DNA origami: Interrogating the nano-landscape of immune receptor activation

Abstract

The immune response is orchestrated by elaborate protein interaction networks that interweave ligand-mediated receptor reorganization with signaling cascades. While the biochemical processes have been extensively investigated, delineating the biophysical principles governing immune receptor activation has remained challenging due to design limitations of traditional ligand display platforms. These constraints have been overcome by advances in DNA origami nanotechnology, enabling unprecedented control over ligand geometry on configurable scaffolds. It is now possible to systematically dissect the independent roles of ligand stoichiometry, spatial distribution, and rigidity in immune receptor activation, signaling, and cooperativity. In this review, we highlight pioneering efforts in manipulating the ligand presentation landscape to understand immune receptor triggering and to engineer functional immune responses.

Introduction

The immune system is an intricate network of lymphoid organs and vessels spanning the human body. As a result of its vast spatial distribution, it has evolved sophisticated signaling pathways mediated by receptor-ligand interactions to control cellular responses and cell fate. This orchestration then dictates the nature of the immune response mounted against invading pathogens or altered self, such as cancer. However, aberrations in the system can contribute to the development of autoimmune disorders, such as multiple sclerosis and type I diabetes. Given the crucial role of ligand-promoted receptor-mediated signaling in maintaining a balance between immunity and tolerance (1), the delineation of the spatiotemporal dynamics of receptor-ligand interactions and their subsequent translation into functional responses has become a key area of study. This is driven by critical technological advancements that allow for the interrogation of the nanoscale events underlying immune receptor activation.

Improvements in live-cell fluorescence imaging facilitated the development of the current receptor activation paradigm, wherein ligand engagement triggers large-scale spatial reorganization, with clustering and exclusion of various immune receptors, coreceptors, and adhesion molecules dictating downstream signaling events (2,3). Notably, utilizing ligand display platforms like polymers, metal nanoparticles, viral-like particles, and artificial planar lipid bilayers (PLBs), studies have established that ligand properties, including affinity, valency, mobility, and spatial patterning influence immune cell signaling, activation, and effector function (4,5,6,7). However, as these platforms lack systematic control over spatial parameters of ligand presentation, there remains a distinct lack of understanding of the precise nanoscale requirements that underlie immune receptor activation, and therefore are critical design parameters for engineering immune-modulating therapeutics such as vaccines. To this end, advances in synthetic materials have been employed to enable nanoscale manipulation of receptor-ligand interactions (8). In particular, the development of DNA origami (9) has proven instrumental in delineating the biophysical aspects of immune receptor triggering and engineering immune cell function (10,11,12,13,14,15,16,17,18).

Here, we discuss key techniques and design considerations in DNA origami and highlight emerging studies that systematically manipulate ligand parameters like rigidity, valency, and spacing to dissect the molecular requirements of immune receptor activation.

DNA origami: Assembly, functionalization, and characterization

In the early 2000s, a series of works introduced DNA as a structural material that can self-assemble into nanomachines of various 2D and 3D structures with high yield (19,20,21). This paved the way to the seminal publication by Rothemund in 2006 in which he coined the term DNA origami and demonstrated its versatility in constructing nanostructures of varying shapes and sizes (9). Assembly, as comprehensively reviewed elsewhere (22), involves the folding of a long single-stranded “scaffold” (commonly derived from the M13 phage genome) by many short synthetic single-stranded oligonucleotide “staples” (Fig. 1 A). Through thermal annealing in magnesium-containing buffers, each staple hybridizes to two or more distant regions in the scaffold, creating strand crossovers to stabilize the overall structure, which can take on a variety of 2D or 3D geometries (9,23). Individual dimensions are constrained by the length of the scaffold, but higher-order superstructures can be created via hierarchical assembly (24).

Figure 1.

DNA origami assembly, functionalization, and deposition. (A) DNA origami is assembled into user-defined shapes by numerous staples that bind two or more regions of the scaffold, creating stabilizing crossover points. (B) Ligand-of-interest can be functionalized post assembly via handle-antihandle, PNA, biotin-streptavidin, DBCO, aptamer, and SNAP tag strategies. (C) DNA nanostructures can be integrated into membrane systems using hydrophobic, electrostatic, and biotin-streptavidin interactions. Image created in BioRender.

