Flexibility of DNA double crossover molecules and construction of DNA graphs.

摘要

Double crossover molecules are DNA structures containing two Holliday junctions connected by two double helical arms. There are several types of double crossover molecules, differentiated by the relative orientations of their helix axes, parallel or antiparallel, and by the number of double helical half-turns (even or odd) between the two crossovers. They are found as intermediates in meiosis and they have been used extensively in structural DNA nanotechnology for the construction of one dimensional and two dimensional arrays and in a DNA nanomechanical device. Whereas the parallel double crossover molecules are usually not well behaved, we have focused on the antiparallel molecules; antiparallel molecules with an even number of half turns between crossovers (termed DAE molecules) produce a reporter strand when ligated, facilitating their characterization in a ligation cyclization assay. Hence, we have estimated the flexibility of antiparallel DNA double crossover molecules by means of ligation-closure experiments. We are able to show that these molecules are approximately twice as rigid as linear duplex DNA.; The feasibility of the molecular computational approach based on the self-assembly of branched DNA junction molecules has been demonstrated by experimentally constructing a DNA graph. The DNA graph chosen for construction corresponds to a prototype system for solving the 3-colorability problem. We used a k-armed branched DNA molecule to represent a vertex of degree k, and a double helical DNA molecule to represent an edge. We also incorporated four restriction sites in each long edge of the graph by means of an extra hairpin fused on to the edge via a three-armed junction. Restricting these sites serves to linearize the graph in a specific way without altering the logical part of the graph. The first step involved self-assembly of component strands to form the vertex and edge building blocks. Then self-assembly of these constructs was allowed to form the graphs, which were subsequently converted to a covalently closed single stranded circle by ligation. Finally the ligated DNA graphs were purified and characterized. By using restriction mapping technique, we are able to prove that the DNA graphs obtained were in correct arrangement.