DNA Brick Crystals with Prescribed Depth

We describe a general framework for constructing two-dimensional crystals with prescribed depth and sophisticated three-dimensional features. These crystals may serve as scaffolds for the precise spatial arrangements of functional materials for diverse applications. The crystals are selfassembled from single-stranded DNA components called DNA bricks. We demonstrate the experimental construction of DNA brick crystals that can grow to micron-size in the lateral dimensions with precisely controlled depth up to 80 nanometers. They can be designed to display user-specified sophisticated three-dimensional nanoscale features, such as continuous or discontinuous cavities and channels, and to pack DNA helices at parallel and perpendicular angles relative to the plane of the crystals. [email protected]; [email protected]. †Equal contribution authors: Ke, Ong, Sun ‡Present address: Wallace H. Coulter Dept. of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322 Author contributions Y.K., L.L.O. and W.S. made equal contributions. Y.K. conceived the project, designed and performed the experiments, analyzed the data, and wrote the paper; L.L.O. and W.S. designed and performed the experiments, analyzed the data, and wrote the paper; J.S. and M.D. performed the cryo-EM and AFM experiments, analyzed the data, and wrote the paper; W.M.S discussed the results and wrote the paper; P.Y. conceived, designed, and supervised the study, interpreted the data, and wrote the paper. Additional information Supplementary information is available in the online version of the paper. Reprints and permission information is available online at http://www.nature.com/reprints. Correspondence and requests for materials should be addressed to P.Y. and Y.K. Competing financial interests The authors declare competing financial interests: a provisional US patent application has been filed. NIH Public Access Author Manuscript Nat Chem. Author manuscript; available in PMC 2015 May 01. Published in final edited form as: Nat Chem. 2014 November ; 6(11): 994–1002. doi:10.1038/nchem.2083. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript The production of two-dimensional materials, particularly crystals with prescribed depths and intricate three-dimensional (3D) features, provides an enabling platform for nanofabrication. For example, these two-dimensional (2D) crystals could be integrated with inorganic nanomaterials for developing complex nanoelectronics1 and photonics systems.2,3 Although thin film structures have been created using either electron/ion beam lithography3 or self-assembly of block co-polymers,4,5 fabricating two-dimensional materials that simultaneously achieve precisely tunable thickness, and prescribed complex surface and internal features (e.g. channels or pores) with sub-5 nm resolution remains challenging.3,6–8 A promising route to address this challenge is structural DNA nanotechnology.9 DNA has been used to create complex discrete shapes9–25 and extended periodic crystals,26–38 including ribbons,33 tubes,27,32,33,35 two-dimensional crystals,18,26–32,36–38 and threedimensional crystals.34 DNA structures can serve as scaffolds for precise patterning of functional moieties (e.g. gold nanoparticles) for electronics and photonics applications.35,39,40 However, in contrast to current organic polymeric films,41 the twodimensional DNA crystals are typically restricted to a single-layer of DNA helices with about 2 nanometer depth. A 3D crystal was previously reported but it grows in all three dimensions with no control in depth and uses a small triangular repeating unit.34 One major categorical gap in constructing atomically precise DNA structures – and, more generally, synthetic molecular structures – is the lack of a general framework for making complex 2D crystals with precisely controlled depth and sophisticated three-dimensional features. Successful construction of such structures could enable a wide range of applications ranging from nanoelectronics and plasmonics to biophysics and molecular diagnosis. Using single-stranded DNA bricks,21,22,33 we describe here a simple, robust, and general approach to engineer complex micron-sized two-dimensional crystals with prescribed depths and complex three-dimensional features with nanometer resolution. In previous reports,26–32,34–38 DNA crystals are typically formed via a two-stage hierarchical process: individual strands first assemble into a discrete building block (often known as a DNA tile) and individual tiles then assemble into crystals. In contrast, DNA brick crystals grow nonhierarchically: the growth of DNA crystals from short, floppy, single-stranded DNA bricks does not involve the assembly of pre-formed discrete multi-stranded building blocks with well-defined shapes. During the brick crystal growth, assembly and disassembly occur by relatively weak intermolecular interactions involving addition or subtraction of a single short strand at a time. We constructed a total of 32 DNA brick crystals. These crystals can grow up to several microns in the lateral dimensions with a prescribed depth up to 80 nanometers, and display sophisticated user-specified nanometer scale three-dimensional features, including intricate cavities, channels, and tunnels (Supplementary Fig. S1). Additionally, the non-hierarchical nature of the assembly permits isothermal formation of the crystals. We illustrated the scaffolding utility of these crystals by functionalizing them with parallel arrays and layers of tightly-packed (1–2 nm spaced) gold nanoparticles. Ke et al. Page 2 Nat Chem. Author manuscript; available in PMC 2015 May 01. