Changes in tissue sodium concentration and sodium relaxation times during the maturation of human knee cartilage: Ex vivo 23 Na MRI study at 10.5 T.

PURPOSE: To evaluate the influence of skeletal maturation on sodium (23 Na) MRI relaxation parameters and the accuracy of tissue sodium concentration (TSC) quantification in human knee cartilage. METHODS: Twelve pediatric knee specimens were imaged with whole-body 10.5 T MRI using a density-adapted 3D radial projection sequence to evaluate 23 Na parameters: B1 + , T1 , biexponential T 2 * $$ {\mathrm{T}}_2^{\ast } $$ , and TSC. Water, collagen, and sulfated glycosaminoglycan (sGAG) content were calculated from osteochondral biopsies. The TSC was corrected for B1 + , relaxation, and water content. The literature-based TSC (TSCLB ) used previously published values for corrections, whereas the specimen-specific TSC (TSCSP ) used measurements from individual specimens. 23 Na parameters were evaluated in eight cartilage compartments segmented on proton images. Associations between 23 Na parameters, TSCLB - TSCSP difference, biochemical content, and age were determined. RESULTS: From birth to 12 years, cartilage water content decreased by 18%; collagen increased by 59%; and sGAG decreased by 36% (all R2 ≥ 0.557). The short T 2 * $$ {\mathrm{T}}_2^{\ast } $$ ( T 2 * S $$ {{\mathrm{T}}_2^{\ast}}_{\mathrm{S}} $$ ) decreased by 72%, and the signal fraction relaxing with T 2 * S $$ {{\mathrm{T}}_2^{\ast}}_{\mathrm{S}} $$ ( fT 2 * S $$ {{\mathrm{fT}}_2^{\ast}}_{\mathrm{S}} $$ ) increased by 55% during the first 5 years but remained relatively stable after that. TSCSP was significantly correlated with sGAG content from biopsies (R2 = 0.739). Depending on age, TSCLB showed higher or lower values than TSCSP . The TSCLB - TSCSP difference was significantly correlated with T 2 * S $$ {{\mathrm{T}}_2^{\ast}}_{\mathrm{S}} $$ (R2 = 0.850), fT 2 * S $$ {{\mathrm{fT}}_2^{\ast}}_{\mathrm{S}} $$ (R2 = 0.651), and water content (R2 = 0.738). CONCLUSION: TSC and relaxation parameters measured with 23 Na MRI provide noninvasive information about changes in sGAG content and collagen matrix during cartilage maturation. Cartilage TSC quantification assuming fixed relaxation may be feasible in children older than 5 years.

A stable, highly concentrated fluorous nanoemulsion formulation for in vivo cancer imaging via 19F-MRI.

Magnetic resonance imaging (MRI) is a routine diagnostic modality in oncology that produces excellent imaging resolution and tumor contrast without the use of ionizing radiation. However, improved contrast agents are still needed to further increase detection sensitivity and avoid toxicity/allergic reactions associated with paramagnetic metal contrast agents, which may be seen in a small percentage of the human population. Fluorine-19 (19F)-MRI is at the forefront of the developing MRI methodologies due to near-zero background signal, high natural abundance of 100%, and unambiguous signal specificity. In this study, we have developed a colloidal nanoemulsion (NE) formulation that can encapsulate high volumes of the fluorous MRI tracer, perfluoro-[15-crown-5]-ether (PFCE) (35% v/v). These nanoparticles exhibit long-term (at least 100 days) stability and high PFCE loading capacity in formulation with our semifluorinated triblock copolymer, M2F8H18. With sizes of approximately 200 nm, these NEs enable in vivo delivery and passive targeting to tumors. Our diagnostic formulation, M2F8H18/PFCE NE, yielded in vivo 19F-MR images with a high signal-to-noise ratio up to 100 in a tumor-bearing mouse model at clinically relevant scan times. M2F8H18/PFCE NE circulated stably in the vasculature, accumulated in high concentration of an estimated 4-9 × 1017 19F spins/voxel at the tumor site, and cleared from most organs over the span of 2 weeks. Uptake by the mononuclear phagocyte system to the liver and spleen was also observed, most likely due to particle size. These promising results suggest that M2F8H18/PFCE NE is a favorable 19F-MR diagnostic tracer for further development in oncological studies and potential clinical translation.

Polyglycerol-Shelled Reduction-Sensitive Polymersome for DM1 Delivery to HER-2-Positive Breast Cancer.