The power of DNA origami resides in its addressability, where staples with known positions can be modified to achieve site-specific ligand functionalization through various chemical and enzymatic methods detailed elsewhere (25) (Fig. 1 B). For example, fluorophores (11,15), lipids (10), small molecules (11,15), and oligonucleotide motifs (16,26) are easily attached to select staples using phosphoramidite chemistry for direct incorporation during origami assembly. Alternatively, the most common post-assembly functionalization approach incorporates single-stranded overhang “handles” in staples, extending from the assembled structure. Handles can then hybridize with complementary “anti-handle” oligonucleotides bearing the ligand-of-interest (13,27,28). However, nanoscale spatial resolution can be compromised with increasing handle length, which is often incorporated to improve the thermodynamic stability of the assembled DNA origami. Another similar, albeit more expensive technique utilizes DNA-binding molecules like peptide nucleic acids (PNA) to attach to overhangs with remarkably high affinity (14). A more accessible strategy involves covalently installing biotin to staples for high-affinity orthogonal interactions with streptavidin (SA), which binds biotinylated ligands to create a more rigid structure (10,12,27). However, given the tetravalent nature of wild-type SA, additional modifications are required to achieve controlled stoichiometry. Moreover, the large size of SA poses spatial constraints that limit functionalization to a fewer number of ligands. Alternatively, dibenzocyclooctyne (DBCO) can be attached to select staples for binding azide-modified ligands (13); and aptamers can be incorporated for selective binding to target molecules (18). Finally, protein tags like SNAP can covalently link ligands to origami platforms, providing greater stability and spatial resolution, albeit sacrificing functionalization efficiency (11). Altogether, the ability to differentially modify select staples renders DNA origami as molecular nanogrids capable of displaying multiple ligands with precise nanoscale distribution.

Beyond functionalization efficacy, the potential impact of design choices on ligand functionality, and consequently cell activation, is of great importance. In a comparative analysis, handle-antihandle strategies resulted in decreased T cell activation compared with PNA and biotin-SA approaches, potentially due to the negatively charged DNA linker impairing T cell receptor (TCR) binding (29). In addition to charge interactions, reduced linker flexibility has been shown to be critical in maintaining ligand spatial rigidity required to discern differences in immune receptor triggering imposed by ligand organization (14,16). Moreover, the importance of ligand height imparted by linker length cannot be understated in the context of receptor-ligand interactions underlying contact-dependent cell-cell communication, where the intermembrane distance may play a critical role in modulating the reorganization of surface molecules. For example, the kinetic-segregation model of TCR triggering implicates the steric exclusion of large phosphatases from closely juxtaposed membrane regions of ligand engagement (30). Indeed, the height of DNA tethers (albeit not in origami structures) has been shown to modulate TCR triggering, where increasing tether length resulted in decreased TCR signaling (31). While ligand height has not been systematically varied in the context of DNA origami, with axial dimensions largely unreported in published designs, varying dimensions imparted by different functionalization strategies present yet another modifiable parameter. In combination with platform and linker flexibility, the impact of overall origami design on functional interface dimensions remains largely uncharacterized in the immunological context. Nonetheless, as seen in nanophotonic applications (32), DNA origami as a revolutionary bottom-up 3D nanofabrication technique allows for unprecedented nanoscale control previously eluding traditional lithography techniques. As DNA origami technology advances toward biomedical application, comprehensive analyses of production efficiency, functionality, lateral and axial dimensions, targeting, and immunogenic properties will prove critical in informing the design paradigm.

As control over stoichiometry and spatial parameters is critical, characterization techniques that can analyze functionalization efficiency and quantify interligand spacing are indispensable. Various visual-based characterization techniques can be used to generate super-resolution images of origami structures to analyze dimensions and quantify the number of ligands and their distribution. For example, atomic force microscopy (AFM) (13) and transmission electron microscopy can visualize protein-functionalized sites by height and contrast differences, respectively, but they are insensitive to small conjugates and often require sample adsorption or dehydration on grids. As such, they are often accompanied by analysis using DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), which utilizes fluorescently labeled oligonucleotides to visualize available addressable sites (33). This enables the evaluation of native origami under aqueous conditions, which becomes pertinent in assessing their nanogeometries when integrated into membrane systems to evaluate cell-cell interactions.

Mimicking cell-cell interactions within membranes

In vivo, many immune receptors signal through juxtacrine cell-cell interactions via the engagement of membrane-associated ligands, which has traditionally been mimicked in vitro using synthetic lipid bilayer systems, including substrate-supported planar lipid bilayers (PLBs) and liposomes. Integration of DNA origami into these systems has been achieved using various techniques reviewed elsewhere (34), allowing for controlled manipulation of membrane-associated ligands on interaction interfaces (Fig. 1 C). The most common strategy exploits hydrophobic molecules like cholesterol for anchorage within lipid membranes (35). Electrostatic interactions have also been employed where zwitterionic lipids, such as phosphatidylcholine, can electrostatically engage DNA origami in the presence of divalent cations like magnesium (36). Alternatively, biotin-functionalized lipids can anchor SA-functionalized DNA origami (37). Notably, the choice of deposition strategy, the number of hydrophobic anchors, the size of the origami, buffer ionic strength, and bilayer composition can influence deposition efficiency (36,38,39).

Another critical consideration in choosing an appropriate deposition method is the lateral mobility of origami platforms within the lipid bilayer. The ability for membrane-associated ligands to freely diffuse has been known to impact immune receptor signaling strength, duration, and functional outcomes by modulating the concomitant reorganization of receptor-ligand complexes. For example, decreased ligand mobility is associated with diminished B cell receptor (BCR) (40) and TCR (41) signaling, demonstrating the importance of characterizing the mobility of membrane-integrated origami platforms, which exhibit average diffusion coefficients ranging from 0.2 to 3.4 μm² s⁻¹ (38,42,43), comparable with that of plasma membrane-associated proteins (44). However, whether changes in platform diffusivity impact immune receptor triggering remains an unexplored avenue of study.

