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DNA origami–based standards for quantitative fluorescence microscopy Jürgen J Schmied, Mario Raab, Carsten Forthmann, Enrico Pibiri, Bettina Wünsch, Thorben Dammeyer & Philip Tinnefeld Institute for Physical and Theoretical Chemistry—NanoBioSciences, Technische Universität Braunschweig, Braunschweig, Germany. Correspondence should be addressed to P.T. ([email protected]).

© 2014 Nature America, Inc. All rights reserved.

Published online 15 May 2014; doi:10.1038/nprot.2014.079

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1–2 d.

INTRODUCTION The past three decades have seen the rapid and successful development of nanotechnology based on nucleic acids, resulting in the introduction of DNA origami, DNA bricks and others1–3. These structures are typically very stable, and their preparation has been robust, with high yields; they therefore have the potential for use in many applications. Applications for DNA origami include drug delivery4, self-assembled electronics5,6 and single-molecule biotechnology7,8 (for reviews, see refs. 9–12). DNA nanotubes can also, for example, be used as alignment medium for NMR spectroscopy structure determination of membrane proteins13. The idea behind DNA origami is to use a long (~7,000–9,000 nt) single-stranded DNA (ssDNA), called a scaffold strand (Box 1), together with ~200 short (~20–50 nt) staple strands (also called oligonucleotides or staples) added in excess, which fold the scaffold in a predetermined way. The position of each staple strand in the fully folded nanostructure is known; therefore, objects such as hairpins, proteins or organic dyes can be positioned with a spatial resolution of ~6 nm (ref. 1). DNA origami becomes even more practical through computer-aided design that helps design DNA origami structures and select staple strands2 as well as evaluate their conformational flexibility14,15. DNA origami for microscopy standards Measuring nanoscale distances was one of the early DNA origami applications16. For this, organic, fluorescent dyes are attached at predefined positions to the DNA origami structures simply by exchanging staple strands with dye-labeled staple strands. New super-resolution techniques have been applied to resolve distances of, e.g., 90 nm between two marks on a rectangular DNA origami16. DNA origami nanorulers have progressed to become a useful tool in microscopy17,18. The rapid development of fluorescence imaging techniques for use in medical and biological sciences means that there is an increased need for validation and direct comparison of methods and instruments by using standardized

samples. Such samples have not been available especially in the recently opened nanometer regime of super-resolution imaging and fluorescence resonance energy transfer (FRET)19–22. Here we present a step-by-step protocol for designing, producing and using fluorescently labeled DNA origami nanostructures as standards for fluorescence imaging. Characterization of optical microscopes Many modern microscopy techniques distinguish themselves by overcoming the diffraction limit of light and offering principally unlimited resolution. These include stimulated emission depletion (STED23) microscopy (including different variants such as pulsed-STED, continuous-wave (cw)-STED24, gated (g)STED25, 3D STED26), structured illumination microscopy (SIM27, and extensions such as saturated SIM28), techniques based on single-molecule localization such as (fluorescence) activation localization microscopy ((F)PALM)29,30, (direct) stochastic optical reconstruction microscopy ((d)STORM)21,31, ground state depletion followed by individual molecule return (GSDIM) 32 or Blink microscopy33 and their extension to 3D (refs. 34–36) or other techniques such as super-resolution optical fluctuation imaging (SOFI)37. These techniques yield beautiful images of cellular structures with unprecedented detail and clarity, and they have started revealing new biological insights of, e.g., cytoskeletal structures38 and viral maturation39,40. We identify three main reasons why these new imaging techniques require detailed and rigorous characterization by using better microscopy standards; see also Figure 1. • At the moment, resolution is usually determined by looking at

cross-sections at arbitrary positions where two filaments (e.g., cytoskeleton labeled with antibodies) are arranged in parallel. Unfortunately, super-resolution images exhibit measurement noise so that individual cross-sections are not meaningful for quantifying microscope resolution, and for cytoskeletal filaments nature protocols | VOL.9 NO.6 | 2014 | 1367

protocol Box 1 | Scaffold strands for DNA origami structures

© 2014 Nature America, Inc. All rights reserved.

In DNA-based nanoengineering, circular ssDNA strands are essential components. They are commonly used as scaffold strands for DNA origami structures, and they function as hierarchic elements in folding reactions. Although enzymatically generated ssDNA of plasmids2 or dsDNA90 have been used in DNA origami folding reactions, the M13mp18 phage–derived ssDNA provides the vast majority of used matrixes for DNA origami structures. It is a filamentous phage that infects Escherichia coli strains with an F′-plasmid (F factor–encoded pili), and it has become, like its many relatives, well established in biotechnology for gene cloning and sequencing within the past 30 years. The genome structure can switch between an intracellular plasmid-like double-stranded replicative form and a ssDNA molecule within the infective phage virions, providing both a basic tool for genetic engineering and a valuable source for circular ssDNA as material for DNA nanotechnology. The double-stranded DNA can be extracted from infected cells, and it allows genetic manipulation and alteration of the size and sequence by deletion or insertion of additional sequence elements by using standard restriction endonucleases91. The corresponding packaged single-stranded (+)-strand progeny DNA molecules can be extracted from PEG-precipitated phage particles. Within our study of DNA origami as fluorescence standards, we use the original p7249 scaffold1 of the M13mp18 phage (ref. 92) (7,249 nt) with a length of ~2.4 µm. Furthermore, we use three recombinant variants with 7,560 (refs. 2,72), 8,064 (refs. 2,72) and 8,634 (ref. 2) nt. Detailed protocols for genetic modification, bacteriophage amplification, purification of phage particles and extraction of ssDNA have been published elsewhere2,13,15,18,91,93. The work with host strain organisms and phages requires a molecular biology lab with relevant permits issued by the local regulatory authority responsible for overseeing work with genetically engineered organisms.

