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The design of dynamic, reconfigurable devices is crucial for the bottom-up construction of artificial biological systems. DNA can be used as an engineering material for the de-novo design of such dynamic devices. A self-assembled DNA origami switch is presented that uses the transition from double- to single-stranded DNA and vice versa to create and annihilate an entropic force that drives a reversible conformational change inside the switch. It is distinctively demonstrated that a DNA single-strand that is extended with 0.34 nm per nucleotide - the extension this very strand has in the double-stranded configuration - exerts a contractive force on its ends leading to large-scale motion. The operation of this type of switch is demonstrated via transmission electron microscopy, DNA-PAINT super-resolution microscopy and darkfield microscopy. The work illustrates the intricate and sometimes counter-intuitive forces that act in nanoscale physical systems that operate in fluids.

Single-Molecule Localization Microscopy (SMLM) has revolutionized light microscopy by enabling optical resolution down to a few nanometer. Yet, localization precision commonly doesn’t suffice to visually resolve single subunits in molecular assemblies or multimeric complexes. Since each targeted molecule contributes localizations during image acquisition, molecular counting approaches to reveal the target copy numbers within localization clusters have been persistently proposed since the early days of SMLM, most of which rely on preliminary knowledge of the dye photo-physics or on a calibration to a reference. Previously, we developed localization-based Fluorescence Correlation Spectroscopy (lbFCS) as an absolute ensemble counting approach for the SMLM-variant DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography), for the first time circumventing the necessity for reference calibrations. Here, we present an extended concept termed lbFCS+ which allows absolute counting of copy numbers for individual localization clusters in a single DNA-PAINT image. In lbFCS+, absolute counting of fluorescent loci contained in individual nanoscopic volumes is achieved via precise measurement of the local hybridization rates of the fluorescently-labeled oligonucleotides (’imagers’) employed in DNA-PAINT imaging. In proof-of-principle experiments on DNA origami nanostructures, we demonstrate the ability of lbFCS+ to truthfully determine molecular copy numbers and imager association and dissociation rates in well-separated localization clusters containing up to ten docking strands. For N ≤ 4 target molecules, lbFCS+ is even able to resolve integers, providing the potential to study the composition of up to tetrameric molecular complexes. Further, we show that lbFCS+ allows to resolve heterogeneous binding dynamics enabling the distinction of stochastically generated and a priori indistinguishable DNA assemblies. Beyond advancing quantitative DNA-PAINT imaging, we believe that lbFCS+ could find promising applications ranging from bio-sensing to DNA computing.

Single-Molecule Localization Microscopy (SMLM) has revolutionized light microscopy by enabling optical resolutions down to a few nanometer. Yet, localization precisions commonly not suffice to visually resolve single subunits in molecular assemblies or multimeric complexes. Since each targeted molecule contributes localizations during image acquisition, molecular counting approaches to reveal the target copy numbers within localization clusters have been continuously proposed since the early days of SMLM, most of which rely on preliminary knowledge of the dye photo-physics or on a calibration to a reference. Previously, we developed localization-based Fluorescence Correlation Spectroscopy (lbFCS) as an absolute ensemble counting approach for the SMLM-variant DNA-Points Accumulation for Imaging in Nanoscale Topography (PAINT), for the first time circumventing the necessity for reference calibrations. Here, we present a revised framework termed lbFCS+ which allows absolute counting of copy numbers for individual localization clusters in a single DNA-PAINT image. In lbFCS+, absolute counting in individual clusters is achieved via precise measurement of the local hybridization rates of the fluorescently-labeled oligonucleotides (‘imagers’) employed in DNA-PAINT imaging. In proof-of-principle experiments on DNA origami nanostructures, we demonstrate the ability of lbFCS+ to truthfully determine molecular copy numbers and imager association and dissociation rates in well-separated localization clusters containing up to six docking strands. We show that lbFCS+ allows to resolve heterogeneous binding dynamics enabling the distinction of stochastically generated and a priori indistinguishable DNA assemblies. Beyond advancing quantitative DNA-PAINT imaging, we believe that lbFCS+ could find promising applications ranging from bio-sensing to DNA computing.

DNA points accumulation in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement super-resolution technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resolution. This improvement now makes DNA-PAINT applicable to high-throughput studies.

