cells that express low-affinity TCRs, which are difficult to detect using tetramers. We anticipate that dorimers could contribute to the investigation of antigen-specific T cells in immune T cell function or immunotherapy applications.

Acceleration of DNA Hybridization Chain Reactions on 3D Nanointerfaces of Magnetic Particles and Their Direct Application in the Enzyme-Free Amplified Detection of microRNA.

Accelerated DNA hybridization chain reactions (HCRs) using DNA origami as a scaffold have received considerable attention in dynamic DNA nanotechnology. However, tailor-made designs are essential for DNA origami scaffolds, hampering the practical application of accelerated HCRs. Here, we constructed the semilocalized HCR and localized HCR systems using magnetic beads (MBs) as a simple scaffold to explore them for the enzyme-free miR-21 detection. The semilocalized HCR system relied on free diffusing one hairpin DNA and MBs immobilized with another hairpin DNA, and the localized HCR system relied on MBs coimmobilized with two hairpin DNAs. We demonstrated that the DNA density on MBs plays a critical role in HCR kinetics and limit of detection (LOD). Among semilocalized HCR systems, MBs with a medium DNA density showed a faster HCR and lower LOD (10 pM) than the diffusive (conventional) HCR system (LOD: 86 pM). In contrast, the HCR further accelerated for the localized HCR systems as the DNA density increased. The localized HCR system with the highest DNA density showed the fastest HCR and the lowest LOD (533 fM). These findings are of great importance for the rational design of accelerated HCRs using simple scaffolds for practical applications.

A dynamic Dab2 keeps myosin VI stably on track

Myosins are actin-based motor proteins known to perform a variety of different mechanical tasks in cells including transporting cargo, generating tension, and linking the cytoskeleton and membrane. Myosins that function as transporters often form complexes with adaptor proteins and vesicular membranes, making it unclear how they transport their cargo through the actin cytoskeletal network. Rai et al. now use single-molecule kinetics, FRET, and DNA origami scaffolds that mimic motor–adaptor complexes to reveal that the myosin VI-Dab2 complex, which is held together weakly and turns over rapidly, can facilitate processive transport without disruption of the cytoskeleton.

Dynamic multimerization of Dab2–Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s−1) of the Dab2 MIR–myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 μM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and “stop-and-go” motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR–myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor–cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies.

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.

Atomistic insight into sequence-directed DNA bending and minicircle formation propensity in the absence and presence of phased A-tracts.

Bending of double-stranded (ds) DNA plays a crucial role in many important biological processes and is relevant for nanotechnological applications. Among all the elements that have been studied in relation to dsDNA bending, A-tracts stand out as one of the most controversial. The "ApA wedge" theory was disproved when a series of linear polynucleotides containing phased 5'-A4T4-3' or 5'-T4A4-3' runs were shown to be bent or straight, respectively, and crystallographic evidence revealed that A-tracts are unbent. Furthermore, some of the smallest dsDNA minicircles described to date (~ 100bp in size) lack A-tracts and are subjected to varying levels of torsional stress. Representative DNA sequences from this experimental background were modeled in atomic detail and their dynamic behavior was simulated over hundreds of nanoseconds using the AMBER force field ParmBSC1. Subsequent analysis of the resulting trajectories allowed us to (i) unambiguously establish the location of the bends in all cases; (ii) identify the structural elements that are directly responsible for the macroscopically detected curvature; and (iii) reveal the importance not only of coherently summing the effects of the bending elements when they are in synchrony with the natural repeat of the helix (i.e. separated by an integral number of helical turns) but also when alternated with a half-integral separation of opposite effects. We conclude that the major determinant of the macroscopically observed bending is the proper grouping and phasing of the positive roll imposed by pyrimidine-purine (YR) steps and the negative or null roll characteristic of RY steps and A-tracts, respectively. This conclusion is in very good agreement with the structural parameters experimentally derived for much smaller DNA molecules either alone or as found in DNA-protein complexes. We expect that this work will pave the way for future studies on drug-induced DNA bending, DNA shape readout by transcription factors, structure of circular extrachromosomal DNA, and custom design of curved DNA origami scaffolds.