Excitingly, DNA origami has also been successfully and reversibly attached to the membrane of living cells, including human fibroblasts, epithelial cells, endothelial cells, leukemic cells, and mouse lymphoma B cells (45). This opens the avenue to more closely mimic in vivo receptor-ligand interactions within heterogeneous and complex membranes.

DNA origami-based interrogation of receptor signaling

The spatial manipulation of immune receptor signaling by DNA nanotechnology emerged in 2007, using DNA dendrimers constructed based on similar design principles as DNA origami (46). To investigate the structural constraints in signal initiation by IgE-bound Fc epsilon receptor I (FcεRI) in mast cells, Sil and colleagues engineered trivalent ligands using Y-shaped DNA spacers to anchor 2,4-dinitrophenyl haptens at varying distances (5–15 nm) apart. Stimulation of rat basophilic leukemia cells revealed distance-dependent FcεRI signaling, with shorter-spaced trivalent ligands (5–10 nm) yielding greater degranulation. Interestingly, at the highest ligand concentration tested, the shortest trivalent ligand triggered reduced degranulation, likely due to increased monovalent binding and thus reduced cross-linking of FcεRI.

While Sil and colleagues correctly predicted the popularized use of DNA nanotechnology in analyzing immune receptor signaling, utilization of Rothemund’s DNA origami in the field would not surface until well over a decade later. Instead, DNA origami’s value as an emerging spatial-manipulation tool in nanoengineering receptor activation was initially established by studies on non-immune receptor triggering. In 2014, Shaw et al. designed DNA origami nanorods to probe the effect of ligand spacing on ephrin receptor Eph2A activation in human breast cancer cells (47). The authors established that lower interligand distance (40 vs. 100 nm) enhanced EphA2 activation and reduced cancer cell invasiveness. In a separate study, Angelin et al. (48) displayed varying numbers (4–12) of epidermal growth factor ligands either sparsely distributed or closely clustered on rectangular DNA origami nanogrids. Their results suggested that increasing ligand stoichiometry and spacing yielded greater epidermal growth factor receptor activation in breast cancer cells.

Initially, the focus of DNA origami as an immunological tool was directed toward its applicability as a drug delivery vehicle with high loading efficiency and biocompatibility (49). Thus, systematic tuning of spatial parameters was overshadowed by a greater need to establish immunogenic properties and cell targeting capabilities. Nevertheless, these studies demonstrated the promising translatability of DNA origami as nanotherapeutics, with highlights in the field demonstrating enhanced Toll-like receptor 9 (TLR9)-dependent cytokine production by CpG-functionalized nanotubes (50) and tetrahedrons (51) with no detectable toxicity in vitro. DNA origami nanotubes were more recently developed as pH-responsive nanovaccines to deliver TLR agonists and tumor antigens to dendritic cell endosomes, resulting in TLR pathway activation and tumor antigen peptide presentation on major histocompatibility complex (MHC) class I that subsequently activated CD8+ T cells to mediate tumor killing in vivo (52). This study, among many, highlights the shift from empirical to rational design principles. Unraveling the spatial parameters of immune receptor activation will undoubtedly prove a critical component of this changing landscape.

Ligand nanogeometries in TCR signaling: From native triggering to adoptive T cell therapy

T cells are extensively studied given their diverse roles as regulatory T cells, helper CD4+ T cells, and cytotoxic CD8+ T cells. Naive T cell activation is initiated by the concurrent triggering of TCR and costimulatory receptor CD28 by peptide-MHC (pMHC) and B7 proteins, respectively, on the surface of antigen-presenting cells (APCs). Upon recognition of pMHCI and pMHCII by CD8+ and CD4+ T cells, respectively, pMHC-bound TCRs reorganize into microclusters surrounded by lymphocyte function-associated antigen 1 (LFA-1) adhesion molecules, with selective exclusion of larger inhibitory coreceptors like CD45 (53). Early TCR signaling events, detailed elsewhere (54), include the phosphorylation of activation motifs in the TCR-CD3 complex by lymphocyte-specific protein tyrosine kinase Lck, which recruits various signaling effectors, like the zeta-chain-associated protein Zap70 kinase. Increases in cytosolic calcium then drive the translocation of transcription factors like nuclear factor of activated T cells (NFAT) and extracellular-signal-regulated kinase (ERK) to direct proliferation and differentiation.

Traditionally, in vitro studies of the spatiotemporal dynamics of TCR activation utilized synthetic supporting substrates like silica and PLBs reconstituted with TCR and coreceptor ligands and adhesion molecules, which are tracked using high-resolution fluorescence imaging. Characterization of the contributions of receptor cluster size, composition, and dynamics was instrumental in developing our current understanding of T cell activation. Building off these studies, DNA origami nanostructures have recently been incorporated into similar in vitro reconstitution surfaces to probe the effects of nanoscale spatial organization on TCR and coreceptor signaling.

