RESUMEN
Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Antígenos CD/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/química , Línea Celular Tumoral , Humanos , Lectinas Tipo C/química , Ligandos , Liposomas/química , Liposomas/metabolismo , Lectinas de Unión a Manosa/metabolismo , Manósidos/química , Manósidos/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Receptores de Superficie Celular/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Binders of langerin could target vaccines to Langerhans cells for improved therapeutic effect. Since langerin has low affinity for monovalent glycan ligands, highly multivalent presentation has previously been key for targeting. Aiming to reduce the amount of ligand required, we rationally designed molecularly defined high-affinity binders based on the precise display of glycomimetic ligands (Glc2NTs) on DNA-PNA scaffolds. Rather than mimicking langerin's homotrimeric structure with a C3-symmetric scaffold, we developed readily accessible, easy-to-design bivalent binders. The method considers the requirements for bridging sugar binding sites and statistical rebinding as a means to both strengthen the interactions at single binding sites and amplify the avidity enhancement provided by chelation. This gave a 1150-fold net improvement over the affinity of the free ligand and provided a nanomolar binder (IC50 =300â nM) for specific internalization by langerin-expressing cells.
Asunto(s)
Antígenos CD/química , ADN/química , Lectinas Tipo C/química , Lectinas de Unión a Manosa/química , Sitios de Unión , Humanos , Células de Langerhans/química , Ligandos , Modelos Moleculares , Conformación MolecularRESUMEN
Asymmetrically branched precision glycooligomers are synthesized by solid-phase polymer synthesis for studying multivalent carbohydrate-protein interactions. Through the stepwise assembly of Fmoc-protected oligo(amidoamine) building blocks and Fmoc/Dde-protected lysine, straightforward variation of structural parameters such as the number and length of arms, as well as the number and position of carbohydrate ligands, is achieved. Binding of 1-arm and 3-arm glycooligomers toward lectin receptors langerin and concanavalin A (ConA) was evaluated where the smallest 3-arm glycooligomer shows the highest binding toward langerin, and stepwise elongation of one, two, or all three arms leads to decreased binding. When directly comparing binding toward langerin and ConA, we find that structural variation of the scaffold affects glycomimetic ligand binding differently for the different targets, indicating the potential to tune such ligands not only for their avidity but also for their selectivity toward different lectins.
Asunto(s)
Antígenos CD/química , Carbohidratos/química , Glicoproteínas/química , Lectinas Tipo C/química , Lectinas de Unión a Manosa/química , Proteínas/química , Antígenos CD/genética , Carbohidratos/síntesis química , Carbohidratos/genética , Concanavalina A/química , Concanavalina A/genética , Concanavalina A/metabolismo , Glicoproteínas/síntesis química , Glicoproteínas/ultraestructura , Humanos , Lectinas Tipo C/genética , Ligandos , Lectinas de Unión a Manosa/genética , Unión Proteica/genética , Conformación Proteica , Proteínas/genética , Proteínas/ultraestructura , Receptores Mitogénicos/química , Receptores Mitogénicos/genéticaRESUMEN
The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.
Asunto(s)
Biopelículas/efectos de los fármacos , Cinamatos/farmacología , Lectinas/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Sulfonamidas/farmacología , Administración Oral , Disponibilidad Biológica , Cinamatos/administración & dosificación , Cinamatos/química , Relación Dosis-Respuesta a Droga , Cinética , Lectinas/metabolismo , Conformación Molecular , Pseudomonas aeruginosa/metabolismo , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/química , TermodinámicaRESUMEN
The C-type lectin receptor Langerin is a glycan-binding protein that serves as an uptake receptor on Langerhans cells and is essential for the formation of Birbeck granules. Whereas most Langerin ligands are recognized by a canonical Ca2+ -dependent binding site, heparins have been proposed to make additional contacts to a secondary, Ca2+ -independent site. Glycan array screening and biomolecular NMR spectroscopy were employed to investigate the molecular mechanism of these interactions. We observed that binding of heparin hexasaccharides to a secondary site did not require the presence of Ca2+ and activated a previously identified intradomain allosteric network of Langerin (thus far only associated with Ca2+ affinity and release). We propose a communication hub between these two binding sites, which sheds new light on modulatory functions of Langerin-heparin interactions.
Asunto(s)
Antígenos CD/química , Heparina/química , Lectinas Tipo C/química , Lectinas de Unión a Manosa/química , Oligosacáridos/química , Regulación Alostérica , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Calcio/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Heparina/metabolismo , Humanos , Células de Langerhans/citología , Células de Langerhans/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Análisis por Micromatrices , Oligosacáridos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Constraining a peptide in its bioactive conformation by macrocyclization represents a powerful strategy to design modulators of challenging biomolecular targets. This holds particularly true for the development of inhibitors of protein-protein interactions which often involve interfaces lacking defined binding pockets. Such flat surfaces are demanding targets for traditional small molecules rendering macrocyclic peptides promising scaffolds for novel therapeutics. However, the contribution of peptide dynamics to binding kinetics is barely understood which impedes the design process. Herein, we report unexpected trends in the binding kinetics of two closely related macrocyclic peptides that bind their receptor protein with high affinity. Isothermal titration calorimetry, 19 Fâ NMR experiments and molecular dynamics simulations reveal that increased conformational flexibility of the macrocycle-receptor complex reduces dissociation rates and contributes to complex stability. This observation has impact on macrocycle design strategies that have so far mainly focused on the stabilization of bioactive ligand conformations.
