NANO LETTERS
High-Resolution Structural Analysis of a DNA Nanostructure by cryoEM
2009 Vol. 9, No. 7 2747-2750
Takayuki Kato,† Russell P. Goodman,§ Christoph M. Erben,§ Andrew J. Turberfield,§ and Keiichi Namba*,†,‡ Graduate School of Frontier Biosciences, Osaka UniVersity, Osaka, Japan, Dynamic Nanomachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan, and Clarendon Laboratory, Department of Physics, UniVersity of Oxford, Oxford, OX1 3PU, U.K. Received April 21, 2009; Revised Manuscript Received May 24, 2009
ABSTRACT Many DNA nanostructures have been produced and a wide range of potential applications have been proposed. However, confirmation of accurate 3D construction is particularly challenging. Here, we demonstrate that cryoEM may be exploited to obtain structural information at sufficient resolution to visualize the DNA helix and reveal the absolute stereochemistry of a 7 nm self-assembled DNA tetrahedron. Structural analysis at such high resolution by cryoEM image analysis is unprecedented for any biological molecule of this size.
The bottom-up approach to nanotechnology allows controllable and reliable construction of three-dimensional (3D) structures on the nanometer scale with near-atomic precision. DNA is an ideal building material on this length scale; Watson-Crick base-pairing interactions can be used to control self-assembly to produce a wide variety of structures, both dynamic and static, discrete and periodic.1,2 Various DNA nanostructures have been designed and produced using this strategy, and a wide range of potential applications by using this strategy have been proposed, including DNA cages3 for drug delivery, protein crystal templates,4 medical diagnosis,5 and DNA computing systems.6-8 For practical applications it is essential to verify that the three-dimensional (3D) structures are accurately produced as designed, which requires high resolution, at least sufficient to resolve the DNA helix. However, examination and confirmation of their accurate 3D constructions are particularly challenging given their extremely small sizes. Structures have been visualized by either atomic force microscopy (AFM)8-11 or electron cryomicroscopy (cryoEM).12-15 AFM is a powerful technique for high-resolution observation of flat structures on flat substrates but less well adapted to the visualization of 3D structures of nonflat molecules such as polyhedra. Threedimensional images of DNA polyhedra have been reconstructed by cryoEM and single particle image analysis to demonstrate that their overall structures are consistent with their designs, but the resolution of the reconstructions is still * To whom correspondence should be addressed. E-mail: keiichi@ fbs.osaka-u.ac.jp. † Osaka University. ‡ Dynamic Nanomachine Project, ICORP, JST. § University of Oxford. 10.1021/nl901265n CCC: $40.75 Published on Web 06/03/2009
2009 American Chemical Society
too low to determine absolute 3D configurations or to distinguish stereoisomers even for relatively large particles with sizes ∼20-80 nm and molecular weights of ∼180 kDa to 2 MDa.12-15 For practical applications, a structure manufactured by selfassembly must correspond to its “blueprint”. In particular, it is important to control the formation of stereoisomers as the basepair connectivity of a DNA nanostructure cannot directly specify which stereoisomer is produced. DNA cage structures can form two conformational diastereomers (interconversion corresponds to turning the structure “inside out”) that have similar chemical and physical properties and are therefore difficult to separate, but in which each functional group is positioned differently. Accurate and reproducible syntheses and the development of methods to discriminate between stereoisomers are essential to future industrial exploitation of this technology. CryoEM and single-particle image analysis are powerful tools for visualizing 3D structures of biological macromolecular assemblies such as icosahedral viruses and ribosomes.16 The resolution of cryoEM 3D image reconstruction can be increased by increasing the number of particle images to be averaged, but the achievable resolution is still dependent upon the particle size because the low contrast and low signal-to-noise ratio (S/N) of raw cryoEM images make it difficult to select particles and accurately align raw images for averaging. This difficulty becomes extremely serious for particles smaller than a critical size of around 10 nm or a molecular weight of 150-200 kDa. For an icosahedral virus with a diameter as large as 75 nm and a molecular weight of 40 MDa, for instance, a
Figure 1. Contrast enhancement by focal pair merging of electron cryomicrographs for particle selection. (a) Micrograph recorded at 40 e-/Å2 electron dose and ∼2.0 µm defocus. (b) Micrograph recorded at 100 e-/Å2 electron dose and ∼5.0 µm defocus from the same area as panel a. (c) Contrast-enhanced image by focal pair merging of panels a and b. Upper right insets show magnified images of a particle cut out from each micrograph. Scale bars, 20 nm.