Notably, TCRs exhibit remarkable sensitivity for cognate pMHC despite transient low-affinity binding and the numerous nonstimulatory endogenous pMHCs on APCs (55). One proposed theory posits that TCR and pMHC present as higher-order structures on the cell surface, evidenced by blue native gel electrophoresis of T cell lysates (56), electron microscopy studies visualizing the T cell surface (57,58), and enhanced TCR clustering induced by preclustered high-affinity anti-TCR antibodies (59). However, this is contradicted by Förster resonance energy transfer (FRET) and fluorescence-based single-molecule imaging studies in live T cells that revealed monomeric TCRs on the cell surface (60), spatially segregated pMHCs within signaling microclusters (60), and TCR activation by singular pMHCs (61). Given the evidence against higher-order ligand organization dictating TCR sensitivity, Hellmeier et al. hypothesized that the spatial proximity of triggered TCRs, rather than pMHCs, is the underlying modulator of TCR sensitivity. To provide evidence, they engineered rectangular DNA origami nanogrids anchored by cholesterol to PLBs to engage primary murine CD4+ T cells (Fig. 2 A) (10). Using biotin-divalent SA interactions, the authors conjugated either high-affinity anti-TCR single-chain antibody fragments (scFv) or low-affinity cognate pMHCs to nanogrids with defined separation distances. Stimulation with scFvs spaced 20 nm apart or less enhanced intracellular calcium and Zap70 recruitment, while increasing the inter-scFv distance to 30 and 48 nm increased the activation threshold, defined as the ligand density required for 50% activated cells. In stark contrast, low-affinity pMHCs with a separation distance of 20 nm or more stimulated robust activation and reducing inter-pMHC spacing to 10 nm increased the activation threshold. Hellmeier et al. proposed a serial triggering model where TCR signaling requires the assembly of a minimum signaling-competent unit composed of two TCRs within 20 nm, which can be formed by either stable binding of closely spaced high-affinity ligands or by a single low-affinity pMHC transiently binding multiple TCRs.

Figure 2.

Manipulating ligand parameters with DNA origami to study TCR signaling. (A) Schematic diagram of experimental set-up with two distance-altering strategies: two ligands (scFv or pMHC) with varying interligand distances on the same rectangular nanogrid or a single ligand on different nanogrids of varying sizes (20 × 30, 54 × 65, or 70 × 100 nm) to impose different nearest-neighbor distances (10). Increasing scFv distance, but not pMHC, beyond 20 nm resulted in decreased TCR signaling. (B) Schematic of a modified CAR-T cell system interacting with rectangular nanogrids functionalized with varying numbers of ligands or four ligands with varying interligand distances (11). Increasing ligand valency resulted in premature termination of ERK signaling. Increasing ligand spacing resulted in decreased ERK activation and slower ERK response. (C) Schematic of aAPCs functionalized with anti-CD28 and varying numbers of pMHC, with each SA site accommodating up to three biotinylated ligands (12). Increasing valency on soluble aAPCs (left) resulted in increased T cell activation and anti-tumor activity and increasing valency on membrane-aAPCs (right) increased TCR binding and dwell time. Image created in BioRender.

In a complementary study, Dong et al. employed a chimeric antigen receptor (CAR) system where the ectodomain of the TCR-like CAR is replaced with a SNAP tag ligated to an oligonucleotide receptor strand (Fig. 2 B) (11). This modification retains normal TCR signaling but allows the CAR to engage complementary oligonucleotides whose binding affinities can be adjusted by altering the number of hybridizing bases. Varying numbers of high-affinity ligands (0–72) were clustered on rectangular DNA origami nanogrids anchored to bovine serum albumin (BSA)-coated glass by biotin-SA interactions. At densities of up to two origami per square micrometer, one- and two-ligand DNA nanogrids did not induce T cell activation as measured by ERK activation, whereas four-ligand nanogrids evoked ERK activation in approximately 30% of cells, suggesting that ligand clustering improves signaling sensitivity. However, further increasing ligand occupancy decreased ERK translocation to the nucleus and induced earlier termination of signaling in CAR-expressing Jurkat cells (human CD4+ T-cell lymphoma), implying that there is a narrow window of local ligand density that is optimal for CAR-TCR signaling. In addition, decreasing interligand spacing on the four-ligand nanogrid induced more potent and earlier ERK signaling, indicating that stoichiometry and spatial patterning affect the threshold and dynamics of T cell activation. Notably, nanogrids co-displaying a central cluster of high-affinity ligands surrounded by low-affinity ligands induced elevated ERK responses, highlighting potential synergistic interactions between ligands of varying affinities.

Dong and colleagues' utilization of DNA-CAR technology presents an exciting system for generating vast ranges of ligand affinities, which is typically limited by the availability of established ligand and receptor mutants. As demonstrated, this enables the systematic probing of how ligand affinity, in conjunction with other biophysical parameters, influences immune receptor triggering. Furthermore, the inherent presentation of oligonucleotides as ligands is compatible with DNA-PAINT characterization.

