nanoscale imaging in dna nanotechnology

Advanced Review

Nanoscale imaging in DNAnanotechnologyRalf Jungmann,1 Max Scheible2 and Friedrich C. Simmel2∗

DNA nanotechnology has developed powerful techniques for the constructionof precisely defined molecular structures and machines, and nanoscale imagingmethods have always been crucial for their experimental characterization. Whileinitially atomic force microscopy (AFM) was the most widely employed imagingmethod for DNA-based molecular structures, in recent years a variety of othertechniques were adopted by researchers in the field, namely electron microscopy(EM), super-resolution fluorescence microscopy, and high-speed AFM. EM isnow typically applied for the characterization of compact nanoobjects and three-dimensional (3D) origami structures, as it offers better resolution than AFM and canbe used for 3D reconstruction from single-particle analysis. While the small size ofDNA nanostructures had previously precluded the application of fluorescencemicroscopic methods, the development of super-resolution microscopy nowfacilities the application of fast and powerful optical methods also in DNAnanotechnology. In particular, the observation of dynamical processes associatedwith DNA nanoassemblies—e.g., molecular walkers and machines—requiresimaging techniques that are both fast and allow observation under nativeconditions. Here single-molecule fluorescence techniques and high-speed AFMare beginning to play an increasingly important role. © 2011 Wiley Periodicals, Inc.

How to cite this article:WIREs Nanomed Nanobiotechnol 2012, 4:66–81. doi: 10.1002/wnan.173

INTRODUCTION

Our ability to produce structures on the nanoscaleis intimately linked to our ability to characterize

them. Without a possibility to ‘check’ whether wehave produced the molecular structure aimed for, wewould not receive the experimental feedback neededto make progress in our nanoscale endeavors. In thepast, researchers most often had to rely on ‘indirect’methods to collect evidence for the correct assemblyof a desired target structure. Of course, the wholerange of analytical chemical methods belongs here,or powerful scattering techniques that have beendeveloped for the elucidation of atomic structure ofmaterials or macromolecules. In many cases, however,these methods do not provide all of the information

∗Correspondence to: [email protected] of Systems Biology, Harvard Medical School, WyssInstitute for Biologically Inspired Engineering, Harvard University,Boston, MA, USA2Department of Physics, ZNN/WSI, Technische UniversitatMunchen, Garching, Germany

one would like to have from a structure, and mostof them do not allow for monitoring dynamicprocesses. Moreover, many of these methods are noteven applicable because the molecule numbers (orconcentrations) in nanoscience are often too small toproduce a measurable signal.

Direct imaging techniques such as scanningprobe microscopy (SPM) and electron microscopy(EM) can, in principle, be used to study singlenanoobjects, and therefore naturally overcomesome of the problems associated with ensemblemeasurements. Imaging techniques therefore generallyplay a prominent role in nanoscale science andtechnology, and they have also been decisive for thedevelopment of DNA nanotechnology.

When Seeman started this field in 1982,1 he envi-sioned the utilization of artificial branched DNA struc-tures for the arrangement of nanoobjects attached tothe DNA—e.g., proteins—in three-dimensional (3D)space. Such ‘DNA crystals’ would be extremely use-ful for structural studies on proteins that are hardto crystallize otherwise. For crystallographic studies,

66 © 2011 Wiley Per iodica ls, Inc. Volume 4, January/February 2012

WIREs Nanomedicine and Nanobiotechnology Nanoscale imaging in DNA nanotechnology

however, large structures with perfect order would berequired, and it took almost 30 years until Seemanachieved his original goal. In the decade followingSeeman’s proposal, a variety of DNA objects were pro-duced in his lab, but were mainly characterized usingbiochemical methods, most often gel electrophore-sis. A major breakthrough for the field came withthe first application of an imaging technique for theanalysis of two-dimensional (2D) DNA assemblies:in 1998, Winfree in collaboration with Seeman forthe first time used an atomic force microscope (AFM)to image 2D lattices made from so-called ‘double-crossover’ (DX) DNA tiles originally developed inSeeman’s lab.2 This is just an example how influentialthe interplay of novel assembly methods and charac-terization techniques can be. The AFM has becomeone of the standard tools in the field ever since andis the ‘workhorse’ for most of the groups working onDNA nanotechnology.

In this review, we will give a short introductioninto the imaging techniques typically used in thefield of DNA nanotechnology. Apart from the‘standard’ techniques such as AFM and EM, wealso cover optical techniques such as super-resolutionfluorescence microscopy.

TECHNIQUES

Atomic Force MicroscopyThe era of SPM began in 1982 with the inventionof the scanning tunneling microscope (STM).3 Thismicroscope allowed researchers for the first timeto visualize matter on the level of single atoms. InSTM, a sharp tip is raster scanned over a surfaceand its distance is controlled using the tunnelingcurrent from tip to surface as a control signal.Experiments are usually performed in vacuum andat low temperatures—requirements, which precludemost applications in biology. Scanning probe imagingof biomolecules on the nanoscale became possiblewith the invention of the AFM in 1986.4 Usingthe STM’s concept of raster scanning a sharp probeacross a surface, AFMs detect surface topographyby directly measuring forces between probe andsurface. The measurements can be performed underambient conditions and samples do not haveto be conductive. Further technical improvementssuch as imaging in liquids5 and dynamic imagingmodes6–9 quickly made AFM one of the mostversatile tools for the characterization of biologicalsamples—and of DNA-based nanostructures inparticular—at high-spatial resolution and near nativeconditions.

