Ligation of DNA Triangles Containing Double Crossover Molecules

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Abstract

An antiparallel DNA double crossover molecule has been incorporated into one edge of a DNA triangle. The goal in this work is to combine a rigid geometrical motif, the triangle, with a rigid DNA motif, the double crossover, incorporating the properties of branched DNA. The triangle has been designed to contain 3 turns of DNA in each edge. The double crossover molecule produces an extra domain along its edge. The extra domain can be restricted by Bbs I to produce two complementary sticky ends separated by 4.5 turns of DNA. Ligation-closure experiments performed on the restricted triangle yield very long reporter strands and no indications of cyclization, suggesting that the double crossover molecule retains its stiffness when incorporated in the triangle. These experiments are evaluated through the use of denaturing gel electrophoresis. The ligation products have been visualized by atomic force microscopy, under nondenaturing conditions. Clear zigzag species can be identified as triangles ligated in one dimension. The images show no systematic distortions such as permanent bends. These visualizations have been confirmed by alternating triangles with simple double crossover molecules. When double crossover molecules containing 4.0 turns are alternated with triangles, the spacing of the vertexes is increased appropriately. When the double crossover molecules contain 4.5 turns, all the vertexes appear on the same side of the linear fragment, confirming the correlation between chemical input and mesoscopic images in the atomic force microscope. We conclude that double crossover molecules can be incorporated into at least one side of a triangle without any apparent effect on their rigidity.

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