ARTICLES NATURETVol 440 16 March 2006 95C to 20C in<2 h. When samples were deposited on mica, only gap; an aspect ratio of 1.05(93.9 nm X 89.5 nm)was expected. By folded DNA structures stuck to the surface while excess staples AFM, 13% of structures were well-formed squares(out of S=45 remained in solution; AFM imaging thus proceeded under buffer observed structures)with aspect ratios from 1.00 to 1.07 and bore the without prior purification. Six different folds were explored; Fig. 2 expected pattern of crossovers(Fig. 2a, upper AFM image). Of the gives their folding paths and their predicted and experimentally remaining structures, -25% were rectangular fragments, and -25% observed DNA structures. Models and staple sequences are given in had an hourglass shape that showed a continuous deformation of the Supplementary Note S3, final designs appear in Supplementary Note crossover lattice(Fig. 2a, lower AFM image). Sequential imaging S12. Experimental methods are given in Supplementary Note S4, documented the stretching of a square into an hourglass, suggesting results described here but not shown are in Supplementary Note S5. that hourglasses were originally squares that stretched upon depositio Of the products imaged by AFM, a particular structure was con- or interaction with the AFM tip. No subsequent designs exhibited sidered qualitatively well-formed if it had no defect(hole or stretching. Other designs had either a tighter 1.5-turn spacing with indentation in the expected outline) greater than 15 nm in diameter. 32-mer staples spanning three helical domains(Fig. 2b-d, f)or r each fold the fraction of well-formed structures, as a percentage smaller domains that appeared to slide rather than stretch(Fig. 2e) of all distinguishable structures in one or more AFM fields, was To test the formation of a bridged seam, a rectangle was designed alculated as a rough estimate of yield. I note that while some (Fig 2b)according to the scheme outlined in Fig. le using 1.5-turn structures classified as well-formed had 15-nm defects, most had crossover spacing, 32-mer staples and a circular scaffold. As seen in no defects greater than 10 nm in diameter. Fig. 2b, the central seam and associated pattern of crossovers was First, a simple 26-helix square was designed (Fig. 2a). The easily visualized(upper AFM image). Rectangles stacked along their had no vertical reversals in raster direction, required a linear vertical edges, often forming chains up to 5 um long(lower AFM and used 2.5-turn crossover spacing. Most staples were 26-m image). The yield of well-formed rectangles was high(90%, S=40), bound each of two adjacent helices as in Fig. Ic, but via 13 bases and so rectangles were used to answer basic questions concerning rather than 8. The design was made assuming a 1. 5-nm inter-helix inter-helix gaps, base-stacking, defects and stoichiometry. AFM drift △AA SAAA A入 A Figure 3 I Patterning and combining DNA origami. a, Model for a pattern lines in e) greatly decrease stacking ().j-m, Two labellings of the sharp representing DNA, rendered using hairpins on a rectangle( Fig. 2b).b, AFM triangle show that each edge may be distinguished. In j-u, pixels fall on a nage. One pixelated DNA turn(-100 nm)is 30x the size of an actual DNA rectilinear lattice. n-u, Combination of iangles into hexagon turn(-3.6nm)and the helix appears continuous when rectangles stack (n, p, q)or lattices(o, r-u) Diagrams(n, o)show positions at which staples appropriately. Letters are 30 nm high, only 6x larger than those writter are extended (coloured protrusions) to match complementary single- sing STM in ref 3: 50 billion copies rather than I were formed. c, d, Model stranded regions of the scaffold(coloured holes). Models(P, r)permit and AFM image, respectively, for a hexagonal pattern that highlights the comparison with data(a, s). The largest lattice observed comprises only early hexagonal pixel lattice used in a-1. e-1, Map of the western hemisphere,scale 1: 2 x 10, on a rectangle of different aspect ratio. d and f were stretched and sheared to correct for afm drift scale ba Normally such rectangles aggregate(h)but 4-T loops or tails on edges(white h, i, I um; q, s-u, 100 300 2006 Nature Publishing Group© 2006 Nature Publishing Group 95 8C to 20 8C in ,2 h. When samples were deposited