Particle fusion for single molecule localization microscopy improves signal-to-noise ratio and overcomes underlabeling, but ignores structural heterogeneity or conformational variability. We present a-priori knowledge-free unsupervised classification of structurally different particles employing the Bhattacharya cost function as dissimilarity metric. We achieve 96% classification accuracy on mixtures of up to four different DNA-origami structures, detect rare classes of origami occuring at 2% rate, and capture variation in ellipticity of nuclear pore complexes., Particle fusion can improve signal-to-noise ratio in single molecule localization microscopy, but is limited by structural heterogeneity. Here, the authors demonstrate an unsupervised classification method that differentiates structurally different DNA origami structures without prior knowledge.

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.

The specificity and simplicity of the Watson–Crick base pair interactions make DNA one of the most versatile construction materials for creating nanoscale structures and devices. Among several DNA-based approaches, the DNA origami technique excels in programmable self-assembly of complex, arbitrary shaped structures with dimensions of hundreds of nanometers. Importantly, DNA origami can be used as templates for assembly of functional nanoscale components into three-dimensional structures with high precision and controlled stoichiometry. This is often beyond the reach of other nanofabrication techniques. In this Perspective, we highlight the capability of the DNA origami technique for realization of novel nanophotonic systems. First, we introduce the basic principles of designing and fabrication of DNA origami structures. Subsequently, we review recent advances of the DNA origami applications in nanoplasmonics, single-molecule and super-resolution fluorescent imaging, as well as hybrid photonic systems. We conclude by outlining the future prospects of the DNA origami technique for advanced nanophotonic systems with tailored functionalities.

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., Probe performance: Using programmable, antigen‐decorated designer DNA origami nanostructures, performance of labeling probes, including antibodies and nanobodies, is tested. The platform reveals that primary‐secondary antibody labeling can only resolve antigens spaced down to 40 nm. The nanostructures enable performance evaluation of a multitude of labeling probes for super‐resolution microscopy.

Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.

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., The design and optimisation of 3D DNA-origami can be a barrier to rapid application. Here the authors design barrel structure of stacked 2D double helical rings with complex surface patterns.

T-cells detect with their T-cell antigen receptors (TCRs) the presence of rare peptide/MHC complexes (pMHCs) on the surface of antigen presenting cells (APCs). How they convert a biochemical interaction into a signaling response is poorly understood, yet indirect evidence pointed to the spatial antigen arrangement on the APC surface as a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. We found that the minimum signaling unit required for efficient T-cell activation consisted of two ligated TCRs within a distance of 20 nanometers, if TCRs were stably engaged by monovalent antibody fragments. In contrast, antigenic pMHCs stimulated T-cells robustly as well-isolated entities. These results identify the minimal requirements for effective TCR-triggering and validate the exceptional stimulatory potency of transiently engaging pMHCs.

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales., The length of single-particle tracking experiments are limited due to photobleaching. Here the authors achieve long-term single-particle tracking with continuous fluorophore exchange in DNA-PAINT and use this to observe DNA origami on lipid bilayers for tens of minutes.

Correlating DNA-PAINT (point accumulation for imaging in nanoscale topography) and single-molecule FRET (Forster resonance energy transfer) enables the multiplexed detection with sub-diffraction optical resolution. We designed pairs of short oligonucleotides, labeled with donor and acceptor fluorophores with various distances generating different FRET efficiencies. The strands can transiently bind to a target docking strand, simultaneous binding of both strands results in FRET signals which yield a super-resolved image via DNA-PAINT imaging. We demonstrate FRET-PAINT by designing and imaging DNA origami, which is a useful tool to establish super-resolution methods. The DNA origami structures were equipped with three target binding sites spaced by 55 nm, a sub-diffraction limited distance, however ensuring that no FRET between the target sites occurs. We resolved the individual binding sites in the donor and acceptor channels, and in addition extracted the FRET efficiency for each site in single and mixed populations. The combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging in conjunction with distance-sensitive readout in the 1 to 10 nm range.

We present a method for extracting high-resolution ordered features from localisation microscopy data by analysis of relative molecular positions in 2D or 3D. This approach allows pattern recognition at sub-1% protein detection efficiencies, in large and heterogeneous samples, and in 2D and 3D datasets. We used this method to infer ultrastructure of the nuclear pore, the cardiomyocyte Z-disk, DNA origami structures and the centriole.

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. We thank Stefan Pettera and Stephan Uebel from the MPIB peptide synthesis service for the production of the peptides and for their advice regarding the peptide modifications.

We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates.