labeled with antibodies the labeling density is not well defined. The labeling density does not have a strong effect to distinguish the distance between two filaments because a distance is measured in one dimension and localizations are summed in the other direction. The labeling density is, however, crucial for arbitrarily shaped objects such as small spherical structures. Owing to the Nyquist criterion, the mean distance between two neighboring dyes has to be half the size a of the smallest detail of the structure that shall be resolved. In general, for an image with dimension d the density of dye molecules has to be about (2/a)d (ref. 41). Therefore, to achieve a spatial resolution of 20 nm, the distance between two neighboring dye molecules has to be 10 nm, which leads to a density of 10,000 dye molecules per square micrometer in this special case. • For many of the innovative (super-resolution) microscopy techniques, resolution needs to be accessed experimentally42. According to the Abbe formula (used for diffraction-limited techniques), optical resolution is limited to roughly half the wavelength of light. Although the Abbe formula could be extended to STED microscopy and related techniques43, resolution often is not simply calculated, e.g., because STED saturation intensities are not easily available and they are strongly wavelength- and dye-dependent. For single-molecule localization approaches44,45, localizing a single molecule with a precision beyond the Abbe limit does not necessarily equate to super-resolution. To achieve super-resolution, many molecules have to be localized successively, and images have to be reconstructed. Therefore, the structures that are investigated have to be labeled densely enough, the photophysics and photochemistry of the fluorescent labels have to be controlled to achieve a reasonable ratio of the off-state to on-state lifetime, the fraction of the molecules identified has to be large enough and so on46,47. In addition, measurement times can be substantially longer than those to measure the localization precision of a single molecule, and thus setup stability and drift become more crucial. In summary, for the new fluorescence imaging techniques, it is not straightforward to determine crucial parameters such as the achievable resolution. • The new microscopy techniques require that a variety of parameters (including laser intensity, acquisition rate, activation laser 1368 | VOL.9 NO.6 | 2014 | nature protocols

settings, image analysis parameters and rendering) are optimized to achieve the specifications of the microscope’s manufacturer. Sample preparation is crucial: this does not only refer to a dense and stable labeling. For techniques that rely on single-molecule detection, impurities have to be strictly avoided. Buffers have to be optimized to meet the specific criteria of the required photophysics, photochemistry and photostability, often involving enzymatic oxygen scavenging, specific reductant or thiol-­containing buffers and so on47. With the huge number of possible error sources, it is crucial that experiments can be performed to allow the researcher and/or microscope’s manufacturer to distinguish instrument-­specific problems from specimen-specific problems. These problems can be addressed by using fluorescence microscopy standards. Advantage of DNA origami A calibration standard requires distinct marks at defined distances. As many super-resolution techniques are based on singlemolecule detection where every molecule counts, the number of molecules per mark has to be rigorously known. Furthermore, all dye molecules should be identical in their properties and should not aggregate with neighboring dye molecules, as this could lead to quenching and altered spectroscopic properties. Switching required for super-resolution could, for example, be compromised. This could result in reduced brightness or alternated blinking kinetics of the dye molecules. Optimally, the arrangement of a defined number of dye molecules at high density should not compromise uniform spectroscopic properties including, e.g., the fluorescence lifetime and spectrum. Top-down lithographical approaches for creating structures with a defined arrangement of dye molecules can cover the required dimensions48, but an accurate positioning of fluorescent molecules by using these techniques remains problematic and is often not parallelizable49. In addition, calibrants created by lithography might not be biocompatible or optically compatible, and they might alter the properties of the fluorescent dyes. Classical chemical synthesis allows better structural control, but although chemical and macromolecular approaches based on self-assembly can form repetitive structures in the required

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size range50,51 they do not allow placement of individual objects over distances relevant for microscopy, such as in the range of 10–400 nm. Biological structures, e.g., nuclear pore complexes40,52,53, exist with the required sizes and defined compositions, and they could therefore be considered for use as internal standards. Because of the fixed geometry of these biological structures, these samples only provide fixed distances and do not allow arrangement of dye molecules in arbitrary geometries. Furthermore, labeling is carried out via antibodies, which generates a substantial distribution of fluorophore numbers per mark. Scaffolded DNA origami, however, enables generation of 2D and 3D objects of defined shape 1,2 in a simple and convenient way, which are easily produced in good yields. DNA origami structures offer the following advantages that make them uniquely suitable for microscopy standards. • For DNA production, scaffold strands of the order of 8,000 bases

are used. This corresponds to roughly 2.7 µm of DNA that can be distributed at will. Dyes can be placed along this strand with a free choice of positions, thus enabling a defined number of fluorescent dye molecules at arbitrary geometries.