Active generation of nanoholes in DNA origami scaffolds for programmed catalysis in nanocavities

DNA origami tiles provide nanostructures for the spatial and temporal control of functional loads on the scaffolds. Here we introduce the active generation of nanoholes in the origami scaffolds using DNAzymes or light as triggers and present the programmed and switchable catalysis in the resulting nanocavities. We engineer “window” domains locked into the origami scaffolds by substrates of the Zn2+-ion- or Pb2+-ion-dependent DNAzymes. Using Zn2+ ions and/or Pb2+ ions, the programmed unlocking of the “window” domains is demonstrated. The tailored functionalization of the origami scaffolds allows the programmed operation of catalytic processes in the confined nanocavities. Also, the “window” domain is integrated into the origami scaffold using photoisomerizable azobenzene-modified locks. The cyclic photoisomerization of the locks between the cis and trans states leads to a reversible opening and closure of the nanoholes and to the cyclic light-induced switching of catalytic processes in the nanocavities.

Vesicle constriction by rings of Janus nanoparticles and aggregates of curved proteins

Membrane constriction and associated scission by proteins and nano structures are crucial to many processes in cellular and synthetic biology. We report mechanical constriction of vesicles by rings of adsorbed Janus nanoparticles that represent synthetic nano structures and mimic contractile proteins, and by aggregates of curved crescents that mimic scaffold proteins. Membrane energetics from Monte Carlo simulations and simulated annealing of the elastic membrane model confirms spontaneous vesicle constriction by aggregates of sufficiently-curved crescents of various lengths and by rings of Janus nanoparticles with a variety of ring lengths, particle sizes, and particle area fractions. We show that shorter rings of smaller particles with higher area fractions reinforce the constriction by increasing the energetic drive towards the constricted vesicle with smaller constriction radius. We demonstrate that vesicle constriction by crescent aggregates strongly depends on the crescent curvature. In contrast to aggregates of sufficiently-curved crescents that are capable of inducing full vesicle constriction, those of near flat crescents with negligible curvature leave the vesicle unconstricted. Our results offer promising perspectives for designing membrane-constricting nano structures such as nanoparticle aggregates and clusters of synthetic curved proteins such as DNA origami scaffolds with applications in synthetic biology. Our findings reveal the significant contribution of highly-curved F-BAR domains to cell division and explain how contractile protein rings such as dynamin GTPase, actomyosin rings, and endosomal sorting complexes required for transport constrict the membrane.

Complex silica composite nanomaterials templated with DNA origami.

Genetically encoded protein scaffolds often serve as templates for the mineralization of biocomposite materials with complex yet highly controlled structural features that span from nanometres to the macroscopic scale1-4. Methods developed to mimic these fabrication capabilities can produce synthetic materials with well defined micro- and macro-sized features, but extending control to the nanoscale remains challenging5,6. DNA nanotechnology can deliver a wide range of customized nanoscale two- and three-dimensional assemblies with controlled sizes and shapes7-11. But although DNA has been used to modulate the morphology of inorganic materials12,13 and DNA nanostructures have served as moulds14,15 and templates16,17, it remains challenging to exploit the potential of DNA nanostructures fully because they require high-ionic-strength solutions to maintain their structure, and this in turn gives rise to surface charging that suppresses the material deposition. Here we report that the Stöber method, widely used for producing silica (silicon dioxide) nanostructures, can be adjusted to overcome this difficulty: when synthesis conditions are such that mineral precursor molecules do not deposit directly but first form clusters, DNA-silica hybrid materials that faithfully replicate the complex geometric information of a wide range of different DNA origami scaffolds are readily obtained. We illustrate this approach using frame-like, curved and porous DNA nanostructures, with one-, two- and three-dimensional complex hierarchical architectures that range in size from 10 to 1,000 nanometres. We also show that after coating with an amorphous silica layer, the thickness of which can be tuned by adjusting the growth time, hybrid structures can be up to ten times tougher than the DNA template while maintaining flexibility. These findings establish our approach as a general method for creating biomimetic silica nanostructures.

Placement of Single Proteins within the SERS Hot Spots of Self-Assembled Silver Nanolenses.