Supramolecular delivery systems with the prolonged circulation, the potential for diverse functionalization, and few toxin-related limitations have been extensively studied. For the present study, we constructed a linear polyglycerol-shelled polymersome attached with the anti-HER-2-antibody trastuzumab. We then covalently loaded the anticancer drug DM1 in the polymersome via dynamic disulfide bonding. The resulted trastuzumab-polymersome-DM1 (Tra-PS-DM1) exhibits a mean size of 95.3 nm and remarkable drug loading efficiency % of 99.3%. In addition to its superior stability, we observed the rapid release of DM1 in a controlled manner under reductive conditions. Compared to the native polymersomes, Tra-PS-DM1 has shown greatly improved cellular uptake and significantly reduced IC50 up to 17-fold among HER-2-positive cancer cells. Moreover, Tra-PS-DM1 demonstrated superb growth inhibition of HER-2-positive tumoroids; specifically, BT474 tumoroids shrunk up to 62% after 12 h treatment. With exceptional stability and targetability, the PG-shelled Tra-PS-DM1 appears as an attractive approach for HER-2-positive tumor treatment.

Dual-Action Heteromultivalent Glycopolymers Stringently Block and Arrest Influenza A Virus Infection In Vitro and Ex Vivo.

Here, we demonstrate the concerted inhibition of different influenza A virus (IAV) strains using a low-molecular-weight dual-action linear polymer. The 6'-sialyllactose and zanamivir conjugates of linear polyglycerol are optimized for simultaneous targeting of hemagglutinin and neuraminidase on the IAV surface. Independent of IAV subtypes, hemagglutination inhibition data suggest better adsorption of the heteromultivalent polymer than homomultivalent analogs onto the virus surface. Cryo-TEM images imply heteromultivalent compound-mediated virus aggregation. The optimized polymeric nanomaterial inhibits >99.9% propagation of various IAV strains 24 h postinfection in vitro at low nM concentrations and is up to 10000× more effective than the commercial zanamivir drug. In a human lung ex vivo multicyclic infection setup, the heteromultivalent polymer outperforms the commercial drug zanamivir and homomultivalent analogs or their physical mixtures. This study authenticates the translational potential of the dual-action targeting approach using small polymers for broad and high antiviral efficacy.

Structural and functional analysis of the roles of influenza C virus membrane proteins in assembly and budding.

Assembly and budding of the influenza C virus is mediated by three membrane proteins: the hemagglutinin-esterase-fusion glycoprotein (HEF), the matrix protein (CM1), and the ion channel (CM2). Here we investigated whether the formation of the hexagonal HEF arrangement, a distinctive feature of influenza C virions is important for virus budding. We used super resolution microscopy and found 250-nm sized HEF clusters at the plasma membrane of transfected cells, which were insensitive to cholesterol extraction and cytochalasin treatment. Overexpression of either CM1, CM2, or HEF caused the release of membrane-enveloped particles. Cryo-electron microscopy of the latter revealed spherical vesicles exhibiting the hexagonal HEF clusters. We subsequently used reverse genetics to identify elements in HEF required for this clustering. We found that deletion of the short cytoplasmic tail of HEF reduced virus titer and hexagonal HEF arrays, suggesting that an interaction with CM1 stabilizes the HEF clusters. In addition, we substituted amino acids at the surface of the closed HEF conformation and identified specific mutations that prevented virus rescue, others reduced virus titers and the number of HEF clusters in virions. Finally, mutation of two regions that mediate contacts between trimers in the in-situ structure of HEF was shown to prevent rescue of infectious virus particles. Mutations at residues thought to mediate lateral interactions were revealed to promote intracellular trafficking defects. Taken together, we propose that lateral interactions between the ectodomains of HEF trimers are a driving force for virus budding, although CM2 and CM1 also play important roles in this process.

A New Support Film for Cryo Electron Microscopy Protein Structure Analysis Based on Covalently Functionalized Graphene.

Protein adsorption at the air-water interface is a serious problem in cryogenic electron microscopy (cryoEM) as it restricts particle orientations in the vitrified ice-film and promotes protein denaturation. To address this issue, the preparation of a graphene-based modified support film for coverage of conventional holey carbon transmission electron microscopy (TEM) grids is presented. The chemical modification of graphene sheets enables the universal covalent anchoring of unmodified proteins via inherent surface-exposed lysine or cysteine residues in a one-step reaction. Langmuir-Blodgett (LB) trough approach is applied for deposition of functionalized graphene sheets onto commercially available holey carbon TEM grids. The application of the modified TEM grids in single particle analysis (SPA) shows high protein binding to the surface of the graphene-based support film. Suitability for high resolution structure determination is confirmed by SPA of apoferritin. Prevention of protein denaturation at the air-water interface and improvement of particle orientations is shown using human 20S proteasome, demonstrating the potential of the support film for structural biology.

