The use of antiparallel DNA double crossover molecules in genetic recombination and DNA nanotechnology

The Holliday junction is a key intermediate in genetic recombinations. Switching the strands between helical arms and cross over arms is known as crossover isomerization; this postulated reversal is thought to be one of the key transformations that the Holliday junction can undergo, because it can lead to changing the products from patch to splice recombinants. Direct evidence is presented that this reaction can indeed occur in Holliday junctions in solution. Double crossover molecules are DNA structures containing two Holliday junctions connected by two double helical arms. A double crossover molecule containing a branched junction has been constructed, constrained not to be in its favored conformation. This junction is released from the double crossover molecule by digestion with restriction endonucleases. It is demonstrated by means of hydroxyl radical autofootprinting that the junction changes crossover isomers spontaneously when released from the double crossover. The possibility that the experimental protocol causes the isomerization is eliminated. Dissociation and interaction with other molecules in solution as contributing to the phenomenon are also excluded. Thus, crossover isomerization is an authentic transformation of Holliday junctions.; Double crossover molecules have been examined from the viewpoint of their potential utility in nanoconstruction. Antiparallel molecules with an even number of half turns between crossovers produce a reporter strand when ligated, so these have been characterized in a ligation cyclization assay. In contrast to other molecules that contain branched junctions, it is found that these molecules cyclize rarely or not at all. The double crossover molecules cyclize no more readily than the linear molecule containing the same sequence as the ligation domain. Both a conventional DAE molecule and one containing a bulged 3-arm branched junction between the crossovers have been tested. The conventional DAE molecule appears to be slightly stiffer, but so few cyclic products are obtained in either case that quantitative comparisons are not possible. Thus, it appears that these molecules may be able to serve as the rigid components that are needed to assemble symmetric molecular structures, such as periodic lattices. They could be combined with DNA triangles and deltahedra in order to accomplish this goal.