This study demonstrates the bottom-up synthesis of silver nanolenses. A robust coating protocol enabled the functionalization of differently sized silver nanoparticles with DNA single strands of orthogonal sequence. Coated particles 10 nm, 20 nm, and 60 nm in diameter were self-assembled by DNA origami scaffolds to form silver nanolenses. Single molecules of the protein streptavidin were selectively placed in the gap of highest electric field enhancement. Streptavidin labelled with alkyne groups served as model analyte in surface-enhanced Raman scattering (SERS) experiments. By correlated Raman mapping and atomic force microscopy, SERS signals of the alkyne labels of a single streptavidin molecule, from a single silver nanolens, were detected. The discrete, self-similar aggregates of solid silver nanoparticles are promising for plasmonic applications.

Membrane sculpting by curved DNA origami scaffolds

Membrane sculpting and transformation is essential for many cellular functions, thus being largely regulated by self-assembling and self-organizing protein coats. Their functionality is often encoded by particular spatial structures. Prominent examples are BAR domain proteins, the ‘banana-like’ shapes of which are thought to aid scaffolding and membrane tubulation. To elucidate whether 3D structure can be uncoupled from other functional features of complex scaffolding proteins, we hereby develop curved DNA origami in various shapes and stacking features, following the presumable design features of BAR proteins, and characterize their ability for membrane binding and transformation. We show that dependent on curvature, membrane affinity and surface density, DNA origami coats can indeed reproduce the activity of membrane-sculpting proteins such as BAR, suggesting exciting perspectives for using them in bottom-up approaches towards minimal biomimetic cellular machineries.

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels.

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K+ channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K+ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels

AbstractIn native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K+ channel proteins are assembled through zinc‐finger protein (ZFP)‐adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP‐fused Kir3 channels and ZFP‐based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K+ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.

Construction of a novel phagemid to produce custom DNA origami scaffolds

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.

DNA computing: Spatially localized DNA domino.

Fast and scalable molecular logic circuits have been constructed by spatially localizing DNA hairpins on DNA origami scaffolds.

Designing Uniquely Addressable Bio-orthogonal Synthetic Scaffolds for DNA and RNA Origami.

Nanotechnology and synthetic biology are rapidly converging, with DNA origami being one of the leading bridging technologies. DNA origami was shown to work well in a wide array of biotic environments. However, the large majority of extant DNA origami scaffolds utilize bacteriophages or plasmid sequences thus severely limiting its future applicability as a bio-orthogonal nanotechnology platform. In this paper we present the design of biologically inert (i.e., "bio-orthogonal") origami scaffolds. The synthetic scaffolds have the additional advantage of being uniquely addressable (unlike biologically derived ones) and hence are better optimized for high-yield folding. We demonstrate our fully synthetic scaffold design with both DNA and RNA origamis and describe a protocol to produce these bio-orthogonal and uniquely addressable origami scaffolds.

Cuboid Vesicles Formed by Frame‐Guided Assembly on DNA Origami Scaffolds

AbstractWe describe the use of a frame‐guided assembly (FGA) strategy to construct cuboid and dumbbell‐shaped hetero‐vesicles on DNA origami nanostructure scaffolds. These are achieved by varying the design of the DNA origami scaffolds that direct the distribution of the leading hydrophobic groups (LHG). By careful selection of LHGs, different types of amphiphiles (both polymer and small‐molecule surfactants) were guided to form hetero‐vesicles, demonstrating the versatility of the FGA strategy and its potential to construct asymmetric and dynamic hetero‐vesicle assemblies with complex DNA nano‐scaffolds.

Cuboid Vesicles Formed by Frame-Guided Assembly on DNA Origami Scaffolds.

We describe the use of a frame-guided assembly (FGA) strategy to construct cuboid and dumbbell-shaped hetero-vesicles on DNA origami nanostructure scaffolds. These are achieved by varying the design of the DNA origami scaffolds that direct the distribution of the leading hydrophobic groups (LHG). By careful selection of LHGs, different types of amphiphiles (both polymer and small-molecule surfactants) were guided to form hetero-vesicles, demonstrating the versatility of the FGA strategy and its potential to construct asymmetric and dynamic hetero-vesicle assemblies with complex DNA nano-scaffolds.