Counts Counts Counts

No. of localizations No. of localizations No. of localizations

© 2014 Nature America, Inc. All rights reserved.

No. of localizations

Figure 1 | Calibration structures for superb c 40 a 98 nm resolution microscopy. (a–o) Comparison of actin filaments (a–c) and DNA origami (d–o) as 20 test structures. Panels b,e show representative super-resolution images and panels a,d show the corresponding TIRF images. Scale bars, 0 1 µm (a,b) and 300 nm (d,e). The super0 50 100 150 200 250 resolution images (g,j,m) show single DNA Position (nm) origami structures with different distances f e d between the two marks. The smallest distance between actin filaments is arbitrary, and it appears usually only once in a superresolution image. This distance can be visualized in a cross-section histogram, by fitting the resulting peaks with the sum of two Gaussian functions and by measuring g h 60 i the distance between the two peaks (c). 101 nm Because of the fact that distances between 80 40 dye molecules can be designed very precisely on DNA origami structures, the distance can 40 20 be decreased systematically to discover the best possible resolution of a super-resolution 0 0 0 50 100 150 0 50 100 150 200 microscope. In this case, the designed distances of Position (nm) Distance (nm) 98 (d,e,g–i), 49 (j–l) and 33 nm (m–o) were j k 60 l 46 nm designed on the rectangular DNA origami by 60 creating two marks. For the measurements in 40 40 j–l and in m–o, a configuration of two marks with seven Alexa Fluor 647 molecules 20 20 per mark (referred to as 2 × 7 configuration) was used, which is shown schematically 0 0 0 50 100 150 200 0 50 100 150 in f. For d,e,g–i), a 2 × 3 Alexa Fluor 647 Distance (nm) Position (nm) configuration was used, which is schematically m n 40 o 31 nm 80 shown in Figure 11c. All samples were labeled externally (see ‘Labeling strategies’ in the INTRODUCTION). The histograms (h,k,n) 20 40 represent histograms of cross-sections similar to that shown in c (respective DNA origami structures are surrounded by white boxes in 0 0 0 50 100 150 0 50 100 150 200 g,j,m). In contrast to super-resolution images Distance (nm) Position (nm) of actin filaments, it is straightforward to create a histogram of several hundred distance measurements with DNA origami structures as shown in i,l,o. The results of the histograms with s.e.m. and number of data points are as follows: 98.0 ± 0.4 nm, n = 327 (i); 46 ± 1 nm, n = 237 (l); and 30.8 ± 0.6 nm, n = 255 (o).

• DNA origami structures are mechanically stable. Even a simple six-helix bundle (6HB) (ref. 54) has a persistence length beyond 1 µm (ref. 55). A 6HB structure with two dye molecules attached at a contour length distance of 386 nm exhibited an average distance of 357 nm that could easily be resolved by a confocal single-molecule–sensitive microscope18. • DNA origami exhibits a dynamic range of 3–400 nm by using a single scaffold strand that can be arbitrarily extended by building higher-order structures13,18,56,57. A single scaffold strand can even be sufficient for 3D super-resolution structures with an intermark distance of ~200 nm (ref. 17). • If each staple is labeled with one fluorophore, a regular pattern of one fluorophore every 6 nm is achieved. The brightness per molecule is not influenced, but the fluorescence intensity scales linearly with the number of fluorescent dyes (Fig. 2; ref. 18). This has been shown for Atto 647N (ref. 18), but it also holds for Oregon Green 488, a dye that is well known for its self-quenching properties58. We studied the intensities of labeled DNA origami structures with different numbers of fluorescent dyes and found a linear dependence of fluorescence intensity and dye number, as is shown in Figure 2a. We assured that the slightly lower intensity of nature protocols | VOL.9 NO.6 | 2014 | 1369

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6 12 18 24 No. of dye molecules

0.1 0 200

400

600

800

Photons per spot

Figure 2 | Brightness standards. (a) Fluorescence brightness standards for DNA origami labeled with varying amounts of Atto 647N18 (red line) and Oregon Green 488 (blue line) dyes. The samples were scanned with an excitation intensity of 1.8 kW cm−2 and an integration time of 0.02 ms nm−1. The buffer used for measurements contained PBS (1× PBS) and 2.5 mM DTT. The slopes are determined mainly by the excitation intensity, and they do not compare the brightness of Atto 647N and Oregon Green 488. (b) Histogram of the photons per spot for a DNA origami labeled with 12 Oregon Green 488 molecules with an inter-dye distance of 12 nm (black) and 6 nm (gray). Insets show sketches of the two different types of DNA origami structures used for this measurement. To achieve a dye-to-dye distance of 6 nm, the 12 dye molecules are positioned in one single column. In the 12-nm dye-to-dye distance, the 12 dye molecules are positioned in two columns, each containing six dye molecules.

