Assembly and characterization of 8-arm and 12-arm DNA branched junctions

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 exploits 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 3-, 4-, 5-, and 6-arm junctions indicates a linear increase in friction constant as the number of arms increases; the 4-arm junction migrates anomalously at 4 degrees 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 8-arm and 12-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.