Imaging ModesIn the most direct AFM imaging mode—contactmode AFM—probe and sample are in continuouscontact with each other during the raster-scan process.Although contact mode allows for relatively high-image speeds and resolution, the constant contact ofprobe and surface leads to high-lateral forces thatcan damage soft samples like DNA. The limitationsof contact mode for imaging biological samples ledto the invention of non-contact imaging in 19876

and finally intermittent contact or tapping modeimaging in 1993.7 In these dynamic modes, thecantilever is excited by a piezocrystal to oscillate ata distance of a few nanometers above the surfaceand interactions with the substrate are detected aschanges in the oscillation amplitude. Intermittentcontact or tapping mode combines the advantagesof contact and non-contact imaging by ‘tapping’ thesample surface during each oscillation, thus obtainingtip-limited resolution while maintaining low lateralforces. Although tapping mode imaging highly reducesdestructive forces in the lateral direction, forcesperpendicular to the sample are relatively high due tocapillary forces induced by a water layer on the samplesurface in ambient conditions. These destructive forcescan be highly reduced when imaging in liquid, whichwas first demonstrated in 1992 for contact5 and 1994for tapping mode.8,9 AFM operation in liquid hasthe important advantage that biological samples likeDNA can be imaged under native conditions, i.e., inbuffer solutions, and therefore tapping mode in liquidtoday is the most widely used imaging mode in DNAnanotechnology.

High-Speed AFMCompared with other imaging techniques such aselectron or fluorescence microscopy (see below),high-resolution AFM imaging is a rather slowand painstaking process. Conventional AFMs needapproximately 10 min to capture a high-resolutionimage of 5 μm size of, e.g., DNA origami structuresusing tapping mode in liquid. While this is usuallysufficiently fast for imaging static nanostructures,most dynamic biomolecular processes occur on afaster time scale and are thus out of reach forconventional AFM systems. In order to speed up AFMimaging, a variety of technical improvements havebeen made in the past decades toward so-called high-speed AFMs.10–12 These instruments are similar indesign to conventional AFMs but various componentssuch as scanners, cantilevers, amplitude detectors, andfeedback electronics have been optimized for higherimaging speeds. Endo et al. first demonstrated imagingof DNA nanostructures using high-speed AFM in

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2010.13 For a more detailed review of biologicalapplications of high-speed AFM, see Ando et al.14

Imaging speeds of up to several frames per secondat resolutions below 10 nm in combination with onlylittle sample preparation effort make high-speed AFMone of the most attractive imaging techniques for DNAnanotechnology today.

Electron MicroscopyEM has played an extremely important role for thedevelopment of nanotechnology in general. It wasfirst demonstrated in 1931 by Ruska and Knoll,15

and previous to the invention of SPM it was theonly imaging method available for structures withdimensions well below the wavelength of visible light.EM utilizes electron beams for imaging, which areextracted in high vacuum from a special cathodeusing high voltage or heat or a combination of both.For imaging, the electron beam is shaped and focusedusing magnetic lenses. The wavelength of the electronsis given by the de Broglie relation and is on the orderof 1 nm. This determines the theoretical resolutionlimit of EM, but in reality this is governed by otherfactors such as the diameter of the electron beam andinteractions with the sample. EM is sub-divided intoscanning electron microscopy (SEM) and transmissionelectron microscopy (TEM). Each has its peculiaritiesand also different fields of application.

Scanning Electron MicroscopyBecause of its special requirements and limitations,SEM has only played a minor role for DNAnanotechnology. In SEM, an electron beam is scannedover a solid sample, and back-scattered electrons arerecorded in a detector. The image is constructed fromthe signals obtained from the different ‘pixels’ ofthe scanning area. While SEM is ideally suited forstudying solid state devices—for which a resolutionof approximately 1 nm can be achieved16—it isonly of restricted utility for DNA nanotechnology.Among the disadvantages of SEM are its requirementfor conductive samples, its operation under vacuumand its relatively low resolution. Structural featuresof DNA nanoassemblies are typically smaller thanthe SEM resolution that can be achieved withthese samples, and the metallization required forimaging masks the finest features. SEM becomesinteresting, when DNA nanostructures are combinedwith inorganic materials such as quantum dots orcarbon nanotubes, or when they are arranged on solidsubstrates using lithographic methods. In these cases,one is not so much interested in the finest details ofthe DNA structures, but rather in their large-scalearrangement.

Transmission Electron MicroscopyThe situation is quite different for TEM. AlthoughTEM also requires high vacuum and complicatedsample treatment, in terms of resolution it beats allother methods discussed here. For this reason, it hasbecome increasingly popular in DNA nanotechnologyin recent years, in particular in the context of 3D DNAnanoobjects. In contrast to SEM, TEM is performedon very thin samples, and only electrons transmittedthrough these are collected with a detector. Thismakes the technique similar to ordinary transmittedlight microscopy, albeit with waves of smallerwavelength. Depending on a variety of factors suchas the acceleration voltage (typically 100–200 kV),the resolution of TEM can be as low as 0.1 nm.Applications of EM on biological samples have along history in structural biology17,18 and many ofthe procedures developed there can be adapted to therequirements of DNA nanotechnology.

