Blunt-ended DNA stacking interactions in a 3-helix motif

Abstract

We demonstrate that intermolecular stacking is capable of forming one-dimensional arrays of a blunt-ended 3-helix DNA motif. The array can be visualized in the atomic force microscopy through conjugated streptavidin nanoparticles. We estimate the strength of the triple stacking interaction to be -8.6 kcal mol(-1).

Fig. 1

Schematic drawings of the DNA BTX molecules. (a) BTX molecules containing sticky ends. The strand structures of the A and B tiles are indicated; the B tile contains a hairpin feature (in green) whose loop contains biotin groups. The sticky ends in the A tile are labeled a, b, c, d, e and f, and they are complementary to the primed sticky ends in the B tile. A GIDEON-generated side view of the BTX molecules showing the 120° angle between DX segments is drawn to the right of the strand structures. (b) The strand structures of the blunt-ended tiles is shown. (c) Regular self-assembly of the sticky-ended BTX tiles. The A and B tiles are shown in GIDEON molecular representations. Streptavidin molecules are shown as yellow circles filled with green that bind to the hairpins. The regular alternation of A and B tiles is visible following sticky-ended cohesion to form a 1D array.

Fig. 2

A non-denaturing gel demonstrating the formation of the BTX molecules. Lane M contains a linear marker ladder separated by 10 nucleotide pairs. Lane 1 contains the blunt-ended B tile and lane 2 contains the blunt-ended A tile. Lane 3 contains the sticky-ended B tile and lane 4 contains the sticky-ended A tile. The hairpin loops retard the mobilities of the B tiles in lanes 1 and 3.

Fig. 3

Atomic force micrographs of blunt and sticky-ended BTX linear arrays. (a) A mixture of sticky-ended BTX A and B tiles lacking streptavidin labels. One-dimensional linear arrays are visible. (b) A mixture of blunt-ended A and B BTX tiles lacking streptavidin labels. These tiles also produce 1D linear arrays. In neither (a) nor (b) are the hairpins clearly visible. (c) An array like those in (a), but labeled with streptavidin. The regular 40 nm repeat of streptavidin markers is visible. (d) Arrays like those in (b), but labeled with streptavidin. Varied spacings between the streptavidin markers can be seen. Scale bars: (a) and (b), 250 nm; (c) and (d) 100 nm.

Fig. 4

Calculation of the Binding Constant. (a) Schematic drawings of the DNA BTX Molecule Used to Measure the Association Constant. The BTX molecule was capped with short T4 loops on one end. The sequence is based on Tile B. (b) An AFM Image of BTX Molecule at a Concentration of 125 nM. The scan size is 1 µm × 1 µm a sufficiently high resolution to distinguish between dimers and monomers. (c) A 3D View of the Rectangle Area within the Image. Arrows point to dimers, which are clearly distinct from monomers. (d) Representative Plot used for K_d Determination. K_d is the inverse slope of the line and is 4.5 × 10⁻⁷ M for the conditions used (25 °C, 10.5 mM Mg²⁺).