Excitingly, Dong and colleagues’ study paved the path toward DNA-based nanosized artificial APCs (aAPCs) that hold great promise for optimizing adoptive T cell therapy, where patient-derived CD8+ T cells are selected for tumor infiltration, expanded ex vivo, and re-infused. To drive T cell expansion, current strategies rely on high-affinity agonist antibodies maximally loaded on biocompatible materials like liposomes, polymers, and microbeads to engage TCRs and costimulatory receptors (62,63). However, Dong and colleagues’ study on CD4+ T cells highlights how reducing antibody load may optimally tune activation kinetics to promote sustained survival and proliferation and avoid strong transient activation associated with apoptosis. To assess the effect of ligand presentation parameters in CD8+ T cell activation, Sun and colleagues generated triangular DNA origami aAPCs decorated with anti-CD28 antibodies and ovalbumin (OVA) pMHC via biotin-SA interactions, with each SA site accommodating up to three ligands (Fig. 2 C) (12). In this design, SA at each vertex was functionalized with anti-CD28 antibodies and varying numbers of SA along the edges (3–12) were ligated with pMHC (9–36 per aAPC) with inter-pMHC distance ranging from 20 to 60 nm. The authors observed increased CD8+ T cell (from OT-1 mice) activation and proliferation with increasing pMHC density and decreasing inter-pMHC spacing. When anchored to ICAM-1 functionalized PLBs using cholesterol, aAPCs with higher pMHC densities enhanced TCR binding and dwell time. Assessing anti-tumor activity by coculturing aAPC-stimulated murine splenocytes with OVA-expressing melanoma cells, the authors observed enhanced cytotoxicity with increased pMHC density. This was recapitulated in vivo where injection of T cells from OT-1 mice alongside 36-pMHC aAPCs induced greatest inhibition of tumor growth and prolonged survival in B16-OVA tumor mice models. Notably, while this study does not provide insights into ex vivo T cell expansion schemes, it does reveal a promising potential for DNA-based aAPC administration directly into patients to stimulate CD8+ T cell responses in situ.

Nanoengineering T cell coreceptor signaling and engagement

While the previous studies discussed focused on TCR-antigen interactions, T cell activation is additionally regulated by various coreceptors. One such inhibitory coreceptor is programmed death-1 (PD-1), which recognizes PD-1 ligand 1 (PD-L1) expressed on most hematopoietic and parenchymal cells (64), with engagement triggering spatial reorganization of PD-1 into microclusters to suppress T cell signaling (65). Normally, this serves to maintain T cell tolerance for self-antigens. However, overexpression of PD-L1 by cancer cells and APCs within tumor microenvironments suppresses the antitumor activity of T cells, marking PD-1 as a key target for cancer immunotherapy (66). To elucidate how PD-L1 organization affects PD-1 clustering, Fang et al. engineered stimulatory and inhibitory DNA origami wireframe sheets functionalized with anti-CD3/CD28 antibodies and PD-L1, respectively, via DBCO-azide interactions (Fig. 3 A) (13). Sheets displaying PD-L1 at varying distances were coimmobilized with agonist sheets on a flat BSA surface to engage PD-1+ Jurkat T cells. The authors observed reduced PD-1 clustering, and suppression of NFAT activity and interleukin-2 (IL-2) production by distantly (202 nm), but not closely (13.6 and 43.5 nm) spaced PD-L1. Given the separation distance between inhibitory and agonistic sheets (minimum 50 nm between PD-L1 and antibodies), this indicates that distal PD-L1 enhances PD-1-mediated T cell inhibition independent of PD-1 and TCR/CD28 proximity, which demands further colocalization analysis. Furthermore, whether the degree of inhibition is impacted by the reduced PD-L1-TCR/CD28 distance imparted by the edge location of PD-L1 in the distant design remains unclear.

Figure 3.

Manipulating T cell coreceptor engagement with DNA origami. (A) Schematic of co-immobilized agonistic and antagonistic rectangular nanogrids. Agonist origami was functionalized with anti-CD28 and anti-CD3 and antagonistic origami was functionalized with a pair of PD-L1 separated by 13.6, 43.5, or 202 nm (13). Increasing PD-L1 distance resulted in decreased NFAT activity, PD-1 cluster size, and T cell activation. (B) Schematic of an adhesion assay with pMHC-presenting RBCs and DNA 10 helix bundle (HB)-presenting beads (27). HB bundles were functionalized with TCR and CD4 separated by varying distances. Increasing separation resulted in reduced pMHC binding and abolished the presence of stiff trimolecular bonds. Image created in BioRender.