To image samples with a TEM, they have to beprepared in a special way. To improve the scatteringof electrons at the object to be imaged, ‘staining’with elements of high atomic number has become astandard method to generate contrast in EM. Oftenuranium salts such as uranyl acetate or formate areused, whose uranyl ions interact electrostatically with(poly)anions like DNA. Two different staining modesare distinguished. In positive staining, material isdeposited onto the objects under investigation, whilein negative staining, the objects are surrounded by thematerial. In the latter case, objects appear bright in theelectron micrograph, while positive staining resultsin dark objects. Staining modifies the sample andhence decreases the resolution of the image and mayalso change the structure of biomolecules comparedwith their native conformation. For preservationof the solution conformation and for ultimateresolution structural studies, cryo-EM is used. Here,the samples are not stained, but frozen and kept atlow temperatures during imaging.

As the TEM image of a single biomolecularobject is usually very noisy, image analysis, and single-particle reconstruction methods play an importantrole in EM imaging.19 To improve image quality,typically images of many objects are super-imposedand then averaged. As the 2D EM images representprojections of the 3D objects under study, the imagesfirst have to be sorted into different classes. Fromthese, one can actually reconstruct a 3D model of theobject. In recent years, single-particle reconstructionmethods have been frequently used to generate 3Drepresentations of self-assembled DNA nanoobjects.

TEM image reconstruction may actually turnout as an important field of application for DNA



nanotechnology. It has been recently shown that DNAnanoassemblies of known structure can be used as a‘frame of reference’ for other biomolecules, whosestructure is yet unknown, and thereby assist theimage analysis process significantly. This has beenused to determine the structure of a non-crystallinemembrane receptor, a G protein, and a complexof both molecules.20 Many researchers in the fieldbelieve that such utilization of DNA nanoassembliesfor structural biology represents a real ‘killer app’ forDNA nanotechnology.

Fluorescence MicroscopyFluorescence is the spontaneous emission of lightfrom atoms or molecules that have been previouslyexcited with light of a smaller wavelength. A largevariety of fluorescent molecules—‘fluorophores’—areavailable today as fluorescent labels and manybioconjugate methods have been developed tospecifically attach these to biomolecules of interest.Fluorescence techniques are ubiquitous in biologicaland biophysical research today, where they areused for the study of molecular interactions, theinvestigation of conformational dynamics, and, ofcourse, for imaging.21

Image quality in light microscopy is character-ized by several factors such as resolution, brightness,and contrast. In classical microscopy, the resolu-tion of an image is limited by the diffraction oflight and is given by Abbes limit of approximatelyλ/(2 NA), where λ is the wavelength of light andNA is the numerical aperture of the objective. Thuswith visible light, the minimum distance between twoobjects that can still be resolved is about 200 nm.To be able to discern structural features within animage, contrast—variations in brightness or color—isrequired. As biological samples often have low con-trast, a variety of contrast-generating methods havebeen developed, for instance, phase contrast imaging.In fluorescence microscopy contrast is generated byselectively staining of biomolecular structures withfluorescent molecules. Using appropriate filters, themolecules under study can be excited and fluorescencelight can be collected to generate an image. In bioimag-ing, using multiple fluorophores and filters allows toimage a variety of cellular structures together andstudy their inter-relations.

Until recently, ordinary fluorescence imaginghas not played a major role in the investigation ofDNA nanostructures, simply because these structureswere usually much smaller than the diffraction limit.This has changed, however, with the advent of super-resolution microscopy or ‘nanoscopy’.

NanoscopyStarting in the early 1990s, a variety of techniqueshave been developed that allow imaging beyond theclassical diffraction limit using far-field fluorescencemicroscopy.22–27 Most implementations of ‘super-resolution’ imaging share the general principle ofswitching molecules between fluorescence ON andOFF states, so that individual molecules can belocalized consecutively. On the basis of the mechanismused for localizing single fluorescence emitters,super-resolution microscopy may be divided intotargeted and stochastic readout schemes.28 In the firstcase, locations of single fluorophores are optically‘targeted’ to actively define ON and OFF states. Aprominent example of such a technique is stimulatedemission depletion (STED) microscopy.22 Using alaser-scanning microscope, each spot of an image isilluminated by an excitation laser overlaid by a second,red-shifted laser with a doughnut-shaped intensityprofile. This second laser de-excites fluorophores inthe doughnut area and thus ‘focuses’ the activeexcitation area below the diffraction limit. STEDmicroscopy currently allows imaging with a lateralresolution of approximately 20 nm. As the samplehas to be scanned, image acquisition time varies frommilliseconds to minutes.

Stochastic Reconstruction TechniquesStochastic reconstruction methods such as photoac-tivated localization microscopy,23 stochastic opti-cal reconstruction microscopy (STORM),24 pointaccumulation for imaging in nanoscale topography(PAINT),25 or blink microscopy (BM)26 rely onstochastic switching of fluorophores between brightand dark states and localization of single moleculeswithin a diffraction-limited area (cf. Figure 1). In fact,localization of single molecules can be achieved withalmost arbitrarily high precision—e.g., in 2003, Yildizet al. utilized a localization technique termed ‘fluores-cence imaging with one nanometer accuracy’ (FIONA)to investigate the motion of myosin V on actinfilaments.29 FIONA works under conditions, whereonly a single molecule emits light within a diffraction-limited area. Then the resulting point-spread function(PSF) of the emitter can be fit by a 2D Gaussian andits center position gives an estimate of the molecule’sposition. The localization precision is mainly influ-enced by the number of collected photons N andscales like.30