resolution better than 4 Å has already been reached by aligning and averaging about 10 000 particle images and also by taking advantage of its intrinsic high symmetry that connects 60 identical copies of one structural unit within each particle; the density map clearly shows main chains and side chains of the subunit polypeptides.17,18 For the bacterial ribosome, which has a size of about 20 nm, a molecular weight of 2.5 MDa and no symmetry, the highest resolution so far achieved is slightly better than 7 Å, at which the RNA backbones and R-helices of component proteins are visible.19,20 A slightly worse but similar resolution has been achieved for a smaller particle; the structure of the human transferrin receptor-transferrin complex, whose size and molecular weight are about 13 nm and 290 kDa, respectively, has been resolved at 7.5 Å resolution.21 The achievable resolution, however, becomes severely lower for much smaller particles. CryoEM image reconstruction of the p53 tumor suppressor protein, whose size and molecular weight are about 10 nm and 180 kDa, respectively, has been carried out but only at 13.7 Å resolution with the density map just allowing the shape of the molecular complex to be visualized.22 Herein, we demonstrate that cryoEM image analysis may be exploited to obtain structural information of sufficient resolution to reveal the absolute 3D configuration of a designed DNA nanostructure. The DNA tetrahedron was assembled by annealing and ligating four 63 mer oligonucleotides as described.11 Each edge is a 7 nm, 20 basepair duplex, and the edges are connected covalently through single unpaired adenosine nucleotides, making it a rigid, triangulated structure that could serve as a building block for larger 3D structures or as a molecular cage.3,11 Figure 1a shows an electron cryomicrograph of the DNA tetrahedron particles embedded in vitreous ice, recorded at a magnification of 142 000×, 2.0 µm underfocus, and an electron dose of 40 e-/Å2. Because of the inherent small size of the particles, they are mostly invisible due to the extremely low contrast and low S/N. Since larger amounts of defocus and electron dose are known to enhance the contrast and S/N, the second micrograph was recorded from the same area of the specimen at 5 µm underfocus and an electron dose of 100 e-/Å2. Despite such large amounts of underfocus 2748
Figure 2. 3D density maps of the DNA tetrahedron revealed by cryoEM image reconstruction. (a) 3D density map at 20 Å resolution produced by using a simple circular mask to isolate particle images. (b) 3D density map at 12 Å resolution produced by using a tight mask determined from the density map shown in panel a. Scale bar, 5 nm.
and electron dose, the contrast and S/N enhancement were not high enough to allow particle image selection (Figure 1b). The contrast and S/N were then further enhanced by applying a focal-pair merging technique.23 The effect of enhancement was rather dramatic (Figure 1c); we were able to determine the positions of about 3000 particles in 278 focal-pair merged micrographs and used this information to pick up particle images from the first micrographs of each pair, that is, those recorded with smaller underfocus and less radiation damage. Figure 2a shows a 3D density map of the DNA tetrahedron reconstructed at 20 Å resolution (at Fourier shell correlation ) 0.5) by using a simple, relatively large circular mask to encircle the particle images. The density map reveals a hollow tetrahedron with each edge 7 nm in length, which is exactly the shape and dimension of the expected structure. However, the resolution is not high enough to visualize the DNA helix and therefore the absolute 3D configuration of the DNA tetrahedron, so discrimination between stereoisomers is not possible. We hypothesized that image noise in the solvent region surrounding the DNA tetrahedron molecule had lead to misclassification and misalignment of the particle images to some extent, resulting in the relatively poor resolution. We therefore applied a solvent flattening technique to reduce the noise in the solvent region and to improve the accuracy of image classification and alignment in the refinement cycle Nano Lett., Vol. 9, No. 7, 2009
Figure 3. Comparison between the 3D density map and structural models of the two expected diastereomers. (a,b) Space-filling representation of atomic models of diastereomers with (a) the minor groove and (b) the major groove facing outward at the edge center. Arrows indicate the major grooves of the double helix, which are resolved in the density map. (c,d) Superposition of the model structures (a,b) and the density map obtained by cryoEM image reconstruction. Scale bar, 5 nm.
