Assembly and Characterization of 8-Arm and 12-Arm DNA Branched Junctions

Abstract

Branched DNA molecules can be assembled into objects and networks directed by sticky-ended cohesion. The connectivity of these species is limited by the number of arms flanking the branch point. To date, the only branched junctions constructed contain six or fewer arms. We report the construction of DNA branched junctions that contain either 8 or 12 double helical arms surrounding a branch point. The design of the 8-arm junction expoits the limits of a previous approach to thwart branch migration, but the design of the 12-arm junction uses a new to principle achieve this end. The 8-arm junction is stable with 16 nucleotide pairs per arm, but the 12-arm junction has been stabilized by 24 nucleotide pairs per arm. Ferguson analysis of these junctions in combination with three, four, five, and six-arm junctions indicates a linear increase in friction constant as the number of arms increases; the four-arm junction migrates anomalously at 4°C., suggesting stacking of its domains. All strands in both the 8-arm and 12-arm junctions show similar responses to hydroxyl radical autofootprinting analysis, indicating that they lack any dominant stacking structures. The stability of the 12-arm junction demonstrates that the number of arms in a junction is not limited to the case of having adjacent identical base pairs flanking the junction. The ability to construct eight-arm and twelve-arm junctions increases the number of objects, graphs and networks that can be built from branched DNA components. In principle, the stick structure corresponding to cubic close packing is now a possible target for assembly by DNA nanotechnology. KeywordsBranched DNA Motifs; N-Connected DNA-Networks; N-Connected DNA Graphs; Hydroxyl Radical Autofootprinting; Gel Electrophoresis; Ferguson Plots DNA branched junctions containing 3 or 4 arms around a single point are seen as ephemeral intermediates in the biological processes of replication 1 and recombination. 2 In addition, immobile versions of these junctions have served as the basis for virtually all of the work done in structural DNA nanotechnology. 3 A variety of DNA structures and motifs have been created using these components, 4 including stick figures whose edges are DNA double helices and whose vertices are the branch points of branched junctions; these include molecules with the connectivities of a cube 5 and a truncated octahedron, 6 as well as an irregular graph. 7 The connectivity of DNA structures of this sort is limited by the number of double helical DNA arms that can flank a branch point.

An Overview of Structural DNA Nanotechnology

“…Individual branched junctions have been constructed containing 3 arms [39] 4 arms [40] 5 arms and 6 arms [41] 8 arms and 12 arms [42]. A variety of studies have shown that the angles between the arms appear to be flexible [39,43,44].…”

DNA Nanotechnology: From Biology and Beyond

“…To this end, a sequence-symmetry minimization algorithm was devised (Seeman 1990), and the resultant predicted structures were realized with chemically synthesized oligonucleotides. The construction of branched DNA junctions flanked by different numbers of arms (Ma et al 1986;Kallenbach et al 1983;Wang et al 1991;Wang and Seeman 2007;Yin et al 2008a) has accordingly been accomplished.…”

DNA nanotechnology-enabled biosensors

“…Later, this family was expanded by three-arm, five-arm, six-arm, eight-arm and even twelve-arm junctions (Ma et al 1986; Wang and Seeman 2007;Wang et al 1991). Based on these arm junctions, DNA tiles were designed by program and fabricated by annealing.…”