For reconstruction of distances below thediffraction limit, a sufficient fraction of moleculeshas to be switched to a fluorescent dark state, whileonly very few molecules are allowed to reside ina bright state during the acquisition of each image



Precise localization ofa single molecule

No localization possible

Temporal separation offluorescence emission

FIGURE 1 | Stochastic readout principle for super-resolution fluorescence microscopy. The localization of a single fluorophore in adiffraction-limited area is possible with high precision by fitting a two-dimensional (2D) Gaussian. If the point-spread function (PSF) of two or moreemitters within a diffraction-limited area overlap, localization is not possible. Using temporal separation of fluorescence emission by stochasticallyswitching fluorescence between ON and OFF states, the locations of the molecules can be reconstructed in a super-resolved image.

frame. This ensures that typically only a singlemolecule emits fluorescence within a diffraction-limited spot. Fluorescence ON and OFF timesand the number of emitted photons per activationare the most important parameters determiningthe resolution that can be obtained. For detailedreviews of current super-resolution techniques seeRefs 28, 31, and 32. As will be discussed below,the utilization of super-resolution microscopy forDNA nanotechnology has started only recently.33,34

In fact, their nanometer-precise addressability makeDNA nanostructures an extremely attractive substratefor super-resolution imaging as well as for theobservation of dynamic processes using single-molecule fluorescence techniques.

VISUALIZATION OF 2D DNAASSEMBLIES

Tile-Based NanoassemblyAs mentioned in the introduction, one of the earlybreakthroughs of structural DNA nanotechnologywas the realization of 2D DNA crystals by Winfreeet al. in 1998.2 These crystals were based on so-called ‘DX’ tiles, in which two parallel DNA doublehelices were linked by strand crossovers similar toHolliday junctions that occur in genetic recombination(Figure 2). These tiles had dimensions of 4 × 16 nmwith a height of 2 nm (the diameter of dsDNA).Via complementary ‘sticky-end’ extensions, these tilescould associate with each other to form nicely ordered2D lattices with extensions of several hundreds ofnanometers. One of the major technological advancesin this work was the first-time utilization of AFMunder liquid to image DNA nanostructures. AFMturned out to be a method matched perfectly to therequirements of DNA nanotechnology—as explainedabove, it allows high-resolution imaging on thetypical length-scale of DNA assemblies and it canbe operated under native conditions. For this reason,

AFM has remained the main characterization methodfor DNA assemblies ever since. AFM has beenused to characterize many other tile-based lattices,such as tilings based on triple-crossover molecules,35

‘4 × 4’,36 ‘tensegrity triangle’ tilings,37 and so forth.Only in a few cases fluorescence microscopy wasused for characterization—e.g., in Ref 38, wherealmost millimeter-sized lattices were assembled usinga variation of the crossbar tiling structure. Herefluorescence microscopy was used to demonstrate theextraordinarily large size of the whole assembly.

Tile-based assemblies were also used as tem-plates for the arrangement of metallic nanoparticlesinto lattices,39 or for the localized deposition of metalfrom solution.36 As metals give good contrast in EM,TEM, or SEM could be used for visualization of thesestructures, respectively.

Scaffolded DNA OrigamiScaffolded DNA origami, first introduced byRothemund in 2006,40 marked another breakthroughin structural DNA nanotechnology. In DNA origami,a long single-stranded viral DNA molecule is ‘folded’into a specific shape using many short ‘staple’strands (cf. Figure 3(a)). As for tile-based assemblies,AFM is the method of choice for imaging of flatorigami structures, as it offers operation under nativeconditions and sub-10 nm resolution. A variety ofshapes from Rothemund’s original publication imagedby tapping mode AFM under liquid are shown inFigure 3(b).

One important feature of DNA origami struc-tures is the fact that each staple strand can be used forthe attachment of other molecular components witha ‘resolution’ of approximately 6 nm—which is thedistance between neighboring ‘molecular pixels’. Todemonstrate the addressability of origami structures,Rothemund used staple strands with dumbbell-shapedhairpin extensions to highlight their position. The rigidhairpin structures produce height contrast in AFM



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FIGURE 2 | A DNA lattice formed from double-crossover (DX) tiles. (a) Architecture of a DX tile: two DNA double helices are connected at twocrossover points. (b) DX tiles can be connected by ‘sticky-end hybridization’ to form extended molecular lattices in two dimensions (2D). (c,d) Atomicforce microscopy (AFM) images in liquid of a 2D DX crystal, scale bars: 300 nm. (Reprinted with permission from Ref 2. Copyright 1998 MacmillanPublishers Ltd)

images and are visible as bright spots compared to theunmodified staple strands of an origami structure.

On the basis of the same principle, in 2008, Keet al. demonstrated the utilization of DNA origamistructures as sensors for the detection of RNA

molecules.42 To this end, DNA origami structureswere modified with an index region containinghairpin-modified staple strands acting as a unique‘barcode’. A single detection site for RNA consisted ofsingle-stranded extensions of two neighboring staple

Stapled DNA

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~ 100 nm

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FIGURE 3 | Design and imaging of single-layer DNA origami structures. (a) Formation of rectangular DNA origami by thermal annealing.(Reprinted with permission from Ref 41. Copyright 2010 Macmillan Publishers Ltd) (b) Schematic representations and atomic force microscopy (AFM)images in liquid from DNA origami structures. In the rightmost structure, single-staple strands are highlighted using dumbbell-shaped hairpinextensions to represent the map of the Americas on an origami scaffold. (Reprinted with permission from Ref 40. Copyright 2006 MacmillanPublishers Ltd)



strands. Each extension was chosen complementary toone half of the RNA target sequence to detect. Uponbinding of the RNA target to the probe, a structurallyrigid RNA/DNA hybrid was formed which could bevisualized using AFM due to its increased height.

