RESUMEN
To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.
Asunto(s)
Proteómica , Imagen Individual de Molécula , ADN , Microscopía Fluorescente/métodos , Neuronas , ProteínasRESUMEN
Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Adenosina Trifosfato/química , Sitios de Unión/genética , Sitios de Unión/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Cromosomas/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Humanos , Complejos Multiproteicos/fisiología , Unión Proteica/fisiología , Subunidades de Proteína/metabolismo , CohesinasRESUMEN
The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.
Asunto(s)
ADN Bacteriano , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , ADN Bacteriano/metabolismo , ADN Bacteriano/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Unión Proteica , Conformación de Ácido Nucleico , ADN/química , ADN/metabolismoRESUMEN
Improving labeling probes for state-of-the-art super-resolution microscopy is becoming of major importance. However, there is currently a lack of tools to quantitatively evaluate probe performance regarding efficiency, precision, and achievable resolution in an unbiased yet modular fashion. Herein, we introduce designer DNA origami structures combined with DNA-PAINT to overcome this issue and evaluate labeling efficiency, precision, and quantification using antibodies and nanobodies as exemplary labeling probes. Whereas current assessment of binders is mostly qualitative, e. g. based on an expected staining pattern, we herein present a quantitative analysis platform of the antigen labeling efficiency and achievable resolution, allowing researchers to choose the best performing binder. The platform can furthermore be readily adapted for discovery and precise quantification of a large variety of additional labeling probes.
Asunto(s)
ADN/química , Nanoestructuras/química , Microscopía FluorescenteRESUMEN
Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-molecule-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, we here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. We benchmark and optimize its binding kinetics in a single-molecule assay and demonstrate its super-resolution capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, we prove the suitability of Peptide-PAINT for cellular super-resolution imaging by visualizing the microtubule and vimentin network in fixed cells.
Asunto(s)
ADN , Oligonucleótidos , Microscopía Fluorescente , Nanotecnología , PéptidosRESUMEN
Innovation in genomics, transcriptomics, and proteomics research has created a plethora of state-of-the-art techniques such as nucleic acid sequencing and mass-spectrometry-based proteomics with paramount impact in the life sciences. While current approaches yield quantitative abundance analysis of biomolecules on an almost routine basis, coupling this high content to spatial information in a single cell and tissue context is challenging. Here, current implementations of spatial omics are discussed and recent developments in the field of DNA-barcoded fluorescence microscopy are reviewed. Light is shed on the potential of DNA-based imaging techniques to provide a comprehensive toolbox for spatial genomics and transcriptomics and discuss current challenges, which need to be overcome on the way to spatial proteomics using high-resolution fluorescence microscopy.
Asunto(s)
ADN , Genómica , Microscopía Fluorescente , ProteómicaRESUMEN
Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.
Asunto(s)
ADN/química , Microscopía Fluorescente/métodos , Proteínas/aislamiento & purificación , ARN/aislamiento & purificación , Simulación por Computador , Cinética , Conformación de Ácido Nucleico , Oligonucleótidos/química , Proteínas/química , ARN/químicaRESUMEN
Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Bacterias/inmunología , ADN/química , Microscopía Fluorescente/métodos , Línea Celular Tumoral , Humanos , Unión ProteicaRESUMEN
Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/química , Conformación de Ácido Nucleico , ADN/metabolismo , Cartilla de ADN/metabolismo , Proteínas de Unión al ADN/química , Transferencia Resonante de Energía de Fluorescencia , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Iones , Cinética , Sustancias Macromoleculares/metabolismo , Nucleótidos/metabolismo , Unión ProteicaRESUMEN
DNA supercoiling crucially affects cellular processes such as DNA replication, gene expression, and chromatin organization. However, mechanistic understanding of DNA supercoiling and the related DNA-processing enzymes has remained limited, mainly due to the lack of convenient experimental tools to probe these phenomena. Here, we report a novel high-throughput single-molecule assay for real-time visualization of supercoiled DNA molecules, named ISD (Intercalation-induced Supercoiling of DNA). We use an intercalating dye to induce supercoiling of surface-attached DNA molecules as well as to visualize coiled-loop structures (i.e., plectonemes) formed on DNA. The technique is solely based on epifluorescence microscopy and requires no mechanical manipulation of the DNA molecules. This new assay allows to track positions and sizes of individual plectonemes and characterize their position-dependent dynamics such as nucleation, termination, and diffusion. We describe the ISD technique and demonstrate its potential by establishing that plectonemes are pinned to a local 10-nucleotide long mispaired sequence along a double-stranded DNA molecule.