of single particle image analysis. In each image alignment cycle we applied a relatively tight mask to the reference 3D volume that is used to align each single-particle image; the mask flattens the density in the solvent region surrounding the DNA tetrahedron to reduce noise that is likely to cause misalignment. The mask was determined from the 3D density map reconstructed in the previous iteration, but at a low contour level to avoid modifying the density of the DNA tetrahedron. The solvent flattening technique was very effective, possibly as a result of the presence of a large, solvent-filled space within the DNA tetrahedron as well as the significant noise reduction in the solvent region surrounding the particle. The resolution of the 3D density map was dramatically improved to 12 Å as shown in Figure 2b. The helical twist of the DNA edges can now be seen clearly, and the exact positions of the B-DNA major grooves can be identified. The DNA tetrahedron has two possible stereoisomers. Figure 3 panels a and b are atomic models of the two conformational diastereomers in which the minor grooves and major grooves of each duplex edge face outward at the edge center, respectively. Indirect, biochemical evidence has previously demonstrated that the diastereomer with the minor grooves facing outward at the edge center (Figure 3a) is the dominant product of the self-assembly process.11 Figure 3 panels c and d are superpositions of the density map obtained by cryoEM image reconstruction and the two structural models shown in Figure 3a,b, respectively. The ridge of high density that winds around each edge of the tetrahedron in the density map corresponds to the minor groove of the double helix, across which the backbones of the oligonucleNano Lett., Vol. 9, No. 7, 2009
otides that form the edge are not resolved. The ridge is bounded by the wider major groove, which is clearly resolved. Close agreement between the expected and the observed positions of the major and minor grooves is evident for the diastereomer with the minor groove facing outward at the edge center (Figure 3a,c). The high resolution of the cryoEM image reconstruction, sufficient to resolve the structure of the DNA helices, allows us to identify the correct diastereomer of the DNA tetrahedron directly and thus to determine its absolute spatial configuration. Examination and confirmation of the absolute spatial configuration of a synthetic DNA nanostructure is an essential step toward its practical application. Although bottom-up nanofabrication by self-assembly is capable of controllable and reliable construction of 3D structures with near-atomic precision, this precision is only useful if it is possible to discriminate between stereoisomers. Different conformational diastereomers, which satisfy the same basepairing constraints that are designed to control assembly and may appear very similar, would produce completely different spatial arrangements of attached functional molecules or particles. For example, quantum dots or proteins conjugated to the outside surface of the tetrahedron shown in Figure 3a,c (the observed diastereomer) would be crammed into the inside of the alternative structure shown in Figure 3b,d. We achieved sufficient resolution to discriminate between the two possible tetrahedra shown in Figure 3 by exploiting two technical developments, one in EM hardware and the other in software for image analysis, that allow us to optimize the resolution of cryoEM. The electron microscope was equipped with a field-emission electron gun, which produces a highly coherent electron beam; a liquid helium-cooled ultrastable specimen stage, by which EM images of high resolution and S/N can be recorded at a relatively high electron dose without significant radiation damage; an incolumn Ω-type energy filter for zero-loss imaging, which further improves S/N by removing inelastically scattered electrons that produce a high background; and a highly sensitive charge-coupled device (CCD) camera that significantly contributes to high-throughput, high-quality image recording. For the efficient recording of a large number of cryoEM images of frozen-hydrated, small, biological molecules embedded in thin vitreous ice film, all of these four features are equally important, but it is noteworthy that the relatively new addition of the last two features, namely the Ω-type energy filter and the CCD camera, played an essential role in the present study. High resolution also required innovative image analysis using focal-pair merging23 and solvent flattening techniques.25 The DNA tetrahedron that we have visualized is the smallest 3D nanostructure made by DNA self-assembly. At 78 kDa, it is also by far the smallest molecule to date for which 3D image reconstruction has been carried out by cryoEM at resolution near 10 Å with the second smallest being around 180 kDa.22 We have achieved resolution sufficient not only to verify its structure but also to discriminate between structurally similar diastereomers, demonstrating that cryoEM is an indispensable tool for the 2749
characterization of 3D nanostructures designed to be selfassembled from biomolecular components. Experimental Section. CryoEM Image Reconstruction. The DNA tetrahedron was prepared as described.11 A 3 µL sample of a solution of DNA tetrahedra in 5 mM MgCl2, 10 mM Tris (pH 8) was applied onto a thin, holey carbon film supported on an EM grid (Quantifoil R1.2/1.3). After the excess solution was blotted by filter paper, the grid was quickly frozen by rapidly plunging it into liquid ethane by using an FEI Vitrobot. The specimen was observed at a temperature close to 4 K using a JEOL JEM-3200FSC electron microscope, which is equipped with a liquid-helium cooled specimen stage, an Ω-type energy filter and a fieldemission electron gun operated at an accelerating voltage of 200 kV. Zero energy-loss images were recorded at a magnification of 142 000× using a TVIPS 4k × 4k slowscan CCD (image pixel size: 1.06 Å/pixel). Paired micrographs were recorded with a defocus between 1.5 and 3.0 µm and an electron dose of 40 e-/Å2 for the first micrograph and a defocus value of 5.0 µm and an electron dose of 100 e-/Å2 for the second. They were merged by using the focalpair command in the EMAN software package.23 The focal pair processing has two steps, alignment of the second micrograph to the first and merging the pairs. For close-tofocus (