In 2009, Steinhauer et al. for the first time usedsuper-resolution fluorescence microscopy to opticallyimage single-layer DNA origami structures.33 In thisstudy, rectangular DNA origami structures weremodified with two single fluorophores at a distance ofapproximately 90 nm (cf. Figure 4). The fluorophoreswere arranged on the origami structure by simplymodifying two staple strands at the 5′ end, i.e.,facing ‘up’. The origami structures also containedbiotinylated staple strands protruding to the oppositeside, i.e., ‘down’. Folded origami structures wereimmobilized on a streptavidin-modified glass slideand imaged using total internal reflection fluorescencemicroscopy (TIRFM). A typical diffraction-limitedimage of these structures is shown in Figure 4(a). InRef 33, a variety of super-resolution techniques wereused to resolve the 90 nm sub-wavelength distanceof the two fluorophores, among them dSTORM, asimplified implementation of the original STORM

method, and BM. In BM, the required dark states ofthe fluorophores for localization-based reconstructionare engineered using a reducing and oxidizing system(ROXS).43 In ROXS, a reducing agent reduces thefluorophore (e.g., the oxazine dye ATTO655) to anon-fluorescence state and an oxidizing agent activatesfluorescence again. By adjusting the ratio of oxidizingand reducing agent the required fluorescence ON andOFF times for super-resolution reconstruction canbe obtained.26 Figure 4(b) shows a typical diffraction-limited image of single origami structures carrying twofluorophores with the super-resolved reconstructionoverlaid. The magnified view reveals two singlefluorophores within each diffraction-limited area.Using BM, the distance between the two fluorophoreson the origami (nominally 89.5 nm) could bedetermined to be 88.2 ± 9.5 nm. As the variationof the measured distances can be almost completelyattributed to the localization precision of the method,DNA origami can be used as a reliable nanoscopicruler for super-resolution microscopy techniques.

In 2010, Jungmann et al. developed a single-molecule assay, termed DNA-PAINT, which allowsfor direct observation of dynamic processes on DNA

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FIGURE 4 | Super-resolution microscopy with DNA origami. (a) Total internal reflection fluorescence (TIRF) image of surface-immobilized DNAorigami containing two ATTO655-labeled staple strands. The positions of the single fluorophores cannot be determined because of their overlappingpoint-spread functions (PSFs). The positions of the fluorophores on the origami nanostructure are shown in the scheme at the bottom, their distanceis 89.5 nm. (b) Using blink microscopy (BM) super-resolution microscopy, the positions of the two fluorophores can be well resolved. The imageshows an overlay of the diffraction-limited TIRF image with a super-resolved reconstruction. The fluorescence time trace obtained from one origamimolecule is shown below the micrograph, highlighting the blinking of the fluorophores. Two magnified views of several structures from (b) reveal twosingle fluorophores. The distance distribution shown at the bottom yields (88.2 ± 9.5 nm).



origami scaffolds using single-molecule fluorescencemicroscopy.34 The method is based on specific,transient binding of short fluorescent DNA oligonu-cleotides to single-stranded ‘docking’ strands protrud-ing from DNA origami structures (cf. Figure 5(a)).Repetitive binding events of fluorescently labeled‘imager’ strands are monitored using TIRFM. Inthe unbound state, only background fluorescence isobserved. Upon binding of a fluorescently labeledstrand from solution, the fluorescence emission fromthis strand is detected (Figure 5(b)). This can be used todetermine association and dissociation rates of single-molecule DNA hybridization events. The fact thattransient binding in DNA-PAINT is based on DNAhybridization also allows for the precise control overfluorescence ON and OFF times—an important pre-requisite for stochastic super-resolution microscopy.The fluorescence ON time can be adjusted by thelength of the imager-docking strand duplex and thefluorescence OFF time can be controlled by the con-centration of imager strands in solution. Figure 5(c)shows the viability of DNA-PAINT super-resolutionmicroscopy by imaging three docking sites on anorigami substrate with a distance of approximately130 nm. The binding sites cannot be distinguishedusing diffraction-limited TIRFM imaging, but afterstochastic reconstruction the three binding sites are

clearly resolved. At its current state, DNA-PAINT isable to resolve distances as low as 30 nm.

The precise control over fluorescence ON andOFF times combined with the specific binding of DNAstrands makes DNA-PAINT applicable over a widerange of samples and imaging conditions. In fact,DNA-PAINT imaging is not restricted to DNA nanos-tructures and can be also applied, e.g., for imaging ofcellular structures by using DNA-antibody conjugates.

CHARACTERIZATION OF 3D DNANANOSTRUCTURES

It is still one of the major challenges for DNAnanotechnology to create structures extending in 3D.This is caused by the lacking structural integrity ofmany of the structures and by their frequently stronginteractions with surfaces (there might actually be 3Dorder in solution that is disrupted upon depositionon a surface). Hence, most of the 3D assembliescharacterized so far are relatively small geometricalobjects, or compact multihelix packings such as in 3Dorigami structures.