the 24 Oregon Green 488 sample is not related to self-quenching. We therefore measured the 12 Oregon Green 488 sample in two different arrangements of fluorescent dyes on the DNA origami. In one arrangement, the minimal distance between dyeattachment points was 12 nm, whereas in the other the distance between adjacent dyes was 6 nm as in the 24-dye sample (see Fig. 2b and inset for arrangement of dyes). Both samples yielded identical fluorescence intensities (Fig. 2b). For all samples, no indication of quenching was observed neither owing to changes of brightness per molecule nor through a change of fluorescence lifetime. A DNA origami with only 12 Atto 647N molecules showed superior and more homogeneous spectroscopic properties than small fluorophore–doped commercial beads18. We thus believe that DNA origami structures offer exceptional properties as brightness standards, i.e., to relate measured intensities to numbers of dyes. They can be used with essentially all dyes that can be attached to DNA as long as DNA itself is not a substantial quencher of the dye’s fluorescence. Higher labeling densities can be realized by increasing the number of labels per staple strand. • DNA origami structures are chemically stable structures that can withstand conditions such as cell lysate and elevated temperature over some time59,60. By photo-crosslinking, DNA origami structures can be further stabilized to remain functional at even higher temperatures61. This makes them also promising structures for in situ standards, e.g., by adding DNA origami nanorulers to the (biological) specimen under study. DNA origami standards have matured from their first presentation16–18 into usable specimens that can be imaged with ease. Figure 1e shows a super-resolution (dSTORM31,62) image of rectangular DNA origami structures with marks consisting of externally labeled Alexa Fluor 647 at a designed distance of 98 nm. Six double spots are visible on an area of 2.5 µm2. Because double spots with defined distances appear hundreds of times on one super-resolution image, their detection can be automated and the distances can be plotted in a histogram (Fig. 1i,l,o). 1370 | VOL.9 NO.6 | 2014 | nature protocols

DNA origami structures for fluorescence microscopy The DNA origami technique enables a virtually unlimited variety of possible structures. Although a manifold of structures could be imagined to be useful as fluorescence standards, a few structures appear particularly suited, and they are discussed in more detail in this protocol. The rectangular 2D DNA origami with dimensions of 100 nm × 70 nm was introduced for the measurements in Figure 1d–f, and it is also displayed in Figure 3 together with an atomic force microscopy (AFM) image. The design (termed NRO for new rectangular origami) is a modification of one of the DNA origami structures presented in Rothemund’s original publication1 (here termed RRO for Rothemund’s rectangular origami) with the difference that internal strain is reduced in the NRO structure by introducing deletions at specified positions65,66. The NRO is simple and easily folded, AFM imaging is facile and it offers a dynamic distance range of up to 100 nm. The disadvantages of remaining flexible16 and having a limited dynamic range can be overcome using DNA bundles such as a 12-helix bundle (12HB) (Fig. 3c). For a 12HB, the distance distribution was not noticeably broadened by structural inhomogeneity because of the increased rigidity17. In addition, the 12HB still offers a high density of labeling along the 220-nm contour, with about one staple strand terminating every nanometer. Even longer distances can be obtained from a single scaffold strand with a 6HB (Fig. 3b)

a

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Very easily, different distances on DNA origami structures are realized, and super-resolution imaging shows that distances of 30 nm are easily resolved by dSTORM16,31. The mean distances of the DNA origami nanorulers shown in Figure 1e,g,j,m are ­uncovered by Gaussian fits. Results for designed distances of 98, 49 and 33 nm with s.e.m. are 98.0 ± 0.4 nm, n = 327 (Fig. 1c); 46 ± 1 nm, n = 237 (Fig. 1d); and 30.8 ± 0.6 nm, n = 255 (Fig. 1e). Owing to the versatility of DNA origami, it is not surprising that researchers have started using them for testing and demonstrating new methods including tip-induced fluorescence quenching with nanometer resolution63 and counting fluorescent molecules by means of photon statistics64.

70 n

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b 0.3

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0

© 2014 Nature America, Inc. All rights reserved.

Atto 647N Oregon Green 488

12 nm

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Counts (norm.)

Photons per spot

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4.54 nm 2.97 nm 0 nm

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Figure 3 | AFM imaging of DNA origami structures. (a–h) Sketches (a–d) and AFM images (e–h) of the different DNA origami structures used in this protocol. Shown are the NRO (a,e), the 6HB (b,f), the 12HB (c,g) and the nanopillar (d,h). The AFM image of the NRO (e) shows six holes (marked with white arrows). At these positions, the staple strands are shortened in order to position the biotin modifications on the bottom of the DNA origami structure. The dye modifications are then positioned on the top, which is at the side of the buffer or embedding medium. The different persistence lengths of 6HB and 12HB are clearly visible in the respective AFM images (f,g). The nanopillar (d,h) has a base with biotin extensions below. Scale bars, 50 nm (e–h). The dimensions given in a–d are typical values of the DNA origami structures measured by AFM.

protocol

© 2014 Nature America, Inc. All rights reserved.