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants.

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4 nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.

Synthesis of two-dimensional triazine covalent organic frameworks at ambient conditions to detect and remove water pollutants.

Synthesis of fully triazine frameworks (C3N3) by metal catalyzed reactions at high temperatures results in carbonized and less-defined structures. Moreover, metal impurities affect the physicochemical, optical and electrical properties of the synthesized frameworks, dramatically. In this work, two-dimensional C3N3 (2DC3N3) has been synthesized by in situ catalyst-free copolymerization of sodium cyanide and cyanuric chloride, as cheap and commercially available precursors, at ambient conditions on gram scale. Reaction between sodium cyanide and cyanuric chloride resulted in electron-poor polyfunctional intermediates, which converted to 2DC3N3 with several hundred micrometers lateral size at ambient conditions upon [2 + 2+2] cyclotrimerization. 2DC3N3 sheets, in bulk and individually, showed strong fluorescence with 63% quantum yield and sensitive to small objects such as dyes and metal ions. The sensitivity of 2DC3N3 emission to foreign objects was used to detect low concentration of water impurities. Due to the high negative surface charge (-37.7 mV) and dispersion in aqueous solutions, they demonstrated a high potential to remove positively charged dyes from water, exemplified by excellent removal efficiency (>99%) for methylene blue. Taking advantage of the straightforward production and strong interactions with dyes and metal ions, 2DC3N3 was integrated in filters and used for the fast detection and efficient removal of water impurities.

Mucin-Inspired Single-Chain Polymer (MIP) Fibers as Potent SARS-CoV-2 Inhibitors.

Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450 kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59 nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0 nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.

Controlling the Assembly of Cellulose-Based Oligosaccharides through Sequence Modifications.

Peptides and nucleic acids with programmable sequences are widely explored for the production of tunable, self-assembling functional materials. Herein we demonstrate that the primary sequence of oligosaccharides can be designed to access materials with tunable shapes and properties. Synthetic cellulose-based oligomers were assembled into 2D or 3D rod-like crystallites. Sequence modifications within the oligosaccharide core influenced the molecular packing and led to the formation of square-like assemblies based on the rare cellulose IVII allomorph. In contrast, modifications at the termini generated elongated aggregates with tunable surfaces, resulting in self-healing supramolecular hydrogels.

Ferumoxytol dynamic contrast enhanced magnetic resonance imaging identifies altered placental cotyledon perfusion in rhesus macaques†.

Identification of placental dysfunction in early pregnancy with noninvasive imaging could be a valuable tool for assessing maternal and fetal risk. Dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) can be a powerful tool for interrogating placenta health. After inoculation with Zika virus or sham inoculation at gestation age (GA) 45 or 55 days, animals were imaged up to three times at GA65, GA100, and GA145. DCE MRI images were acquired at all imaging sessions using ferumoxytol, an iron nanoparticle-based contrast agent, and analyzed for placental intervillous blood flow, number of perfusion domains, and perfusion domain volume. Cesarean section was performed at GA155, and the placenta was photographed and dissected for histopathology. Photographs were used to align cotyledons with estimated perfusion domains from MRI, allowing comparison of estimated cotyledon volume to pathology. Monkeys were separated into high and low pathology groups based on the average number of pathologies present in the placenta. Perfusion domain flow, volume, and number increased through gestation, and total blood flow increased with gestation for both low pathology and high pathology groups. A statistically significant decrease in perfusion domain volume associated with pathology was detected at all gestational ages. Individual perfusion domain flow comparisons demonstrated a statistically significant decrease with pathology at GA100 and GA145, but not GA65. Since ferumoxytol is currently used to treat anemia during human pregnancy and as an off-label MRI contrast agent, future transition of this work to human pregnancy may be possible.

Design and Functional Analysis of Heterobifunctional Multivalent Phage Capsid Inhibitors Blocking the Entry of Influenza Virus.

Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qß) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qß conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.

A Nanohook-Equipped Bionanocatalyst for Localized Near-Infrared-Enhanced Catalytic Bacterial Disinfection.