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Design and assembly of DNA-brick crystals Crystal design is based on previous discrete three-dimensional DNA-brick structures.22 A DNA brick is a 32-nucleotide (nt) strand with four 8-nt binding domains, and can be modeled as a LEGO-like brick (Fig. 1a). In a one-step annealing reaction, DNA bricks – each with a distinct sequence – assemble into a prescribed structure by binding to their designated neighbors. Implementing “connecting” bricks between discrete structures yields DNA-brick crystals. The design strategy is illustrated using a 6H (helix) × 6H (helix) × 24B (basepair) cuboid structure that can be programmed to grow along three orthogonal axes (Fig. 1b). To achieve homo-multimerization along the Z-axis (i.e. parallel to helical axes), the first layer of domains are modified to be complementary to the last layer of domains. Homomultimerization along the X-axis or Y-axis is achieved by including bricks that each has two of its domains bound to one face of the cuboid and the other two domains to the opposing face. See Supplementary Fig. S2 for detailed strand connecting patterns. The crystals are designed to form via non-hierarchical growth: individual bricks (rather than preformed multi-brick blocks) are directly incorporated into the crystal (Fig. 1c). We constructed four groups of crystals: (1) Z-crystals: one-dimensional “DNA-bundle” crystals extending along the Z-axis (Fig. 1d); (2) X-crystals: one-dimensional crystals extending along the X-axis; (3) ZX-crystals: two-dimensional “multilayer” crystals extending along the Z-axis and the X-axis (Fig. 1e); (4) XY-crystals: two-dimensional “DNA-forest” crystals extending along the X-axis and Y-axis (Fig. 1f). Using different designs of repeating units, DNA crystals with prescribed depths and features, such as pores, channels, and tunnels, can be made (Fig. 1g to 1i). Here we define a channel as a surfaceexposed cavity extending across multiple repeating units, a pore as a hole across a single repeating unit, and a tunnel as a series of concatenated pores. A crystal is named as “[the growth direction(s)]-[the dimensions of the repeating-unit]-[the shape of the unit]”. For instance, an “XY6H×6H×24B-cuboid” crystal is a two-dimensional XY-crystal with a cuboid-shaped 6H×6H×24B repeating unit. Like discrete DNA-brick structures,22 the sequences for DNA-brick crystals were randomly generated. All crystals used 10.67 basepair (bp)/turn reciprocal twist density which is slightly under-wound compared to the 10.5 bp/turn of natural B-form DNA. Each crystal was assembled by mixing 100 nM of each unpurified DNA brick strand in 40 mM MgCl2, without careful adjustment of strand stoichiometry. After 72-hour or 168-hour one-pot annealing, assembled crystals were imaged using transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), or atomic force microscopy (AFM) without further purification. See Methods for details. One-dimensional DNA-bundle crystals (Z-crystals) Both solid Z-crystals (Fig. 2a–f) and Z-crystals with tunnels (Fig. 2g–i) were successfully constructed. Ke et al. Page 3 Nat Chem. Author manuscript; available in PMC 2015 May 01. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Solid Z-crystals with different cross-sectional shapes We first constructed three solid Z-crystals with distinct square-shaped cross-sections (6H×6H×32B, 8H×8H×32B, and 10H×10H×32B; Fig. 2a–c). We then demonstrated crystals with more complex cross-sections: a Z-8H×8H×128B-spiral crystal with a surface helical channel along the Z-axis (Fig. 2d), a Z-43H×32B-triangle crystal (Fig. 2e), and a Z-44H×32Bhexagon crystal (Fig. 2f). The spiral channel was clearly visible in the TEM image of the Z-8H×8H×128B-spiral crystal. However, many broken structures were also observed for this spiral crystal (Supplementary Fig. S6). Z-crystals with tunnels Three Z-crystals with tunnels were tested (Fig. 2g–i). The cross-section of the Z-56H×32Btunnel is an 8H×8H square with a 2H×4H rectangle removed from the center (Fig. 2g). The Z-108H×32B-tunnel has a 12H×12H square cross-section with a 6H×6H hole (Fig. 2h). The Z60H×64B-tunnel crystal contains a 2H×2H tunnel along the Z-axis and 8H×2H×24B pores that intersect the 2H×2H tunnel every 64bp along the Z-axis (Fig. 2i). TEM images of the Z-60H×64B-tunnel showed many splintered structures containing only half of the designed DNA helices, likely reflecting the weakening effect of the periodic 8H×2H×24B pores on the connections between the top and bottom halves of the structures along the Y-axis. All Z-crystals displayed a global right-handed twist, which likely resulted from the stress generated by the underwound design.17,42 Zoomed-out TEM images of Z-crystals are included in Supplementary Figs. S3 to S11. One-dimensional X-crystals We constructed two 1D-crystals that extended along the X-axis: an X-6H×6H×64B-cuboid crystal (Fig. 2j) and an X-32H×64B-pore crystal (Fig. 2k). Both appeared well-formed and grew up to a few hundred nanometers in length in TEM images. See Supplementary Fig. S12 for larger images. Two-dimensional DNA-multilayer crystals (ZX-crystals) Solid ZX-crystals (Fig. 3a–d), ZX-crystals with channels, pores, and tunnels (Fig. 3e–h), and an offset ZX-crystal (Fig. 3i) were successfully constructed.