The assembly of synthetic, controllable molecular mechanical systems is one of the goals of nanotechnology. Protein-based molecular machines, often driven by an energy source such as ATP, are abundant in biology. It has been shown previously that branched motifs of DNA can provide components for the assembly of nanoscale objects, links and arrays. Here we show that such structures can also provide the basis for dynamic assemblies: switchable molecular machines. We have constructed a supramolecular device consisting of two rigid DNA 'double-crossover' (DX) molecules connected by 4.5 double-helical turns. One domain of each DX molecule is attached to the connecting helix. To effect switchable motion in this assembly, we use the transition between the B and Z forms of DNA. In conditions that favour B-DNA, the two unconnected domains of the DX molecules lie on the same side of the central helix. In Z-DNA-promoting conditions, however, these domains switch to opposite sides of the helix. This relative repositioning is detected by means of fluorescence resonance energy transfer spectroscopy, which measures the relative proximity of two dye molecules attached to the free ends of the DX molecules. The switching event induces atomic displacements of 20-60 A.

Controlled mechanical movement in molecular-scale devices has been realized in a variety of systems-catenanes and rotaxanes, chiroptical molecular switches, molecular ratchets and DNA-by exploiting conformational changes triggered by changes in redox potential or temperature, reversible binding of small molecules or ions, or irradiation. The incorporation of such devices into arrays could in principle lead to complex structural states suitable for nanorobotic applications, provided that individual devices can be addressed separately. But because the triggers commonly used tend to act equally on all the devices that are present, they will need to be localized very tightly. This could be readily achieved with devices that are controlled individually by separate and device-specific reagents. A trigger mechanism that allows such specific control is the reversible binding of DNA strands, thereby 'fuelling' conformational changes in a DNA machine. Here we improve upon the initial prototype system that uses this mechanism but generates by-products, by demonstrating a robust sequence-dependent rotary DNA device operating in a four-step cycle. We show that DNA strands control and fuel our device cycle by inducing the interconversion between two robust topological motifs, paranemic crossover (PX) DNA and its topoisomer JX2 DNA, in which one strand end is rotated relative to the other by 180 degrees. We expect that a wide range of analogous yet distinct rotary devices can be created by changing the control strands and the device sequences to which they bind.

Paranemic crossover (PX) DNA is a four-stranded coaxial DNA structure containing a central dyad axis that relates two flanking parallel double helices. The strands are held together exclusively by Watson-Crick base pairing. The key feature of the structure is that the two adjacent parallel DNA double helices form crossovers at every point possible. Hence, reciprocal crossover points flank the central dyad axis at every major or minor groove separation. This motif has been modeled and characterized in an oligonucleotide system; a minor groove separation of 5 nucleotide pairs and major groove separations of 6, 7, or 8 nucleotide pairs produce stable PX DNA molecules; a major groove separation of 9 nucleotide pairs is possible at low concentrations. Every strand undergoes a crossover every helical repeat (11, 12, 13 or 14 nucleotides), but the structural period of each strand corresponds to two helical repeats (22, 24, 26 or 28 nucleotides). Non-denaturing gel electrophoresis shows that the molecules are stable, forming well-behaved complexes. PX DNA can be produced from closed dumbbells, demonstrating that the molecule is paranemic. Ferguson analysis indicates that the molecules are similar in shape to DNA double crossover molecules. Circular dichroism spectra are consistent with B-form DNA. Thermal transition profiles suggest a premelting transition in each of the molecules. Hydroxyl radical autofootprinting analysis confirms that there is a crossover point at each of the positions expected in the secondary structure. These molecules are generalized Holliday junctions that have applications in nanotechnology.

Specific cohesion of DNA molecules is key to the success of work in biotechnology, DNA nanotechnology and DNA-based computation. The most common form of intermolecular cohesion between double helices is by sticky ends, but sticky ends generated by naturally occurring restriction enzymes may often be too short to bind large constructs together. An alternative form of binding is available through the paranemic crossover (PX) motif. Each of the two components of a PX motif can be a DNA dumbbell or other topologically closed species. Alternate half-turns of the dumbbell are paired intramolecularly. The intervening half-turns are paired with those of the opposite component. We demonstrate the efficacy of PX cohesion by showing that it can result in the 1:1 binding of two triangle motifs, each containing nearly 500 nucleotides. The cohesion goes to completion, demonstrating an alternative to binding nucleic acid molecules through sticky ends.

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX, has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attributes and usages of PX-DNA-its design, characteristics, applications, and potential biological relevance-and aims to accelerate the understanding of PX-DNA motif in its many roles and manifestations.