DNA ObjectsEarly DNA objects such as Seeman’s famous DNAcube44 lacked structural stability and only had the

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FIGURE 5 | DNA-point accumulation for imaging in nanoscale topography (PAINT) concept. (a,b) DNA origami structures can easily be modifiedwith single-stranded extensions of staple strands. A complementary imager strand carrying a fluorophore can transiently bind from solution to theseextension strands on the surface-immobilized origami, thus producing an apparent blinking as shown in the intensity versus time trace. Apart fromutilizing the directly obtained fluorescence ON and OFF times for the analysis of hybridization kinetics and dynamic processes on the single-moleculelevel, the precise tunability of these times allows for localization-based reconstruction microscopy. (c) The positions of three extended staple strandsat a distance of approximately 130 nm on an origami structure cannot be resolved with diffraction-limited total internal reflection (TIRF) microscopy.Using DNA-PAINT and localization-based reconstruction, the staple positions can be well resolved showing the designed distance.



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FIGURE 6 | Electron microscopy (EM) of a three-dimensional (3D) DNA object. (a) Molecular model of a DNA tetrahedron. (b) 3D density mapsobtained by single-particle reconstruction from cryo-EM images at 20 A resolution, and (c) at 12 A resolution—the length of the scale bar is 5 nm.(Reprinted with permission from Ref 47. Copyright 2009 American Chemical Society)

connectivity of their ideal geometrical counterparts.Because of their intrinsic structural stability,tetrahedral structures were natural target objectsfor 3D assemblies that could actually be visualized.One of the first examples in this direction was therealization of a DNA-based octahedron by Shih andJoyce in 2004,45 which was based on intramolecularfolding of a long single-stranded DNA molecule (ina sense a predecessor of Rothemund’s scaffoldedorigami method). Because of its small diameter ofonly 20 nm, the structure was visualized by cryo-EMand single-particle reconstruction methods. In 2005,Goodman et al. created a simple DNA tetrahedroncomposed of only four strands of DNA, with aside length of 10 nm. In the original publication,46

the structure was visualized using AFM, and alsoits mechanical properties were probed with theAFM. However, due to the small size of thetetrahedron, the AFM was clearly at its resolutionlimit. Later, the same structure was investigated indetail using cryo-EM (Figure 6).47 To date this isactually the smallest biomolecular structure that wasreconstructed from cryo-EM images at a resolution of1 nm!

In recent years, researchers demonstrated otherimpressive 3D objects such as platonic bodies madefrom DNA48,49 or DNA/RNA hybrids,50 and alsorigid RNA cubes,51 and it has become common tocharacterize these structures both using AFM andTEM. Because of strong interactions with the chargedmica surfaces that are typically used as substrates forAFM imaging, 3D objects are often ‘flattened’ out inAFM, while they retain their structure in EM.49

DNA Helix BundlesDNA helix bundles represent, simply speaking, rolledup 2D lattices and only have small extensions

in two of their dimensions. There have beendemonstrations of nanotubular structures composedof DNA tiles,40 or simply DNA helices52–55 withdiameters ranging between 10 and 50 nm and lengthsof several micrometers. Most of these structures werecharacterized by AFM and sometimes also TEM.56

One notable exception is the work by Fygensonand coworkers,57 in which video microscopy wasapplied to study the dynamics of fluorescently labeledDNA nanotubes. Similar as in biophysical experimentson dsDNA or cytoskeletal filaments, studies of theBrownian dynamics of DNA nanostructures can beused to determine, e.g., their persistence length.58 Inaddition to ‘tile-based’ DNA nanotubes, a variety ofother helix bundle types were realized recently usingthe DNA origami technique.

3D OrigamiIn 2009, Douglas et al.59 demonstrated that DNAorigami can be extended to the third dimension by‘stacking’ of 2D DNA origami slices. In contrast toRothemund’s 2D patterns, the new design was basedon a ‘honeycomb lattice’, in which neighboring DNAhelices were connected together at 120◦ angles (cf.Figure 7(a)). It turned out that the resulting massiveblocks of DNA were best imaged by TEM—severalTEM micrographs of such DNA monoliths are shownin Figure 7(a).

Later, Shih and Yan published an even morecompact 3D origami design based on a squarelattice.60 Several multilayer structures were built withthis technique, ranging from 2 × 21 to 8 × 8 blocks(number of layers × number of helices). A schematicof a 6 × 12 block is shown in Figure 7(b) along withcorresponding TEM micrographs. In addition to TEMimaging of negatively stained particles, in Ref 60 alsocryo-EM was used to investigate the origami structuresunder ‘native’ conditions.