Asunto(s)
ADN Superhelicoidal/química , Fluorescencia , Difusión , Conformación de Ácido NucleicoRESUMEN
The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.
RESUMEN
Super-resolution imaging is becoming a commonly employed tool to visualize biological targets in unprecedented detail. DNA-PAINT is one of the single-molecule localization microscopy-based super-resolution imaging modalities allowing the ultra-high-resolution imaging with superior multiplexing capabilities. We discuss the importance of patterned DNA nanostructures in demonstrating the capabilities of DNA-PAINT and the design of various combinations of imager-docking strand pairs for imaging. Central to the implementation of DNA-PAINT imaging in a biological context is the generation of docking strand-conjugated binders against the target molecules. Several researchers have developed a variety of labelling probes for improving resolution while also providing multiplexing capabilities for the broader application of DNA-PAINT. This review provides a comprehensive summary of the repertoire of labelling probes used for DNA-PAINT in cells and the strategies implemented to chemically modify them with a docking strand.
Asunto(s)
ADN , Nanoestructuras , Microscopía Fluorescente/métodos , ADN/química , Nanoestructuras/química , Imagen Individual de MoléculaRESUMEN
The DNA double helix structure is stabilized by base-pairing and base-stacking interactions. However, a comprehensive understanding of dinucleotide base-stacking energetics is lacking. Here we combined multiplexed DNA-based point accumulation in nanoscale topography (DNA-PAINT) imaging with designer DNA nanostructures and measured the free energy of dinucleotide base stacking at the single-molecule level. Multiplexed imaging enabled us to extract the binding kinetics of an imager strand with and without additional dinucleotide stacking interactions. The DNA-PAINT data showed that a single additional dinucleotide base stacking results in up to 250-fold stabilization for the DNA duplex nanostructure. We found that the dinucleotide base-stacking energies vary from -0.95 ± 0.12 kcal mol-1 to -3.22 ± 0.04 kcal mol-1 for C|T and A|C base-stackings, respectively. We demonstrate the application of base-stacking energetics in designing DNA-PAINT probes for multiplexed super-resolution imaging, and efficient assembly of higher-order DNA nanostructures. Our results will aid in designing functional DNA nanostructures, and DNA and RNA aptamers, and facilitate better predictions of the local DNA structure.
Asunto(s)
ADN , Nanoestructuras , ADN/química , Emparejamiento Base , Nanotecnología/métodos , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we visualize plectonemes (extended intertwined DNA structures formed upon supercoiling) on individual DNA molecules. Remarkably, our experiments show that the DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific sequences. We develop a physical model that predicts that sequence-dependent intrinsic curvature is the key determinant of pinning strength and demonstrate this simple model provides very good agreement with the data. Analysis of several prokaryotic genomes indicates that plectonemes localize directly upstream of promoters, which we experimentally confirm for selected promotor sequences. Our findings reveal a hidden code in the genome that helps to spatially organize the chromosomal DNA.
Asunto(s)
ADN Bacteriano/química , ADN Superhelicoidal/química , Plásmidos/química , Secuencia de Bases , Biotina/química , Carbocianinas/química , ADN Bacteriano/genética , ADN Superhelicoidal/genética , Colorantes Fluorescentes/química , Microscopía Fluorescente , Compuestos Orgánicos/química , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Estreptavidina/químicaRESUMEN
It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation and processive extension of DNA loops by yeast condensin in real time. Our findings constitute unambiguous evidence for loop extrusion. We observed that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1500 base pairs per second, using the energy of adenosine triphosphate hydrolysis. Condensin-induced loop extrusion was strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/química , ADN/química , Complejos Multiproteicos/química , Conformación de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/química , Imagen Individual de Molécula/métodos , Adenosina Trifosfato/química , Hidrólisis , Factores de TiempoRESUMEN
The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.