Figure 4 | Flowchart of the procedure. The flowchart schematically shows the steps described in the procedure section and references the respective steps and/or boxes. The steps mentioned in the light-blue boxes are explained in detail in the PROCEDURE. The boxes in gray show the different DNA origami structures that can be used or mention further possible imaging methods.

12HB

NRO

caDNAno, ordering staples and scaffold

that enables distances of up to 360 nm. For this structure, however, the rigidity is reduced and bending is observed (see AFM image in Fig. 3). Recent microscopy developments aim for isotropic imaging and focus on increasing the resolution in the axial direction24,67, e.g., by engineering the point spread function35,36,68–71. Constructing a DNA origami from a single scaffold with intermark distances >60 nm in the axial direction (which is a typical precision for localizing molecules in 3D) requires introducing anisotropy because an object of high symmetry such as a cube or a tetrahedron would not be high enough. We therefore designed a DNA nanopillar (Fig. 3d) that consists of a 12HB with a broadened base17. To place the nanopillar upright on the coverslip, we introduced 15 biotin-modified staple strands in the base and selectively immobilized the DNA nanopillar on a BSAbiotin-neutravidin surface. Overview of the procedure The workflow for making and using DNA origami structures as microscopy standards is illustrated in Figure 4. Depending on the desired intermark distance and microscopy technique, a DNA origami structure is selected. For many instances, this will be the NRO, a DNA bundle or for 3D applications a structure such as a nanopillar (Fig. 3). Based on the caDNAno file of a chosen structure, one selects the associated scaffold (Table 1), the staples and the positions for dye modifications on the DNA origami (Box 2; Figs. 5–7). One has to decide on the labeling strategy (see ‘Labeling strategies’ below) and order the staple strands. Next, the DNA origami structures are synthesized and purified (Steps 1–33). After immobilization on appropriately prepared surfaces (Step 34), the measurement is carried out with the respective microscopy method. Here we describe this procedure with the example of dSTORM imaging31 (Steps 35–44). Finally, the data are analyzed, which can be done automatically with CAEOBS (computer-aided evaluation of origami-based standards; Steps 45–51) software. CAEOBS can be downloaded from our website at https://www.tu-braunschweig.de/pci/forschung/tinnefeld. For troubleshooting, see Table 2.

6HB

Nanopillar

· Boxes 1 and 2

Folding

External labeling

· Steps 1–4 · Steps 15–18

Purification

Quality-control: AFM, gel

· Steps 5–13 · Steps 19–33 · Box 3

· Step 34

Immobilization

Imaging STED

SIM

Confocal

...

2D/3D dSTORM

· Steps 35–44

· Steps 45–51 · Box 4

Data analysis

In the following, the steps outlined in Figure 4 are explained in more detail. DNA origami design with caDNAno software The design of DNA origami structures from scratch is much facilitated through free or open-source software such as caDNAno 2 and CanDo14. caDNAno provides a graphical user interface that helps to design the 3D shape of DNA origami structures or helps to modify existing designs72. CanDo14 provides an online platform that determines the 3D shape of DNA origami designs based on a caDNAno file, and it calculates the mechanical stability of structures in solution. Here the user can upload and submit the respective caDNAno file; the user then receives an e-mail with

Table 1 | Information concerning the four DNA origami structures that we present as microscopy standards.

DNA origami

MgCl2 concentration (folding)

MgCl2 concentration (immobilization, Imaging)

Scaffold

Folding program

Ref.

12HB

p8064

Program 1

16 mM

100 mM

Derr et al.74

6HB

p7560

Program 1

14 mM

500 mM

Schreiber et al.73

NRO

p7249

Program 2

12.5 mM

Figure 5

Woo et al.65

Nanopillar

p8634

Program 1

14 mM

500 mM

Schmied et al.17

The respective scaffold strands are shown in column two. Two different folding programs are used, which are explained in Step 4. The proposed MgCl2 concentration during the folding process has to be verified for each new ordered set of staple strands, as mentioned in the text. The recommended MgCl2 concentration during the immobilization and imaging process and the references to the respective publications with further information are listed in fifth and sixth columns.

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protocol Box 2 | Modification of DNA origami structures with caDNAno ● TIMING 1–2 h

© 2014 Nature America, Inc. All rights reserved.

