RESUMEN
DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.
Asunto(s)
ADN/química , Imagenología Tridimensional , Conformación de Ácido Nucleico , Dimerización , Modelos MolecularesRESUMEN
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
Optical super-resolution microscopy is rapidly changing the way imaging studies in the biological and biomedical sciences are conducted. Due to the unique capability of achieving molecular contrast using fluorescent labels and sub-diffraction resolution down to a few tens of nanometers, super-resolution is developing as an attractive imaging modality. While the increased spatial resolution has already enabled structural studies at unprecedented molecular detail, the wide-spread use of super-resolution approaches as a standard characterization technique in biological laboratories has thus far been prevented by mainly two issues: (1) Intricate sample preparation and image acquisition and (2) costly and complex instrumentation. We here introduce a combination of the recently developed super-resolution technique DNA-PAINT (DNA points accumulation for imaging in nanoscale topography) with an easy-to-replicate, custom-built 3D single-molecule microscope (termed liteTIRF) that is an order of magnitude more economic in cost compared to most commercial systems. We assay the performance of our system using synthetic two- and three-dimensional DNA origami structures and show the applicability to single- and multiplexed cellular imaging.
RESUMEN
Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.
Asunto(s)
ADN/química , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Imagen Individual de Molécula/métodos , Fibroblastos , Células HeLa , Humanos , Imagenología Tridimensional/instrumentación , Hibridación Fluorescente in Situ , Sustancias Macromoleculares/análisis , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Oligonucleótidos/química , ARN/química , Imagen Individual de Molécula/instrumentación , Coloración y EtiquetadoRESUMEN
To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from â¼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.
Asunto(s)
ADN/química , Hipocampo/citología , Inmunoconjugados/química , Microscopía Confocal/métodos , Neuronas/ultraestructura , Imagen Óptica/métodos , Mapeo de Interacción de Proteínas/métodos , Animales , Encéfalo/ultraestructura , Células Cultivadas , Colorantes Fluorescentes/química , Hipocampo/ultraestructura , Ratones , Microscopía Fluorescente/métodos , Neuronas/citología , Retina/citología , Retina/ultraestructura , Coloración y Etiquetado/métodos , Sinapsinas/análisis , Sinaptofisina/análisisRESUMEN
Fluorescence microscopy allows specific target detection down to the level of single molecules and has become an enabling tool in biological research. To transduce the biological information to an imageable signal, we have developed a variety of fluorescent probes, such as organic dyes or fluorescent proteins with different colors. Despite their success, a limitation on constructing small fluorescent probes is the lack of a general framework to achieve precise and programmable control of critical optical properties, such as color and brightness. To address this challenge, we introduce metafluorophores, which are constructed as DNA nanostructure-based fluorescent probes with digitally tunable optical properties. Each metafluorophore is composed of multiple organic fluorophores, organized in a spatially controlled fashion in a compact sub-100-nm architecture using a DNA nanostructure scaffold. Using DNA origami with a size of 90 × 60 nm2, substantially smaller than the optical diffraction limit, we constructed small fluorescent probes with digitally tunable brightness, color, and photostability and demonstrated a palette of 124 virtual colors. Using these probes as fluorescent barcodes, we implemented an assay for multiplexed quantification of nucleic acids. Additionally, we demonstrated the triggered in situ self-assembly of fluorescent DNA nanostructures with prescribed brightness upon initial hybridization to a nucleic acid target.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Nanoestructuras/química , Fenómenos Biofísicos , Microscopía Fluorescente , Nanotecnología , Conformación de Ácido Nucleico , Ácidos Nucleicos/químicaRESUMEN
Counting molecules in complexes is challenging, even with super-resolution microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative points accumulation in nanoscale topography (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the number of single-molecule FISH probes bound to an mRNA target.
Asunto(s)
ADN/química , ADN/ultraestructura , Aumento de la Imagen/métodos , Hibridación Fluorescente in Situ/métodos , Microscopía Fluorescente/métodos , Animales , Colorantes Fluorescentes/química , Humanos , Microscopía Confocal/métodos , Simulación del Acoplamiento Molecular , Análisis de Secuencia de ADN , Programas InformáticosRESUMEN
Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Nanotecnología/métodos , Fluorescencia , Nanoestructuras/química , Proteínas/químicaRESUMEN
Synthetic polymers are ubiquitous in the modern world, but our ability to exert control over the molecular conformation of individual polymers is very limited. In particular, although the programmable self-assembly of oligonucleotides and proteins into artificial nanostructures has been demonstrated, we currently lack the tools to handle other types of synthetic polymers individually and thus the ability to utilize and study their single-molecule properties. Here we show that synthetic polymer wires containing short oligonucleotides that extend from each repeat can be made to assemble into arbitrary routings. The wires, which can be more than 200â nm in length, are soft and bendable, and the DNA strands allow individual polymers to self-assemble into predesigned routings on both two- and three-dimensional DNA origami templates. The polymers are conjugated and potentially conducting, and could therefore be used to create molecular-scale electronic or optical wires in arbitrary geometries.
Asunto(s)
ADN/química , Nanoestructuras/química , Nanotecnología/métodos , Polímeros/química , ADN de Cadena Simple/química , Nanoestructuras/ultraestructura , Nanocables/química , Nanocables/ultraestructura , Conformación de Ácido Nucleico , Oligonucleótidos/química , Polivinilos/químicaRESUMEN
A small and compact DNA cube with zeptoliter volume is constructed by means of a generalized DNA brick concept using short synthetic oligonucleotides with varying lengths. By mimicking design principles from the DNA origami technique, the DNA cube offers higher stability and assembly yields compared to other approaches. Its potential application as nanoscale fluorescent probe is demonstrated using super-resolution imaging.
Asunto(s)
ADN/química , ADN/ultraestructura , Colorantes Fluorescentes/síntesis química , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la PartículaRESUMEN
DNA self-assembly has produced diverse synthetic three-dimensional polyhedra. These structures typically have a molecular weight no greater than 5 megadaltons. We report a simple, general strategy for one-step self-assembly of wireframe DNA polyhedra that are more massive than most previous structures. A stiff three-arm-junction DNA origami tile motif with precisely controlled angles and arm lengths was used for hierarchical assembly of polyhedra. We experimentally constructed a tetrahedron (20 megadaltons), a triangular prism (30 megadaltons), a cube (40 megadaltons), a pentagonal prism (50 megadaltons), and a hexagonal prism (60 megadaltons) with edge widths of 100 nanometers. The structures were visualized by means of transmission electron microscopy and three-dimensional DNA-PAINT super-resolution fluorescent microscopy of single molecules in solution.
Asunto(s)
ADN/química , Nanoestructuras/química , Conformación de Ácido Nucleico , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Peso Molecular , NanotecnologíaRESUMEN
Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.