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FIGURE 7 | Three-dimensional (3D) origami. (a) A 3D structure based on a honeycomb lattice is constructed by stacking of two-dimensional (2D)DNA origami slices (top). Transmission electron microscopy (TEM) micrographs confirm the successful formation of origami structures (bottom).(Reprinted with permission from Ref 59. Copyright 2009 Macmillan Publishers Ltd) (b) An alternative approach to form 3D origami structures basedon a square lattice cross section (top), and corresponding TEM images (bottom). (Reprinted with permission from Ref 60. Copyright 2009 AmericanChemical Society) (c) A nanoscale box made from 2D DNA origami with a controllable lid (top). Atomic force microscopy (AFM) images of the box inliquid show unfolded, lid-closed, and lid-opened states (middle). Single-particle analysis of cryo-EM images allows for a 3D reconstruction of the DNAbox. The theoretical model on the left agrees well with the surface representation of the cryo-EM map (bottom). (Reprinted with permission from Ref61. Copyright 2009 Macmillan Publishers Ltd)

Also in 2009, Andersen et al. took anotherapproach toward 3D structures by arranging planarDNA origami sheets into the form of a nanoscalebox61 (Figure 7(c)). Notably, they designed the boxwith a controllable lid, which could be openedand closed upon addition or removal of ‘lock’ and‘key’ strands via a strand displacement mechanism.Andersen et al. used a whole battery of analytictechniques to study the properties of the box, e.g.,dynamic light scattering and also small-angle X-ray scattering. As nanoscale imaging techniques,AFM, cryo-EM, and also single-molecule fluorescencemicroscopy were applied.62 AFM could be used toimage both open and closed states of the box, whereascryo-EM facilitated single-particle reconstruction ofthe box. As shown in Figure 7(c), a 3D map generatedfrom the cryo-EM data showed good correspondencewith a theoretical model.

EM offers a wealth of structural informationabout 3D bio-nanostructures and is therefore used

in essentially all recent publications on 3D origami.EM studies were performed on twisted and curvedshapes,63 tensegrity structures,64 and curved spherical-or flask-like shells.65

DNA CrystalsAs mentioned in the introduction, one of the originalgoals of the founder of the field, Nadrian Seeman,was to produce macroscopic crystals from DNA. In2010, he and his coworkers came up with a simpledesign that generated millimeter-sized crystals thatcould be used for X-ray crystallographic studies.66

From the X-ray diffraction pattern, an all-atom modelcould be reconstructed with a resolution of 4 A. Thecrystals were built from triangular structures that wereformed by three crossover structures at the vertices.This structure is not planar and extension of its helicesby sticky-end hybridization to other triangles leads toa 3D crystal structure. Important for the successful



assembly of a highly ordered structure were two mainingredients: the rigid triangular building block itself,and the utilization of very short sticky ends of only2 nt length. Notably, due to the macroscopic nature ofthe crystals they were actually visible to the naked eye!

DYNAMICAL STRUCTURES ANDPROCESSES

One of the ultimate goals of nanotechnology isthe realization of artificial molecular machines and‘robots’, and their operation within autonomous,dynamical molecular systems. While there is alreadya large body of work on nucleic acid-based molecu-lar switches and machines,67 most of the structuralchanges and movements realized so far were too smallto be imaged directly. DNA origami now offers aversatile substrate for the construction of systems,in which the dynamics of biomolecules and artificialnanomachines may be studied on a larger scale, andin a well-controlled setting. However, real-time obser-vation of dynamical processes involving biomoleculesprecludes many of the techniques traditionally used forthe characterization of DNA nanostructures—theseare either too slow, or they are not compatible withthe reaction conditions required. Hence, the realiza-tion of large-scale dynamical DNA nanosystems notonly represents the frontier of research regarding theirconstruction, but it also requires the most advancedimaging techniques available.

Imaging Chemical and BiochemicalProcesses on OrigamiUnlike reactions in well-stirred chemical reactors,chemical processes in live cells often occur localized,within small compartments and in a spatially orches-trated manner. It is therefore of considerable interestto study the influence of spatial organization onchemical reaction systems, and also to attempt the con-struction of artificial chemical ‘reaction centers’—e.g.,to optimize synthetic pathways or energy transductionbetween neighboring components. One first exampleof a spatially organized system was recently demon-strated by Voigt et al.68 who monitored the progressof chemical reactions on DNA origami on the single-molecule level. To this end, streptavidin proteins wereattached to origami structures via chemically cleavablelinkers, and the effect of cleaving agents on the assem-blies was investigated by AFM. With ‘traditional’AFM, however, the reactions could only be assessed‘in hindsight’, i.e., without temporal resolution.

In addition to the fabrication of spatiallycontrolled reaction systems, DNA self-assembly also

facilitates the controlled placement of binding sites forbiomolecules, which may be used to study the dynam-ics of biomolecular processes under well-characterizedconditions. In a series of ground-breaking exper-iments, Sugiyama and coworkers recently utilizedhigh-speed AFM to image biochemical processes inreal time. Using DNA origami ‘frame’ structures intowhich DNA molecules were incorporated that con-tained the recognition sequences for a DNA methyltransferase,13 they were able to directly image theattachment of the enzyme to DNA and its subse-quent search for its binding site. With a similar setupthey were able to monitor the dynamic formationand disruption of intermolecular G quadruplex struc-tures upon addition or removal of potassium ions,69

and also the action of DNA-based excision repairenzymes.70

DNA WalkersOne of the most challenging goals of DNAnanotechnology is the realization of artificialmolecular motors. Since 2004, a variety ofmolecular ‘walker’ concepts have been put forward,71

but typically their operation was only indirectlydemonstrated by gel electrophoresis or fluorescencespectroscopy. It took until 2010 to see the firstnanoscale images of DNA walkers, when Lund et al.presented a ‘molecular robot’ walking on a DNAorigami-based track.72 The walker consisted of astreptavidin core with one capture and three catalyticlegs made from DNA73 (Figure 8(a)). When the walkeris bound to the DNA origami track via complementarysubstrate strands, the catalytic legs (containing the8–17 deoxyribozyme) cleave the substrates andthereby reduce their lengths. This destabilizes theassociation of the walker with the track and it passeson to the next substrate. By patterning a ‘path’ ofsubstrate molecules onto the DNA origami structure,the otherwise diffusive movement of the walker ismade directional. The motion of the walker could bemonitored by AFM imaging as shown in Figure 8(a),where its position is clearly visible in the heightcontrast image at different times. In Ref 72, thespeed of the walker was also determined using single-molecule fluorescence measurements, from whichposition-time trajectories could be constructed.