Here, we would like to highlight another emerging applicability of DNA origami in unraveling T cell mechanotransduction and the physical forces underlying receptor cooperativity. The coreceptor CD4 is thought to enhance antigen recognition by TCRs through CD4-MHCII interactions stabilizing TCR-pMHC binding. However, previous attempts at interrogating proposed mechanisms were hindered by the extremely low-affinity interaction between CD4 and native MHCII, which were notoriously difficult to capture (67). To interrogate the organizational requirements behind TCR-CD4 cooperativity, Rushdi et al. constructed DNA origami nanorods functionalized with CD4 and TCR at varying distances apart (6–100 nm) via strand hybridization and biotin-SA interactions, respectively (Fig. 3 B) (27). Nanorods were attached to a magnetic bead to make controlled contacts with pMHC-presenting red blood cells. The authors observed increased adhesion probability with small TCR-CD4 separation distances (6 and 13 nm), which, with mathematical modeling, revealed the appearance of stiff trimolecular bonds (0.58 pN/nm). These results suggested a proximity-dependent cooperativity model and presented DNA origami-based 2D kinetic assays as a compelling tool for dissecting the spatial requirements of immune receptor cooperativity.

Nanogeometries in B cell activation: From antibody spatial tolerance to nanovaccines

B cells exhibit similar activation patterns as T cells with a notable difference in antigen-binding, where BCRs exhibit both monovalent and bivalent interactions via two antigen-binding sites. The mechanism of early B cell activation remains an area of active investigation, with competing models proposing antigen-triggered BCR clustering, dissociation, and kinetic changes as underlying reorganizational events (68). Nonetheless, the sequence of biochemical events is well-established wherein antigen encounter triggers the phosphorylation of activation motifs in the BCR-associated Igα/β heterodimer. This leads to increases in intracellular calcium and subsequent antigen internalization and presentation to CD4+ T cells to acquire additional signals for activation.

Notably, BCRs recognize a larger repertoire of antigens in both soluble and APC-presented formats, inviting the use of varying antigen presentation platforms for in vitro studies, including synthetic supporting substrates and soluble particulates. These studies established the multivalent immunogen vaccine strategy in enhancing B cell activation (69), but the independent role of antigen stoichiometry and spatial arrangement remains elusive with DNA origami-based studies focusing more on antigen interactions with antibodies, rather than the BCR. In 2019, Shaw et al. engineered DNA origami helix bundles and bricks displaying hapten antigens at varying distances (3–44 nm) to dissect the spatial tolerance of different antibody isotypes by surface plasmon resonance (Fig. 4 A) (70). The authors observed a striking bivalent affinity peak with 16-nm-spaced antigens for all IgG subclasses, with affinity dropping sharply at interantigen distances of 17 nm and beyond. Moreover, an engineered monomeric IgM exhibited bivalent binding to antigens separated by a larger range (3–29 nm), with strong binding from 3 to 17 nm. The greater spatial tolerance may be crucial in IgM-BCR’s role in the activation of typically lower-affinity naive B cells to initiate the humoral immune response. The spatial tolerance of IgG is recapitulated by Zhang et al., who generated triangular DNA origami displaying digoxin separated by distances ranging from 3 to 20 nm to mimic the uneven distribution of epitope spikes on viruses (Fig. 4 B) (71). Using high-speed AFM, the authors observed bivalent engagement of epitopes within 16 nm, but, unlike Shaw’s study, optimal antibody engagement occurred at 10 nm. While differences in characterization techniques and antigen linker lengths may lead to discrepancies, both studies nonetheless beautifully illustrated the importance of nanoscale epitope arrangement for intuiting antigen-antibody interactions. While these interrogations do not tackle B cell activation, the findings contributed to informing nanopattern testing in Veneziano et al.’s assessment of membrane-bound BCR triggering (14).

Figure 4.

Investigating antibody binding and B cell activation with DNA origami. (A) Schematic of DNA helix bundles and bricks decorated with two antigens separated by varying distances, with 16 nm yielding optimal bivalent binding by IgG and IgM (70). (B) Schematic of triangular origami decorated with antigen pairs separated by varying distances, with 10 nm yielding optimal bivalent binding by IgG (71). (C) Schematic of DNA icosahedrons decorated with varying numbers of eOD-GT8 antigens, and DNA icosahedrons and helix bundles functionalized with two and five antigens, respectively, separated by varying distances (14). Increasing antigen valency and spacing resulted in enhanced B cell activation up to a plateau. Image created in BioRender.

