Unidirectional scaffold-strand arrangement in DNA origami

Since the origin of DNA nanotechnology over 30 years ago, branched DNA tiles have been developed to construct a variety of DNA nanostructures. Among the unique building blocks that have been demonstrated, rigid, antiparallel double-crossover (DX) tiles have had great significance as both the unit motif in the first two-dimensional DNA crystal reported and as the basic repeating unit in most DNA origami structures. In contrast, parallel DX tiles have yet to be developed, despite that they were initially reported more than 20 years ago, most likely because their assembly yields are not comparable to anti-parallel DX tiles. Herein we demonstrate construction of DNA origami architectures based on modified parallel DX tiles, in which a single-crossover linkage, rather than reciprocal crossovers, is present at each junction point between neighboring helices. The yields of these novel origami structures are similar to their counterparts that are constructed based on antiparallel DX tile units containing double-reciprocal crossovers at each junction point. We demonstrate that a unidirectional arrangement of the scaffold strand can be used in the assembly of a variety of 2D and 3D DNA origami. This new design will greatly expand the diversity of DNA origami achieved and enable their further assembly into larger structures. The DX tiles, defined by Fu and Seeman in 1993, each contain two independent crossover points that join two adjacent DNA helices to form a rigid tile with the axes of the helices between the crossovers arranged roughly coplanar. Five types of DX tiles were initially reported: two antiparallel and three parallel molecules that differed in the relative orientation of the helical domains, the arrangement of the constituent strands, and the distance between the crossover points. DAE and DAO molecules (double-crossover antiparallel molecules with an even number or odd number of half turns between crossover points along the same two helices) were later applied to DNA origami designs with great success, largely because these tiles contain two unperturbed DNA strands of opposing polarity that are easily linked to form a scaffold strand. In that case, the antiparallel scaffold is wound back and forth in a raster fill pattern to form a particular shape, through the use of hundreds of staple strands. In contrast, DPE (double-crossover parallel molecules with an even number of half turns between crossover points) molecules were not thoroughly explored for the construction of higher-order structures. Compared to antiparallel DX molecules, parallel DX molecules are generally regarded as less stable presumably because of stronger electrostatic repulsion between the opposing DNA backbones. Later, Sherman and co-workers reported that strand end-pinning and misfolding caused by the structural bias of nominally flexible junctions might affect the proper structural formation of parallel DX molecules. Breaking the continuity of one of the two strands that comprise each junction in a DPE tile at the junction point will produce parallel, double-helical units with single-crossover linkages between the helices. Such modified DPE tiles provide a direct solution to both the possible steric hindrance at the crossover points and the kinetic trap noted by Sherman and co-workers. Herein, we demonstrate that the unperturbed strands in these modified DPE tiles can be linked to form a long scaffold strand that is directed by a collection of staple strands to form DNA origami structures. The scaffold strand in adjacent helices possesses the same 5’–3’ polarity and we therefore refer to these structures as parallel helix (PH) origami. Assembling PH origami required that we develop unique, coiled-scaffold folding paths rather than the typical raster fill patterns adopted by the scaffold in DNA origami structures with antiparallel helices (AH). We also designed and assembled hybrid DNA origami structures that contain both parallel and antiparallel scaffold regions. The DAE, DPE, and modified DPE tiles are shown in Figure 1A–C for comparison. The single-stranded DNA (depicted in gray) remains unperturbed at all junction points and the relative polarities of these two strands determine whether the molecules are classified as antiparallel or parallel. The remaining DNA strands are shown in various colors and correspond to the staple strands that direct/hold the linear strands in a coplanar arrangement. The staple strands in the antiparallel tiles (Figure 1A) reverse direction at each crossover point while those in the parallel tiles (Figure 1B) do not. Note that the backbones of the strands at the crossover points in the parallel tiles are directly opposite one another, which imparts a certain degree of instability to the parallel tiles. Thus, in the modified DPE tiles (Figure 1C), a single-crossover linkage replaces the typical reciprocal [*] Dr. D. Han, S. Jiang, A. Samanta, Prof. Y. Liu, Prof. H. Yan The Biodesign Institute, Arizona State University Tempe, AZ 85287 (USA) E-mail: hao.yan@asu.edu