Asunto(s)
Péptidos/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Sitios de Unión , Calorimetría , Ciclización , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Overexpression of the CREB-binding protein (CBP), a bromodomain-containing transcription coactivator involved in a variety of cellular processes, has been observed in several types of cancer with a correlation to aggressiveness. We have screened a library of nearly 1500 fragments by high-throughput docking into the CBP bromodomain followed by binding energy evaluation using a force field with electrostatic solvation. Twenty of the 39 fragments selected by virtual screening are positive in one or more ligand-observed nuclear magnetic resonance (NMR) experiments. Four crystal structures of the CBP bromodomain in complex with in silico screening hits validate the pose predicted by docking. Thus, the success ratio of the high-throughput docking procedure is 50% or 10% if one considers the validation by ligand-observed NMR spectroscopy or X-ray crystallography, respectively. Compounds 1 and 3 show favorable ligand efficiency in two different in vitro binding assays. The structure of the CBP bromodomain in the complex with the brominated pyrrole 1 suggests fragment growing by Suzuki coupling.
Asunto(s)
Derivados del Benceno/química , Proteína de Unión a CREB/antagonistas & inhibidores , Compuestos Heterocíclicos/química , Proteína de Unión a CREB/química , Simulación por Computador , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Enlace de Hidrógeno , Ligandos , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Dominios Proteicos , Bibliotecas de Moléculas PequeñasRESUMEN
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.
Asunto(s)
Formación de Anticuerpos , Glicoproteína de la Espiga del Coronavirus , Vacunas de Partículas Similares a Virus , Humanos , Animales , Ratones , Anticuerpos Bloqueadores , Vacunas de Partículas Similares a Virus/genética , Anticuerpos Neutralizantes , ADN , Anticuerpos AntiviralesRESUMEN
DNA origami has emerged as a powerful method to generate DNA nanostructures with dynamic properties and nanoscale control. These nanostructures enable complex biophysical studies and the fabrication of next-generation therapeutic devices. For these applications, DNA origami typically needs to be functionalized with bioactive ligands and biomacromolecular cargos. Here, we review methods developed to functionalize, purify, and characterize DNA origami nanostructures. We identify remaining challenges, such as limitations in functionalization efficiency and characterization. We then discuss where researchers can contribute to further advance the fabrication of functionalized DNA origami.
RESUMEN
Multivalent antigen display is a well-established principle to enhance humoral immunity. Protein-based virus-like particles (VLPs) are commonly used to spatially organize antigens. However, protein-based VLPs are limited in their ability to control valency on fixed scaffold geometries and are thymus-dependent antigens that elicit neutralizing B cell memory themselves, which can distract immune responses. Here, we investigated DNA origami as an alternative material for multivalent antigen display in vivo, applied to the receptor binding domain (RBD) of SARS-CoV2 that is the primary antigenic target of neutralizing antibody responses. Icosahedral DNA-VLPs elicited neutralizing antibodies to SARS-CoV-2 in a valency-dependent manner following sequential immunization in mice, quantified by pseudo- and live-virus neutralization assays. Further, induction of B cell memory against the RBD required T cell help, but the immune sera did not contain boosted, class-switched antibodies against the DNA scaffold. This contrasted with protein-based VLP display of the RBD that elicited B cell memory against both the target antigen and the scaffold. Thus, DNA-based VLPs enhance target antigen immunogenicity without generating off-target, scaffold-directed immune memory, thereby offering a potentially important alternative material for particulate vaccine design.
RESUMEN
Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not previously been characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following therapeutically relevant dosage of unmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.
RESUMEN
Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not been previously characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following a therapeutically relevant dosage of nonmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver and kidney biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.
Asunto(s)
Nanopartículas , Ácidos Nucleicos , Ratones , Animales , Distribución Tisular , ADN/química , Ácidos Nucleicos/química , Ácidos Nucleicos/uso terapéutico , Nanopartículas/toxicidad , Nanopartículas/químicaRESUMEN
Viruslike particles (VLPs) fabricated using wireframe DNA origami are emerging as promising vaccine and gene therapeutic delivery platforms due to their programmable nature that offers independent control over their size and shape, as well as their site-specific functionalization. As materials that biodegrade in the presence of endonucleases, specifically DNase I and II, their utility for the targeting of cells, tissues, and organs depends on their stability in vivo. Here, we explore minor groove binders (MGBs) as specific endonuclease inhibitors to control the degradation half-life of wireframe DNA origami. Bare, unprotected DNA-VLPs composed of two-helix edges were found to be stable in fetal bovine serum under typical cell culture conditions and in human serum for 24 h but degraded within 3 h in mouse serum, suggesting species-specific endonuclease activity. Inhibiting endonucleases by incubating DNA-VLPs with diamidine-class MGBs increased their half-lives in mouse serum by more than 12 h, corroborated by protection against isolated DNase I and II. Our stabilization strategy was compatible with the functionalization of DNA-VLPs with HIV antigens, did not interfere with B-cell signaling activity of DNA-VLPs in vitro, and was nontoxic to B-cell lines. It was further found to be compatible with multiple wireframe DNA origami geometries and edge architectures. MGB protection is complementary to existing methods such as PEGylation and chemical cross-linking, offering a facile protocol to control DNase-mediated degradation rates for in vitro and possibly in vivo therapeutic and vaccine applications.