To generate custom DNA origami–based standards, the positioning and selection of modified staple strands is crucial. Here we describe the selection and modification of an example 12HB with caDNAno72 version 2.2.0, run within Autodesk Maya 2012. If 3D view is not required or Maya is not available, caDNAno can be run as a stand-alone version as well. More information and tutorials on the software are available via the internet, e.g., at http://www.youtube.com/watch?v=EabqNaYAI7o. Distance calculation For the design of customized DNA origami rulers with dyes in a defined distance, the theoretical distances are calculated first. Along the helix, a distance of 0.34 nm per base pair is considered (see example below). On structures such as the NRO, we additionally take an interhelical center-to-center distance of 3 nm into account1,15,18. Note that the precise distance depends not only on the position of the staple strand in the structure but more precisely on the location of the label on the staple strand, e.g., at the 5′ or 3′ end, and it is thus represented best by the respective square or arrow heads in a caDNAno file.   The calculated distance provides a good estimate of the intermark distance that will be measured on the microscope. The accurate distance, however, depends on factors such as the specific DNA origami, the immobilization methods and the buffer conditions. In particular, the concentration of bivalent cations such as Mg2+ is known to have a strong influence on the folding, stability and structure of nucleic acid nanostructures. Although successful folding commonly requires exact concentrations of Mg2+ in the range of 10–20 mM, the folded structures are comparably robust and remain folded as long as a minimal salt concentration is sustained59,60. We, however, observe an influence of the magnesium concentration on the distances between marks in DNA origami nanorulers, especially at low magnesium concentrations. To exemplify this influence, we measured the dependence of the Mg2+ concentration on the intermark distance for the NRO immobilized via BSA-biotin-neutravidin (Fig. 5). When the marks are placed on opposing corners of the NRO (Fig. 5a), we find a reduction of the intermark distance from 97 nm to 81 nm upon increasing the Mg2+ concentration from 0 to 500 mM. Interestingly, when the marks are placed along one double-stranded DNA along the central axis of the DNA origami (see sketch in Fig. 5b), the distance between the marks increases by ~4 nm (Fig. 5b). This different behavior can be explained by the different flexibility along and perpendicular to the DNA origami axis, which is defined by the orientation of the double-stranded DNA helices.   Determination of the accurate distances between marks always requires traceability to SI units involving calibrated micrometers, piezo stages or interferometric measurements. Importantly, calibrations are not frequently necessary because measured distances can be reproduced under identical conditions. The data of Figure 5 were, for example, measured on different days with one DNA origami surface preparation for each data point. Procedure Design of modified DNA origami structures 1. Install Autodesk Maya 2012 (free for academic users, registration required), Python 2.7.2 and caDNAno 2.2.0 as described on http://cadnano.org/. Take care to choose the matching versions for your operating system of caDNAno and Maya. Start Autodesk Maya and the caDNAno interface. 2. Load a caDNAno file in .json format (File → Open). Enlarge the path panel (left, bottom) (Fig. 6a), which provides the systematic view of scaffold and staples, and zoom in (mouse-wheel). 3. Activate the ‘Seq’-tool (Add sequence) and click on the scaffold strand to select the appropriate scaffold sequence (here p8064) from the pop-up window in the standard or custom tab, click on the 5′ end of the scaffold strand and confirm (Apply). The sequences of the scaffold and all staples are now displayed in the scheme with the 5′ break points marked as squares and the 3′ break points marked as arrows. Moving the mouse over the strands, positions are displayed in the left bottom corner in the format ‘helix number [base position] length: length of selected DNA molecule’. 4. Choose the staple strands to be labeled (the actual modifications can be introduced in an exported table containing all staples before ordering). For surface-exposed modifications (e.g., biotin for surface immobilization), choose a strand that is close to the surface of the DNA origami structure. To select the appropriate positions, we recommend having a look at the slice panel where the 12 helices of the structure are numbered 0–11. Activation of helices 12–23 in the slice panel (in order to display virtual neighboring helices) facilitates the identification of potential positions for surface-exposed modifications (Fig. 6b). 5. By clicking on a staple strand in the path panel, possible crossovers to the virtual helices are displayed in gray. A staple strand that has its 5′ or 3′ end close to crossing over to one of the virtual helices is suited for the introduction of a surface-exposed modification. For example, choose staple 4[251]/4[229] and click to display possible crossovers to virtual helix 23 at its 5′ end (Fig. 6a). 6. Color the selected staple by activating the ‘Paint’ tool, and click on the staple. The selected position can also be seen in the 3D panel (Fig. 6c). 7. Proceed accordingly with all strands that you wish to modify. 8. To export all staple strand sequences in a table click ‘Export’, enter a filename and save it in (*.csv) format. 9. Open a spreadsheet application and click on data tab and import external data, choose the text format and import the file, choosing commas to separate columns. 10. You now have generated a table with all staples displayed in the following columns: start ‘start helix number [base position]’, end ‘end helix number[base position]’, sequence, length and color. Click ‘Select all’ and sort according to color in order to find the marked staples. The sequence can be modified by introducing additional linker bases or longer sequences for external labeling. For external labeling (see the ‘Labeling strategies’ section in the INTRODUCTION), a length of the dye-modified labeling strand and the respective (continued) 1372 | VOL.9 NO.6 | 2014 | nature protocols

protocol Box 2 | (continued)

