Construction, Analysis, Ligation, and Self-Assembly of DNA Triple Crossover Complexes

Abstract

This paper extends the study and prototyping of unusual DNA motifs, unknown in nature, but founded on principles derived from biological structures. Artificially designed DNA complexes show promise as building blocks for the construction of useful nanoscale structures, devices, and computers. The DNA triple crossover (TX) complex described here extends the set of experimentally characterized building blocks. It consists of four oligonucleotides hybridized to form three double-stranded DNA helices lying in a plane and linked by strand exchange at four immobile crossover points. The topology selected for this TX molecule allows for the presence of reporter strands along the molecular diagonal that can be used to relate the inputs and outputs of DNA-based computation. Nucleotide sequence design for the synthetic strands was assisted by the application of algorithms that minimize possible alternative base-pairing structures. Synthetic oligonucleotides were purified, stoichiometric mixtures were annealed by slow cooling, and the resulting DNA structures were analyzed by nondenaturing gel electrophoresis and heat-induced unfolding. Ferguson analysis and hydroxyl radical autofootprinting provide strong evidence for the assembly of the strands to the target TX structure. Ligation of reporter strands has been demonstrated with this motif, as well as the self-assembly of hydrogen-bonded two-dimensional crystals in two different arrangements. Future applications of TX units include the construction of larger structures from multiple TX units, and DNA-based computation. In addition to the presence of reporter strands, potential advantages of TX units over other DNA structures include space for gaps in molecular arrays, larger spatial displacements in nanodevices, and the incorporation of well-structured out-of-plane components in two-dimensional arrays.

Reducing Facet Nucleation during Algorithmic Self-Assembly

“…The strands for each lattice (50 nM per strand) were annealed from 90 to 10°C over 10 h, held at 10°C for 2 h, and then melted back to 90°C at the same rate. For both lattices, we observed ( Figure 12 in Supporting Information) a reversible transition between 45 and 70°C, where tile formation has been observed previously, 3,17,24 and a hysteretic transition at lower temperatures, where lattices formed. Formation of both lattices occurred around 16°C and melted between 25 and 37°C, indicating a significant kinetic barrier to homogeneous nucleation.…”

Architecture with GIDEON, a program for design in structural DNA nanotechnology

“…For instance, various DX and TX molecules are, perhaps, the most commonly used SDN motifs. 9,16 They are generally taken to be flat, but precise modeling with GIDEON reveals, in many cases, deviations from planarity. Figures 7 and 8 show precision SDN models including the 6° tilt of bases known to occur in B-DNA.…”

“…It was vital in the design of Double Crossover (DX) molecules, and facilitated the design of a wide variety of other motifs and nanomechanical devices. 9,[16][17][18][19][20][21][22] As larger and more complex SDN constructs are developed, however, the need for alternate methods of modeling has become apparent. Large physical models become distorted, and even unstable under their own weight, and they are not easily reduced below a size scale dictated by the jacks used to join the straws together.…”

DNA Self-Assembly For Constructing 3D Boxes

“…Seeman's visionary work [8] in 1982 pioneered the molecular units used in self-assembly of such objects. More than a decade later, double-crossover (DX) molecules were proposed by Fu and Seeman [3] and triple-crossover (TX) molecules by LaBean et al [5] as DNA self-assembly building blocks. Laboratory efforts have been successful in generating interesting three-dimensional (3D) molecular structures, including the small cube of Chen and Seeman [1].…”