Asunto(s)
Nanoestructuras , Ratones , Humanos , Animales , Conformación de Ácido Nucleico , ADN , Endonucleasas , Desoxirribonucleasa IRESUMEN
DNA origami is a powerful nanomaterial for biomedical applications due in part to its capacity for programmable, site-specific functionalization. To realize these applications, scalable and efficient conjugation protocols are needed for diverse moieties ranging from small molecules to biomacromolecules. Currently, there are no facile and general methods for in situ covalent modification and label-free quantification of reaction conversion. Here, we investigate the postassembly functionalization of DNA origami and the subsequent high-performance liquid chromatography-based characterization of these nanomaterials. Following this approach, we developed a versatile DNA origami functionalization and characterization platform. We observed quantitative in situ conversion using widely accessible click chemistry for carbohydrates, small molecules, peptides, polymers, and proteins. This platform should provide broader access to covalently functionalized DNA origami, as illustrated here by PEGylation for passivation and HIV antigen decoration to construct virus-like particle vaccines.
Asunto(s)
ADN , Nanoestructuras , ADN/química , Nanoestructuras/química , Nanoestructuras/ultraestructura , Conformación de Ácido NucleicoRESUMEN
Neuronal synapses contain hundreds of different protein species important for regulating signal transmission. Characterizing differential expression profiles of proteins within synapses in distinct regions of the brain has revealed a high degree of synaptic diversity defined by unique molecular organization. Multiplexed imaging of in vitro rat primary hippocampal culture models at single synapse resolution offers new opportunities for exploring synaptic reorganization in response to chemical and genetic perturbations. Here, we combine 12-color multiplexed fluorescence imaging with quantitative image analysis and machine learning to identify novel synaptic subtypes within excitatory and inhibitory synapses based on the expression profiles of major synaptic components. We characterize differences in the correlated expression of proteins within these subtypes and we examine how the distribution of these synapses is modified following induction of synaptic plasticity. Under chronic suppression of neuronal activity, phenotypic characterization revealed coordinated increases in both excitatory and inhibitory protein levels without changes in the distribution of synaptic subtypes, suggesting concerted events targeting glutamatergic and GABAergic synapses. Our results offer molecular insight into the mechanisms of synaptic plasticity.
Asunto(s)
Plasticidad Neuronal , Sinapsis , Animales , Hipocampo , Neuronas , Imagen Óptica , Ratas , Transmisión SinápticaRESUMEN
Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy number, spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clinical vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to ~25-30 nm monotonically increases B-cell receptor activation. Moreover, scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos B/inmunología , ADN/ultraestructura , Activación de Linfocitos/inmunología , Nanoestructuras/ultraestructura , Vacunas contra el SIDA , Animales , Antígenos Virales/química , Antígenos Virales/ultraestructura , Línea Celular , ADN/química , Femenino , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Ratones , Nanoestructuras/química , Conformación de Ácido Nucleico , Transducción de SeñalRESUMEN
Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a ß-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.
Asunto(s)
Reactores Biológicos/virología , ADN de Cadena Simple/biosíntesis , Escherichia coli/metabolismo , Microbiología Industrial/métodos , Bacteriófago M13/genética , Reactores Biológicos/economía , ADN de Cadena Simple/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/virología , Microbiología Industrial/economía , Nanotecnología/economía , Nanotecnología/métodos , Plásmidos/genéticaRESUMEN
Structural DNA nanotechnology is beginning to emerge as a widely accessible research tool to mechanistically study diverse biophysical processes. Enabled by scaffolded DNA origami in which a long single strand of DNA is weaved throughout an entire target nucleic acid assembly to ensure its proper folding, assemblies of nearly any geometric shape can now be programmed in a fully automatic manner to interface with biology on the 1-100-nm scale. Here, we review the major design and synthesis principles that have enabled the fabrication of a specific subclass of scaffolded DNA origami objects called wireframe assemblies. These objects offer unprecedented control over the nanoscale organization of biomolecules, including biomolecular copy numbers, presentation on convex or concave geometries, and internal versus external functionalization, in addition to stability in physiological buffer. To highlight the power and versatility of this synthetic structural biology approach to probing molecular and cellular biophysics, we feature its application to three leading areas of investigation: light harvesting and nanoscale energy transport, RNA structural biology, and immune receptor signaling, with an outlook toward unique mechanistic insight that may be gained in these areas in the coming decade.
Asunto(s)
Biofisica/métodos , ADN/química , Biomimética , ADN/metabolismo , ARN/química , ARN/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de SeñalRESUMEN
Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+ myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy.
RESUMEN
Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.