Novel bionanocatalysts have opened a new era in fighting multidrug-resistant (MDR) bacteria. They can kill bacteria by elevating the level of reactive oxygen species (ROS) in the presence of chemicals like H2 O2 . However, ROSs' ultrashort diffusion distance limit their bactericidal activity. We present a nanohook-equipped bionanocatalyst (Ni@Co-NC) with bacterial binding ability that shows robust ROS-generating capacity under physiological H2 O2 levels. The Ni@Co-NC's pH-dependent performance confines its effects to the biofilm microenvironment, leaving healthy tissue unaffected. Furthermore, it can generate heat upon NIR laser irradiation, enhancing its catalytic performance while achieving heat ablation against bacteria. With the Ni@Co-NC's synergistic effects, bacterial populations fall by >99.99 %. More surprisingly, the mature biofilm shows no recurrence after treatment with the Ni@Co-NC, demonstrating its tremendous potential for treating MDR bacterial related infections.

Graphene-Assisted Synthesis of 2D Polyglycerols as Innovative Platforms for Multivalent Virus Interactions.

2D nanomaterials have garnered widespread attention in biomedicine and bioengineering due to their unique physicochemical properties. However, poor functionality, low solubility, intrinsic toxicity, and nonspecific interactions at biointerfaces have hampered their application in vivo. Here, biocompatible polyglycerol units are crosslinked in two dimensions using a graphene-assisted strategy leading to highly functional and water-soluble polyglycerols nanosheets with 263 ± 53 nm and 2.7 ± 0.2 nm average lateral size and thickness, respectively. A single-layer hyperbranched polyglycerol containing azide functional groups is covalently conjugated to the surface of a functional graphene template through pH-sensitive linkers. Then, lateral crosslinking of polyglycerol units is carried out by loading tripropargylamine on the surface of graphene followed by lifting off this reagent for an on-face click reaction. Subsequently, the polyglycerol nanosheets are detached from the surface of graphene by slight acidification and centrifugation and is sulfated to mimic heparin sulfate proteoglycans. To highlight the impact of the two-dimensionality of the synthesized polyglycerol sulfate nanosheets at nanobiointerfaces, their efficiency with respect to herpes simplex virus type 1 and severe acute respiratory syndrome corona virus 2 inhibition is compared to their 3D nanogel analogs. Four times stronger in virus inhibition suggests that 2D polyglycerols are superior to their current 3D counterparts.

Graphene Sheets with Defined Dual Functionalities for the Strong SARS-CoV-2 Interactions.

Search of new strategies for the inhibition of respiratory viruses is one of the urgent health challenges worldwide, as most of the current therapeutic agents and treatments are inefficient. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic and has taken lives of approximately two million people to date. Even though various vaccines are currently under development, virus, and especially its spike glycoprotein can mutate, which highlights a need for a broad-spectrum inhibitor. In this work, inhibition of SARS-CoV-2 by graphene platforms with precise dual sulfate/alkyl functionalities is investigated. A series of graphene derivatives with different lengths of aliphatic chains is synthesized and is investigated for their ability to inhibit SARS-CoV-2 and feline coronavirus. Graphene derivatives with long alkyl chains (>C9) inhibit coronavirus replication by virtue of disrupting viral envelope. The ability of these graphene platforms to rupture viruses is visualized by atomic force microscopy and cryogenic electron microscopy. A large concentration window (10 to 100-fold) where graphene platforms display strongly antiviral activity against native SARS-CoV-2 without significant toxicity against human cells is found. In this concentration range, the synthesized graphene platforms inhibit the infection of enveloped viruses efficiently, opening new therapeutic and metaphylactic avenues against SARS-CoV-2.

Detection and viability of murine NK cells in vivo in a lymphoma model using fluorine-19 MRI.

Natural killer (NK) cell therapies are being increasingly used as an adoptive cell therapy for cancer because they can recognize tumor cells in an antigen-independent manner. While promising, the understanding of NK cell persistence, particularly within a harsh tumor microenvironment, is limited. Fluorine-19 (19 F) MRI is a noninvasive imaging modality that has shown promise in longitudinally tracking cell populations in vivo; however, it has not been studied on murine NK cells. In this study, the impact of 19 F labeling on murine NK cell viability and function was assessed in vitro and then used to quantify NK cell persistence in vivo. While there was no noticeable impact on viability, labeling NK cells with 19 F did attenuate cytotoxicity against lymphoma cells in vitro. Fluorescent microscopy verified 19 F labeling in both the cytoplasm and nucleus of NK cells. Lymphoma-bearing mice were given intratumoral injections of 19 F-labeled NK cells in which signal was detectable across the 6 day observation period via 19 F MRI. Quantification from the composite images detected 78-94% of the initially injected NK cells across 6 days, with a significant decrease between Days 3 and 6. Postmortem flow cytometry demonstrated retention of 19 F intracellularly within adoptively transferred NK cells with less than 1% of 19 F-containing cells identified as tumor-associated macrophages that presumably ingested nonviable NK cells. This work demonstrates that 19 F MRI offers a specific imaging platform to track and quantify murine NK cells within tumors noninvasively.