We report the development of multiplexed cellular super-resolution imaging using DNA-barcoded binders., Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells.

Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary ‘blinking’ without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation., The use of TIRF microscopy for DNA-PAINT experiments is limited by inhomogeneous illumination. Here the authors show that quantitative analysis of single-molecule TIRF experiments can be improved by using a segment-wise analysis approach and overcome by using a beam-shaping device to give a flat-top illumination profile.

DNA nanostructures offer the possibility to mimic functional biological membrane components due to their nanometer-precise shape configurability and versatile biochemical functionality. Here we show that the diffusional behavior of DNA nanostructures and their assembly into higher order membrane-bound lattices can be controlled in a stop-and-go manner and that the process can be monitored with super-resolution imaging. The DNA structures are transiently immobilized on glass-supported lipid bilayers by changing the mono- and divalent cation concentrations of the surrounding buffer. Using DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy, we confirm the fixation of DNA origami structures with different shapes. On mica-supported lipid bilayers, in contrast, we observe residual movement. By increasing the concentration of NaCl and depleting MgCl2, a large fraction of DNA structures restarts to diffuse freely on both substrates. After addition of a set of oligonucleotides that enables three Y-shaped monomers to assemble into a three-legged shape (triskelion), the triskelions can be stopped and super-resolved. Exchanging buffer and adding another set of oligonucleotides triggers the triskelions to diffuse and assemble into hexagonal 2D lattices. This stop-and-go imaging technique provides a way to control and observe the diffusional behavior of DNA nanostructures on lipid membranes that could also lead to control of membrane-associated cargos.

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.

DNA-PAINT is an optical super-resolution microscopy method that can visualize nanoscale protein arrangements and provide spectrally unlimited multiplexing capabilities. However, current multiplexing implementations based on, for example, DNA exchange (such as Exchange-PAINT) achieves multitarget detection by sequential imaging, limiting throughput. Here, we combine DNA-PAINT with single-molecule FRET and use the FRET efficiency as parameter for multiplexed imaging with high specificity. We demonstrate correlated single-molecule FRET and super-resolution on DNA origami structures, which are equipped with binding sequences that are targeted by pairs of dye-labeled oligonucleotides generating the FRET signal. We futher extract FRET values from single binding sites that are spaced just ∼55 nm apart, demonstrating super-resolution FRET imaging. This combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging with low background and opens the door for accurate distance readout in the 1-10 nm range.

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.

Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density.

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., Existing methods for nanoscale visualization of biological targets in thick samples require complex hardware. Here, the authors combine the standard spinning disk confocal (SDC) microscopy with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) to image proteins, DNA and RNA deep in cells.

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.

Programmable DNA-based metafluorophores allow simultaneous tagging and imaging with more than 100 virtual colors., 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.

Structural DNA nanotechnology is revolutionizing the ways researchers construct arbitrary shapes and patterns in two and three dimensions on the nanoscale. Through Watson-Crick base pairing, DNA can be programmed to form nanostructures with high predictability, addressability, and yield. The ease with which structures can be designed and created has generated great interest for using DNA for a variety of metrology applications, such as in scanning probe microscopy and super-resolution imaging. An additional advantage of the programmable nature of DNA is that mechanisms for nanoscale metrology of the structures can be integrated within the DNA objects by design. This programmable structure-property relationship provides a powerful tool for developing nanoscale materials and smart rulers.

Engineering Larger DNA Structures Several approaches now exist for the self-assembly of DNA into nanostructures. For example, three-arm DNA tripods can be assembled into larger wireframe polyhedra, but for the most complicated shapes, assembly yields can be low, apparently because the flexibility of smaller tripods allows for misassembly. Iinuma et al. (p. 65 , published online 13 March) now show that larger, stiffer tripods that have controlled arm lengths and interarm angles can be designed to form a wide variety of open wireframe polyhedra—including tetrahedra, cubes, and hexagonal prisms, with edges 100 nanometers in length.