To demonstrate the functional relevance of antigen organization for naive B cell activation, Veneziano and colleagues constructed DNA origami icosahedrons displaying varying copies (1–10) of engineered outer domains (eOD-GT8) found on human immunodeficiency viruses (HIV) with varying spacing (3–22 nm) (Fig. 4 C). Upon soluble stimulation, maximum B cell activation was observed with 5 or more copies of antigen. Furthermore, a monotonic increase in intracellular calcium was observed with increasing interantigen distances, with signaling appearing to plateau at distances beyond 22 nm. This was recapitulated using DNA origami helix bundles displaying eOD-GT8 up to 80 nm apart, well beyond the bivalent spatial tolerance of IgM, leading the authors to suggest that monovalent engagement of non-local, distal BCRs can also drive the initiation of BCR signaling. Notably, this model assumes that IgM-BCRs are monomerically distributed on the cell surface. However, super-resolution imaging studies have provided evidence that, at least to some degree, IgM-BCRs exist in preformed nanoscale clusters with an average radius of approximately 60–150 nm (72,73). Thus, it is possible that engagement of BCRs within a single nanocluster by distal antigens is sufficient to trigger signaling. Future studies integrating inter-antigen spacing by DNA origami and BCR organization by super-resolution imaging will provide key insights into the mechanism of BCR triggering. Interestingly, replacement of rigid helix bundles with flexible linkers abolished the observed differences in activation imparted by spatial segregation, implicating platform rigidity as an essential physical property for robust B cell activation. The authors proposed a spatially mediated positive feedback system upon BCR clustering, where each antigen can recruit a specific number of BCRs within a specific radius. Thus, proximal monovalent antigens reduce total BCR recruitment due to steric exclusion; and as antigen separation increases to a point of non-overlap, a signaling plateau is subsequently reached. Notably, increased spacing of lower affinity antigen eOD-GT5 stimulated similar monotonic increases in signaling but with overall lower plateaus. This demonstrated the potential differences in the degree to which a single factor contributes to B cell activation, wherein valency may outweigh antigen spacing for lower-affinity interactions, highlighting the importance of continued systematic analysis of physical design parameters and how they translate to functional differences in immunogenicity. Furthermore, future studies on the copatterning of membrane-associated antigens and coreceptor ligands will prove critical in fleshing out rational vaccine design guidelines.

Nanogeometries in FcƴR-mediated phagocytosis

Macrophages destroy pathogens and unhealthy cells through Fc gamma receptor (FcγR)-mediated phagocytosis of IgG-opsonized targets, which is a key mechanism of action for many antibody-based cancer immunotherapies (74). Interestingly, macrophages exhibit an all-or-none engulfment response requiring a critical antibody threshold across the target to be reached, activating multiple FcγRs to drive the closure of the phagocytic cup (75). FcγR dynamics resemble what has been discussed for adaptive immune receptors, albeit on a smaller scale, where IgG engagement leads to receptor phosphorylation and the formation of signaling-competent nanoclusters that recruit phosphoinositide 3 kinase, which produces the secondary messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (76).

To address how ligand stoichiometry and spacing modulate FcγR-mediated phagocytosis, Kern et al. developed a CAR system similar to Dong’s study where the extracellular ligand binding domain of FcγR was replaced by a SNAP-tag covalently attached to a receptor strand (Fig. 5 A) (15). DNA origami nanogrids were then deposited on PLBs via biotin-neutravidin interactions, displaying varying numbers (0–72) of ligand strands to stimulate CAR-expressing murine leukemia-derived RAW 264.7 macrophages and human leukemia-derived THP-1 monocytes. Maximal engulfment was observed for both cell types when DNA nanogrids contained eight or more ligands. Varying the separation distance between four ligands (3.5–38.5 nm), the authors observed enhanced receptor phosphorylation, PIP3 production, and probability of engulfment initiation and completion with distances of 7 nm or less. These results suggest that ligand-mediated FcγR nanoclustering may enable target discrimination between highly opsonized targets and low-density background signals, such as soluble antibodies.

Figure 5.

Manipulating innate immune receptor activation with DNA origami. (A) Schematic of a modified Fcγ-CAR system interacting with rectangular nanogrids functionalized with varying numbers of ligands or with four ligands with varying interligand distances (15). Increasing valency and decreasing distance resulted in enhanced phagocytosis. (B) Schematic of triangular nanogrids decorated with CpG pairs separated by varying distances, with or without linkers of varying lengths (16). Increasing distance and length resulted in decreased TLR9 activation. (C) Schematic of DNA icosahedrons or pentagonal bipyramids decorated with varying numbers of CpG at varying distances apart (17). Increasing valency and decreasing spacing resulted in enhanced TLR9 activation. Image created in BioRender.

At this point, we would like to highlight DNA origami geometry as another critical design consideration pertinent to the internalization of these nanostructures by phagocytes and B cells. Bastings et al. demonstrated that compact DNA nanostructures with low aspect ratios, like spheroids and icosahedrons, are better internalized by dendritic cells and non-phagocytic endothelial and epithelial cells (77). Thus, they may hold greater applicability in vivo compared with the more commonly employed rectangular nanogrids in mimicking cell-cell interactions in vitro. However, compact nanostructures have limited dimensions on which interligand distances can be varied within the same plane of presentation, revealing a current challenge in translating design principles underlying ligand geometry across differently shaped platforms in vivo.

Nanogeometries in intracellular TLR9 signaling

The studies discussed so far analyzed surface receptor engagement by extracellular ligands. However, a large population of intracellular innate immune receptors exist within acidic endosomes, including TLR9 that recognize CpG motifs. Upon pH-dependent binding of two CpG motifs in acidic late endosomes, TLR9 dimerizes and activates, with reported separation distances of 7 nm from crystallography data (26). This triggers signaling cascades that lead to increased surface expression of costimulatory molecules like CD83 and CD40 as well as the release of interferons and pro-inflammatory cytokines like IL-6 (78).