Mucin-Inspired, High Molecular Weight Virus Binding Inhibitors Show Biphasic Binding Behavior to Influenza A Viruses.

Multivalent binding inhibitors are a promising new class of antivirals that prevent virus infections by inhibiting virus binding to cell membranes. The design of these inhibitors is challenging as many properties, for example, inhibitor size and functionalization with virus attachment factors, strongly influence the inhibition efficiency. Here, virus binding inhibitors are synthesized, the size and functionalization of which are inspired by mucins, which are naturally occurring glycosylated proteins with high molecular weight (MDa range) and interact efficiently with various viruses. Hyperbranched polyglycerols (hPGs) with molecular weights ranging between 10 and 2600 kDa are synthesized, thereby hitting the size of mucins and allowing for determining the impact of inhibitor size on the inhibition efficiency. The hPGs are functionalized with sialic acids and sulfates, as suggested from the structure of mucins, and their inhibition efficiency is determined by probing the inhibition of influenza A virus (IAV) binding to membranes using various methods. The largest, mucin-sized inhibitor shows potent inhibition at pm concentrations, while the inhibition efficiency decreases with decreasing the molecular weight. Interestingly, the concentration-dependent IAV inhibition shows a biphasic behavior, which is attributed to differences in the binding affinity of the inhibitors to the two IAV envelope proteins, neuraminidase, and hemagglutinin.

Impact of ferumoxytol magnetic resonance imaging on the rhesus macaque maternal-fetal interface†.

Ferumoxytol is a superparamagnetic iron oxide nanoparticle used off-label as an intravascular magnetic resonance imaging (MRI) contrast agent. Additionally, ferumoxytol-uptake by macrophages facilitates detection of inflammatory sites by MRI through ferumoxytol-induced image contrast changes. Therefore, ferumoxytol-enhanced MRI holds great potential for assessing vascular function and inflammatory response, critical to determine placental health in pregnancy. This study sought to assess the fetoplacental unit and selected maternal tissues, pregnancy outcomes, and fetal well-being after ferumoxytol administration. In initial developmental studies, seven pregnant rhesus macaques were imaged with or without ferumoxytol administration. Pregnancies went to term with vaginal delivery and infants showed normal growth rates compared to control animals born the same year that did not undergo MRI. To determine the impact of ferumoxytol on the maternal-fetal interface (MFI), fetal well-being, and pregnancy outcome, four pregnant rhesus macaques at ~100 gestational day underwent MRI before and after ferumoxytol administration. Collection of the fetoplacental unit and selected maternal tissues was performed 2-3 days following ferumoxytol administration. A control group that did not receive ferumoxytol or MRI was used for comparison. Iron levels in fetal and MFI tissues did not differ between groups, and there was no significant difference in tissue histopathology with or without exposure to ferumoxytol, and no effect on placental hormone secretion. Together, these results suggest that the use of ferumoxytol and MRI in pregnant rhesus macaques does not negatively impact the MFI and can be a valuable experimental tool in research with this important animal model.

Adaptive Flexible Sialylated Nanogels as Highly Potent Influenza A Virus Inhibitors.

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand-receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC50 values in low picomolar concentrations and also blocks the virus entry into MDCK-II cells.

Sialyl-LacNAc-PNA⋠DNA Concatamers by Rolling-Circle Amplification as Multivalent Inhibitors of Influenza†A Virus Particles.

The surfaces of influenza A virus (IAV) particles are packed with hundreds of homo-trimeric hemagglutinins (HAs). Monovalent sugars have low affinity for HA, but distance-optimized bivalent sialyl-LacNAc (SLN) conjugates bind it with 103 -fold enhanced potency. Herein, we describe the oligomerization of distance-optimized bivalent binders by branched and linear hybridization on long repetitive DNA templates. The most effective complexes fully inhibited IAVs at a DNA template concentration of 10-9 m. Although a 10-2 m concentration of free trisaccharide ligand is required for full inhibition of the virus, DNA templating enables a 104 -fold reduction in the amount of sugar required. Notably, hybridization-induced rigidification of the DNA templates increased the serospecificity. Cryo-TEM analysis revealed that both spaghetti-type linear forms and cotton-ball-like clusters are able to bridge several adjacent HA molecules on the IAV surface. Programmed self-assembly of ligand-nucleic acid conjugates on long DNA templates might provide generic access to target-specific, high-affinity binders of proteins on globular objects such as cells and viruses.