Furthermore, as shown by Zhang and co-workers, where longrange PCR was used for the production of single-stranded scaffolds of up to 26 kb, [ 17 ] alternative scaffold sources enable the assembly of larger single structures while avoiding often low-yield hierarchical assembly procedures. [ 18–20 ] All these methods extend the DNA origami technique considerably but also rely on various time consuming and tedious modifi cation steps of either naturally occurring or synthetic template DNA such as several purifi cation steps, PCR, strand separation, enzymatic digestion and/or modifi cation. In addition, for many promising applications of the DNA origami technique – such as nanomedicine or drug delivery – a large quantity of the material is needed. Scaling up the assembly process and maximizing the assembly yield while reducing both labor and cost remains a major challenge for the future development of the fi eld. [ 21 ]

Push Me, Release, Pull You In eukaryotic cells, nearly all long-distance transport of cargos is carried out by the microtubule-based motors kinesin and dynein. These opposite-polarity motors move cargos bidirectionally so that they reach their cellular destinations with spatial and temporal specificity. To understand transport by motor ensembles, Derr et al. (p. 662 , published online 11 October; see the Persective by Diehl ) used a DNA scaffold for building an artificial cargo that could be programmed to bind different numbers and types of molecular motors with defined geometry. A cargo with multiple copies of the same motor was transported with minimal interference, suggesting that similar-polarity motors can coordinate without the need for additional cellular factors. However, ensembles of opposite-polarity motors frequently engaged in a sort of “tug of war,” which could only be resolved by releasing one motor from the microtubule track. Thus, within the cell, it is likely that regulation is required for bidirectional transport.

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with

Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to 10-20 nm in size to be imaged. However, the direct optical observation of individual molecular targets (similar to 5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography) a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridization on synthetic DNA nanostructures. We examined the effects of a high photon count, high blinking statistics and an appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves

Recent advances in super-resolution microscopy have allowed the circumvention of the classical diffraction limit of light, and demonstrated the ability of imaging macromolecular protein complexes with single particle averaging methods. However, the ability to obtain fluorescence images within a single molecular complex that allows the study of molecular heterogeneity and low copy number complexes is still challenging, and remains an important goal for the future development of super-resolution microscopy. We here report early results towards this goal of “single-molecule digital imaging” using DNA-PAINT. We introduced three technical advances, including better localization accuracy by obtaining more photons per binding/blinking event, a novel template-based drift correction algorithm, and wide-range tuning of blinking duty cycles for high-density imaging. We demonstrated our approach to obtain molecular-scale resolution on a synthetically designed DNA origami nanostructure, which is made of closely packed (spaced 5 nm apart) DNA molecules with imaging sites, arranged in a “digital” grid pattern. Using small molecule-based oligonucleotide labeling or genetically encoded tags, our imaging technology could achieve molecular resolution within single-molecule complexes. It could be applied to a wide range of biological and biomedical applications including the analysis of densely packed cell surface receptor clusters, cytoplasmic signaling pathways, or genomic organization and short-range interactions.

DNA origami is a revolutionary method for the assembly of molecular nanostructures from DNA with precisely defined dimensions and with an unprecedented yield. This can be utilized to arrange nanoscale components such as proteins or nanoparticles into pre-defined patterns. For applications it will now be of interest to arrange such components into functional complexes and study their geometry-dependent interactions. While commonly DNA nanostructures are characterized by atomic force microscopy or electron microscopy, these techniques often lack the time-resolution to study dynamic processes. It is therefore of considerable interest to also apply fluorescence microscopic techniques to DNA nanostructures. Of particular importance here is the utilization of novel super-resolved microscopy methods that enable imaging beyond the classical diffraction limit.

A variety of polymerization methods for the assembly of elongated nanoribbons from rectangular DNA origami structures are investigated. The most efficient method utilizes single-stranded DNA oligonucleotides to bridge an intermolecular scaffold seam between origami monomers. This approach allows the fabrication of origami ribbons with lengths of several micrometers, which can be used for long-range ordered arrangement of proteins. It is quantitatively shown that the length distribution of origami ribbons obtained with this technique follows the theoretical prediction for a simple linear polymerization reaction. The design of flat single layer origami structures with constant crossover spacing inevitably results in local underwinding of the DNA helix, which leads to a global twist of the origami structures that also translates to the nanoribbons.

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. We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support.

Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20 × higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.

DNA origami is one of the most promising recent developments in DNA self-assembly. It allows for the construction of arbitrary nanoscale patterns and objects by folding a long viral scaffold strand using a large number of short “staple” strands. Assembly is usually accomplished by thermal annealing of the DNA molecules in buffer solution. We here demonstrate that both 2D and 3D origami structures can be assembled isothermally by annealing the DNA strands in denaturing buffer, followed by a controlled reduction of denaturant concentration. This opens up origami assembly for the integration of temperature-sensitive components.