on mica, only folded DNA structures stuck to the surface while excess staples remained in solution; AFM imaging thus proceeded under buffer without prior purification. Six different folds were explored; Fig. 2 gives their folding paths and their predicted and experimentally observed DNA structures. (Models and staple sequences are given in Supplementary Note S3, final designs appear in Supplementary Note S12. Experimental methods are given in Supplementary Note S4, results described here but not shown are in Supplementary Note S5.) Of the products imaged by AFM, a particular structure was con￾sidered qualitatively ‘well-formed’ if it had no defect (hole or indentation in the expected outline) greater than 15 nm in diameter. For each fold the fraction of well-formed structures, as a percentage of all distinguishable structures in one or more AFM fields, was calculated as a rough estimate of yield. I note that while some structures classified as well-formed had 15-nm defects, most had no defects greater than 10 nm in diameter. First, a simple 26-helix square was designed (Fig. 2a). The square had no vertical reversals in raster direction, required a linear scaffold, and used 2.5-turn crossover spacing. Most staples were 26-mers that bound each of two adjacent helices as in Fig. 1c, but via 13 bases rather than 8. The design was made assuming a 1.5-nm inter-helix gap; an aspect ratio of 1.05 (93.9 nm £ 89.5 nm) was expected. By AFM, 13% of structures were well-formed squares (out of S ¼ 45 observed structures) with aspect ratios from 1.00 to 1.07 and bore the expected pattern of crossovers (Fig. 2a, upper AFM image). Of the remaining structures, ,25% were rectangular fragments, and ,25% had an hourglass shape that showed a continuous deformation of the crossover lattice (Fig. 2a, lower AFM image). Sequential imaging documented the stretching of a square into an hourglass, suggesting that hourglasses were originally squares that stretched upon deposition or interaction with the AFM tip. No subsequent designs exhibited stretching. Other designs had either a tighter 1.5-turn spacing with 32-mer staples spanning three helical domains (Fig. 2b–d, f) or smaller domains that appeared to slide rather than stretch (Fig. 2e). To test the formation of a bridged seam, a rectangle was designed (Fig. 2b) according to the scheme outlined in Fig. 1e using 1.5-turn crossover spacing, 32-mer staples and a circular scaffold. As seen in Fig. 2b, the central seam and associated pattern of crossovers was easily visualized (upper AFM image). Rectangles stacked along their vertical edges, often forming chains up to 5 mm long (lower AFM image). The yield of well-formed rectangles was high (90%, S ¼ 40), and so rectangles were used to answer basic questions concerning inter-helix gaps, base-stacking, defects and stoichiometry. AFM drift Figure 3 | Patterning and combining DNA origami. a, Model for a pattern representing DNA, rendered using hairpins on a rectangle (Fig. 2b). b, AFM image. One pixelated DNA turn (,100 nm) is 30£ the size of an actual DNA turn (,3.6 nm) and the helix appears continuous when rectangles stack appropriately. Letters are 30 nm high, only 6£ larger than those written using STM in ref. 3; 50 billion copies rather than 1 were formed. c, d, Model and AFM image, respectively, for a hexagonal pattern that highlights the nearly hexagonal pixel lattice used in a–i. e–i, Map of the western hemisphere, scale 1:2 £ 1014, on a rectangle of different aspect ratio. Normally such rectangles aggregate (h) but 4-T loops or tails on edges (white lines in e) greatly decrease stacking (i). j–m, Two labellings of the sharp triangle show that each edge may be distinguished. In j–u, pixels fall on a rectilinear lattice. n–u, Combination of sharp triangles into hexagons (n, p, q) or lattices (o, r–u). Diagrams (n, o) show positions at which staples are extended (coloured protrusions) to match complementary single￾stranded regions of the scaffold (coloured holes). Models (p, r) permit comparison with data (q, s). The largest lattice observed comprises only 30 triangles (t). u shows close association of triangles (and some breakage). d and f were stretched and sheared to correct for AFM drift. Scale bars: h, i, 1 mm; q, s–u, 100 nm. ARTICLES NATURE|Vol 440|16 March 2006 300