The groups of Turberfield and Sugiyama showedanother walker system that also utilized a planarDNA origami structure as track74 (Figure 8(b)). Thewalker is based on a single DNA strand binding tocomplementary stator strands on the track movingby toehold-mediated strand displacement. The toe-hold is generated by a nicking enzyme, which cutsthe stator strands as soon as a walker-stator-duplex



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FIGURE 8 | DNA walkers and assembly lines. (a) A DNA spider walks on a prescriptive track on a planar DNA origami structure. High-resolutionfluorescence microscopy is used to monitor the movement of the walker (top). Atomic force microscopy (AFM) allows the observation of specificactions at the start, on the track and at the stop site (bottom, sites marked with the green arrow respectively). (Reprinted with permission from Ref72. Copyright 2010 Macmillan Publishers Ltd) (b) A DNA walker proceeds by toehold-mediated strand displacement on a linear DNA track (top).Kymographs constructed from AFM images at different times allow analysis of the progress of the walker. The histogram obtained from the heightprofiles reveals the walker’s stepping motion (bottom). (Reprinted with permission from Ref 74. Copyright 2011 Macmillan Publishers Ltd)(c) Molecular assembly line. A transporter made from DNA can take up cargo from three loading sites. The loading sites consist of two-state DNAmachines that can either present or hide their cargo. The cargos themselves are combinations of differently sized gold nanoparticles (left). AFMimaging in air shows the uptake of all three cargos by the transporter (right), whereas transmission electron microscopy (TEM) micrographs show theresults for all eight possible combinations of the cargo (bottom). (Reprinted with permission from Ref 75. Copyright 2010 Macmillan Publishers Ltd)

is built. Beside ensemble fluorescence measurementsthey used AFM imaging to show the movement ofthe walker (Figure 8(b)). As conventional AFM istoo slow to detect single steps of the walker they

applied high-speed AFM allowing the observationof discrete steps between two neighboring stators ina kymograph. Assuming the highest point on eachAFM profile as the walker position yields distances of



7.4 ± 1.0 nm between two adjacent stators. Thisvalue is in good accordance with the value of 6 nmexpected from the origami design.

Nanomechanical Devices and AssemblyLinesIn addition to ‘molecular robots’ and machines,another vision of nanotechnology is the realization ofmolecular-scale ‘assembly lines’. Most impressively,one first example of such a system could recentlybe demonstrated by Seeman and coworkers.75 Theassembly line was composed of a DNA transporterand three switchable two-state DNA machines placedonto a planar DNA origami substrate (Figure 8(c)).By switching any of the DNA machines into the ‘ON’state, cargo (here differently sized gold nanoparticles)could be transferred from machine to transporter.By moving from machine to machine, the transporterwas able to pick up any of eight possible combinationsof the cargos. Figure 8(c) shows AFM images of theuptake and steps of the transporter. The results of theeight possible assembly programs are shown as TEMmicrographs.

CONCLUSION

In the past years, we have witnessed an excitingnew development in DNA nanotechnology—DNAorigami. DNA origami is a powerful technique forthe assembly of almost arbitrarily shaped nanoscaleobjects. These objects may be modified sequencespecifically with organic or inorganic materials, andhence customized for the requirements of the appli-cations at hand. Origami is currently adopted by a

growing number of researchers who aim at appli-cations as diverse as nanophotonics, nanochemistry,drug delivery, structural biology, synthetic biology, orbasic biophysics.

While AFM has been the dominant nanoscaleimaging technique in DNA nanotechnology for almosta decade, many of the new structures and applicationsrequire additional characterization tools. Multilayer(3D) origami is mainly investigated with EM, andseveral groups are starting to realize the potentialof modern super-resolution fluorescence microscopicmethods. Interestingly, DNA nanotechnology is begin-ning to ‘pay back’ to imaging technology, and thismay actually be one of the major applications forDNA-based nanostructures. The precise control overmatter on the nanometer scale allows the construc-tion of objects with known shape and dimensionsthat may serve as calibration standards for nanoscaleimaging techniques. For example, it has been shownthat origami structures can be used to calibrate thedistances between fluorophores for optical super-resolution methods.33 The structure of a DNA objecthas been reconstructed from cryo-EM at an extraordi-nary accuracy,47 and DNA objects may in the futureassist in the determination of yet unknown molecularstructures.20

The new frontier for DNA nanotechnology is therealization of dynamic, spatially extended nanoscalesystems such as artificial reaction centers,68 molec-ular robotic systems,72 molecular transporters, andassembly lines.75 For these, fast imaging techniquesare required, for which the samples can be kept undernative conditions. It is clear that single-molecule flu-orescence and super-resolution techniques as well asvideo rate AFM will play an increasingly importantrole in DNA nanotechnology in the years to come.

ACKNOWLEDGMENTWe gratefully acknowledge funding by the Nanosystems Initiative Munich (NIM).

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