the results, usually within a couple of hours or even minutes. As excellent introductions to using these software packages exist and a substantial number of suitable DNA origami structures are already published1,17,57,65,73,74, including staple sequences and caDNAno files, we focus here on using those existing structures. The caDNAno files for the DNA origami structures used in this protocol are available as Supplementary Methods. Labeling strategies There are two different ways to position organic fluorescent dyes on a DNA origami structure, which we describe in the following by explaining the pros and cons in each case (see also Fig. 7 for a schematic of the different labeling techniques). 1. The most direct and obvious way to label a DNA origami is to modify the respective staple strand at the 3′ or 5′ end with the respective dye (of course, internal modifications are also possible but less common). The yield of labeling of the DNA origami is a combination of the degree of labeling for the modified staple and the yield of incorporation of this staple. This method is called ‘internal labeling’ in this protocol. 2. A cheaper way to label DNA origami structures especially if >4 staples shall be labeled is to attach a ~20-nt-long binding site at the 5′ or 3′ end of the respective staple and to hybridize a complementary labeling strand modified with the respective dye. This approach will be called ‘external labeling’. The main advantage of this strategy is that staples without dye modifications are cheaper by a factor of ~10, and only one labeled staple strand needs to be purchased. The drawback is that the labeling yield of the DNA origami is additionally reduced by the yield of hybridizing the dye-modified staple to the binding sites on the DNA origami structure. There are two options regarding how the labeling strand can be hybridized to the DNA origami structure. The first option is to add the labeling strand for the folding process. In this case, the labeling strand has to be added in excess over the staple strands for the binding sites. The second option involves hybridization of the labeling strand after folding and purification of the DNA origami structures. Here the folded DNA origami structures are diluted in folding buffer together with the labeling strand, and they are incubated at 37 °C for 2 h. As large amounts of expensive dye-modified staple strands are necessary for the first option, we present only the second option in this protocol.

Commonly, DNA origami structures are immobilized on a BSA-biotin-neutravidin surface, which makes it necessary to attach biotins on one side, preferably the opposite side of the dyes to prevent the dyes from unspecific interactions with the surface or surface-bound proteins. The immobilization occurs after the purification step and before imaging the samples. The biotins have to be distributed equally over the area/length of the DNA origami structure. In particular, in the case of DNA origami structures with an internal strain like the rectangular structures (RRO and NRO), it is crucial to attach biotin molecules in each corner of the DNA origami structures. This allows immobilizing the DNA origami structures flat on the surface. Examples of biotin positions can be found in the caDNAno files in the Supplementary Methods. As biotin-labeled staple strands are cheaper than dye-labeled strands, it is advisable

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© 2014 Nature America, Inc. All rights reserved.

complementary strand of at least 20 nt is recommend. You can now order the selected oligonucleotide with or without modifications, for example, with a 5′-biotinylation or dye modification.  CRITICAL STEP Avoid direct contact of organic dyes with electron donors such as guanine, as this can lead to quenching and thereby reduced brightness of the dye, as well as to changed photophysical properties in general94,95. We therefore recommend including a linker of three or four adenine or thymine nucleotides if possible.  CRITICAL STEP For external labeling and for ordering the labeling strand, be aware that DNA binds in an antiparallel manner, which means that the 5′ end of one strand binds to the 3′ end of another strand. Therefore, make sure that the sequence and polarity of the labeling strand fit to the respective binding sites on the DNA origami structure.  CRITICAL STEP We recommend leaving out the staple strands at the ends of the DNA origami structure in order to avoid blunt-end stacking65. Blunt-end stacking leads to DNA origami dimers or even multimers, yielding a gel band that runs behind the monomer band and thus lowering the concentration or the total amount of DNA origami monomers after purification. 11. To receive a structure labeled at the specific position, proceed accordingly with all strands that you wish to modify, and replace the unmodified (negative handle) by the modified (positive handle) staples in the folding reaction mixture.

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Figure 5 | Distance dependence on Mg2+ concentration. Mg2+ dependence of measured distance between two marks on the NRO for different designs. The marks contain three Alexa Fluor 647 dyes each, and they are imaged by dSTORM. The positions of the marks are shown in the sketches. The theoretical distances are 98 nm for the diagonal attachment of marks and 70 nm when the marks are attached along one DNA double-strand. The black and gray horizontal lines indicate the theoretical distances. The measured distances are smaller than those designed, because the geometry of the NRO is not perfectly flat and it depends on the Mg2+ concentration. The influence is much smaller and with opposite sign for dye positions along a single DNA double-strand (compare with Fig. 11). The DNA origami standards are easily imaged with well-reproducible results for all concentrations. For each data point, a new DNA origami surface was prepared by immobilizing at the same Mg2+ concentration as that used for the measurement. Measuring 500–3,000 structures per data point yields a sub-nanometer s.e.m. nature protocols | VOL.9 NO.6 | 2014 | 1373

protocol Figure 6 | caDNAno-assisted modification of DNA origami structures. caDNAno program screenshots of the 12HB DNA origami structure. (a) caDNAno path view with scaffold p8064 colored blue, staples in gray, example staple 4[251] in yellow and possible positions for surface-exposed labels (crossovers to helix 23) in gray. Buttons referenced in the box are highlighted in red. (b) Slice view of the 12HB with helices 0–11 (12HB) in yellow and virtual neighboring helices in pale yellow. (c) 3D view with staple strands used for dye attachment colored in red and a front view of 12HB helices 0–11 (inset).