To investigate how the spatial organization of CpG motifs impacts TLR9 activation, Comberlato et al. created DNA origami disks with staple strands presenting CpG-containing oligonucleotides with a separation distance of 7 or 38 nm (Fig. 5 B) (16). Upon internalization by RAW 264.7 macrophages, the authors observed enhanced upregulation of CD83 and CD40 and IL-6 release induced by CpG spaced 7 nm apart, supporting previous crystallography results (26). Interestingly, introducing flexible DNA linkers between the platform and CpG abolished observable differences imparted by ligand spacing. Furthermore, increasing linker length resulted in progressively lower activation, supporting a potential requirement for ligand rigidity in robust TLR9 activation.

In a complementary study, Du et al. engineered wireframe DNA origami pentagonal bipyramids (PBs) and icosahedrons with staple strands displaying varying numbers (0-40) of CpG motifs (Fig. 5 C) (17). Notably, increasing number of CpGs stimulated a monotonic increase in TLR9 activation in origami-transfected human embryonic kidney (HEK)-Blue cells. The authors then varied the separation distance (7–20 nm) between 10 ligands and observed maximal TLR9 activation at 7 nm, consistent with Comberlato et al.’s study. Interestingly, upon further assessment of spatial distributions beyond the optimal 7 nm (10–25 nm apart) using only five ligands, no significant differences in TLR9 activation were observed, suggesting low TLR9 binding tolerance for suboptimal interligand distances. To further highlight the importance of platform geometry, Du and colleagues demonstrated that similar geometries, such as PBs and octahedrons, induced similar levels of TLR9 activation while more distinct geometries, such as tetrahedrons and icosahedrons, resulted in significantly different TLR9 activation. Notably, icosahedrons induced the highest level of TLR9 activation. However, as these observations were yielded from DNA origami construct internalization by HEK-Blue cells in the presence of transfection reagents, potential differences in uptake efficiency by professional phagocytes like macrophages may impact the efficacy of different constructs in stimulating immune cells in vivo.

Nanogeometries of immune responses against SARS-CoV-2

The translatability of DNA origami can be highlighted by its recent use in nanoengineering ligand geometries to elicit protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To assess how the stoichiometry and distribution of receptor-binding domain (RBD) spikes on SARS-CoV-2 influences viral infection and immune cell activation, Zhang et al. designed viral-like DNA origami soccer-ball frameworks displaying varying numbers (10–90) of RBDs via aptamer functionalization (18). The authors observed enhanced binding to host receptor angiotensin converting enzyme 2 expressed in HEK293T cells with increasing numbers of RBDs, plateauing at 60 RBDs. Furthermore, enhanced binding for more accessible (equatorial) and densely packed (unipolar) RBDs were observed compared with evenly and bipolar distributed RBDs. Looking at immune activation, as few as 20 evenly distributed RBDs elicited upregulation of CD40 in RAW264.7 macrophages. Surprisingly, even distribution resulted in greater macrophage activation compared with all other arrangements, revealing a dichotomy in the spatial requirements for infection and immune cell activation and a possible geometrically-based mechanism of immune evasion.

Expanding further upon the utility of DNA origami as a nanovaccine in vivo, Oktay et al. developed PB structures functionalized with RBD antigen and/or CpG adjuvants on opposite faces via DNA hybridization (28). BALB/c mice were immunized with the nanovaccine, with a booster shot 3 weeks post initial immunization. Six weeks post injection, the most robust neutralizing antibodies were detected in mice vaccinated with nanovaccines functionalized with both RBD and CpG. Similarly, exposure of RBD-CpG nanovaccine immunized human ACE2-expressing mice to live SARS-CoV-2 showed 100% survival rate. Furthermore, the levels of RBD-specific antibodies 2 months post immunization were similar to that of human clinical trials with mRNA vaccines (79), demonstrating the potential of DNA-based nanovaccines (79). Thus, the systematic delineation of design principles will prove critical in driving the future of nanoengineering receptor patterning to maximize the elicitation of protective immunity.

Final remarks

The unprecedented control over the nanoscale landscape of biomolecules marks DNA origami as a groundbreaking tool for unraveling the biophysical properties underlying immune receptor signaling. Integration of these platforms into membrane systems offers tremendous potential for manipulating cell-cell interactions with nanoscale precision. As we begin to address basic questions on the biophysical parameters driving immune receptor activation, rigorous reporting of methodology and the standardization of characterization techniques will prove critical in facilitating reproducibility. Alongside continued progress in fundamental analyses in vitro, defining the trafficking, immunogenicity, and stability of these platforms in vivo will drive the synthesis of rational design guidelines as DNA origami trends toward clinical application as novel nanotherapeutics.

Author contributions

Y.H. and B.T. wrote the manuscript.

Editor: Meyer Jackson.