a

© 2014 Nature America, Inc. All rights reserved.

to use internal labeling for the biotin modifications even when external labeling is possible as well.

b

Folding DNA origami structures The folding reaction is carried out in a tube with 100 µl of folding mixture, which contains the scaffold DNA, modified and unmodified staple strands, and ions and buffer for stabilization of the pH value and structure of the DNA origami. The scaffold DNA and custom DNA oligonucleotides are commercially available, and they can be used without further purification. General information concerning the scaffold strand is provided in Box 1. The basic idea of the DNA origami technique requires that every scaffold strand has access to all required staple strands. In the folding assay, the staples are therefore added in excess over the scaffold concentration, thus resulting in the high yield of correctly folded structures of >90%. It has been shown empirically that the yield of correctly folded DNA origami structures saturates when the staples are at a molar excess of more than fivefold 1. Nevertheless, especially for crucial staple strands, an excess of 10–100-fold is applied. As typical scaffold concentrations in the folding mix are in the range of 10 nM, each staple strand is added in a final concentration of 50–1,000 nM. An example of a folding recipe is shown in Step 3, assuming a folding volume of 100 µl and an initial oligonucleotide concentration of 100 µM. Divalent cations such as magnesium are essential helpers in DNA origami folding reactions. They counteract the negative charges of the DNA-backbone phosphates and reduce repulsion between the scaffold and staple strands to promote proper

c folding. In addition, magnesium stabilizes the stacked form of DNA Holliday junctions, which is a key element of DNA origami. Typical concentrations of magnesium chloride are in the 10–20 mM range, and they need to be determined by a folding concentration screening followed by gel electrophoretic analysis in order to find the best conditions (Fig. 8). For pH stabilization during the folding process, a folding buffer is applied, which is usually prepared in 10× concentration and contains 50 mM Tris base and 10 mM EDTA (pH 8). NaCl can be added to a final concentration of 50 mM, as it seems to decrease the amount of undesired aggregation, but this is not obligatory2,15. The folding process is conducted in a PCR tube in a PCR machine, and it involves fast heating to ~80 °C and controlled hybridization by slow and stepwise cooling to ~30 °C within hours or even days. The high starting temperature melts unspecific base pairings to prevent undesired side products. It was shown recently, by using a real-time PCR machine, that the actual folding occurs in a very small temperature range and within only ~2 h or even a couple of minutes75. This opens the possibility to reduce the folding time tremendously through folding at a constant temperature. As the temperature at which folding occurs depends on the specific DNA origami structure, it has to be identified empirically by folding at temperatures between 45 and 60 °C for 2 h, followed by agarose gel electrophoresis and AFM quality controls (Fig. 8).

BSA Biotin Neutravidin DNA origami with binding slots

Dyes

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Figure 7 | Immobilization and labeling of DNA origami structures. A 12HB DNA origami on a BSA-biotin-neutravidin surface is shown. The 12HB structure carries two dye labels. The cylindrical extensions on the 12HB symbolize the breadboard character of DNA origami structures. This raster of binding slots can be used to immobilize the DNA origami via a BSA-biotin-neutravidin-biotin linker, as well as for labeling the structure with dye molecules. Internal labeling (green dye) and external labeling (red dye) are shown here. In internal labeling, the dye is linked to a staple strand, which is incorporated into the DNA origami structure during folding. External labeling involves hybridization of dye-modified labeling strands to complementary staple strands sticking out of the DNA origami structure.

protocol

© 2014 Nature America, Inc. All rights reserved.

Figure 8 | Quality control of DNA origami structures. Agarose gel electrophoresis and AFM are used for quality control. Folding quality is dependent on the MgCl2 concentration. NRO DNA origami structures are folded with MgCl2 concentrations ranging from 0 to 20 mM, and a quality control was performed by agarose gel electrophoresis. A 2% (wt/vol) gel was run at 60 V in 0.5× TBE buffer with 11 mM MgCl2 for 240 min. The example shows incomplete structures resulting from 2 mM MgCl2 and properly formed structures at 12 mM MgCl2, as confirmed by AFM imaging. Scale bars, 50 nm. Usually the quality of folding is reflected by the brightness, sharpness and migration of the gel band. M, GeneRuler 1kb plus DNA ladder; sc, scaffold p7249 (11.5 µl (100 nM)).

Purification and quality control The folded DNA origami structures need to be separated from the excess of staple strands before they can be used for the actual measurements. The dye-labeled oligonucleotides would give a high background signal or would bind to the surface nonspecifically and thus impair measurements. Two common techniques among other possibilities are used for purification. A fast (

DNA origami-based standards for quantitative fluorescence microscopy.

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new t...
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