132 Impact of sticky end length on the diffraction of self-assembled DNA crystals

Our laboratory has reported a self-assembled 3-D crystal based on a DNA tensegrity triangle. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry consisting of three helices whose axes are directed along three linearly independent directions (1). The triangles form a crystalline lattice stabilized via sticky ends (2). The length of the sticky ends reported previously was two nucleotides (nt) GA:TC. Although diffracting to 4 Å resolution at the APS-ID19 beam line, they diffract only to 4.9 Å at the NSLS-X25 beam line. In the current study, we have analysed the effect of sticky end length and sequence on crystal formation and the resolution of the X-ray diffraction pattern on NSLS-X25. Tensegrity triangle motifs having 1-, 2-, and 3-nt sticky ends have all formed crystals. X-ray diffraction data from the same beam line revealed that the crystal resolution was somewhat better for the 2-nt sticky end having an AA:TT base pair (4.75 Å) than GA:CT and CC:GG (8.0 Å). Moreover, the 1-nt sticky end (C:G) yielded a diffraction pattern whose resolution (3.5 Å) compared favorably with all the three 2-nt sticky end systems. However, the triangle motif having a 1-nt sticky end with an A:T base pair did not yield any crystals. For motifs with 3-nt sticky ends, the sequence GAG:CTC produced small crystals (10–20?μm), while larger crystals (150?μm) were obtained with the sequences TAG:ATC and TAT:ATA. Our results indicate that not only do the lengths and sequences of the sticky ends define the interactions between motifs, but they also have an impact on the resulting resolution. We expect redesigned assemblies to form 3-D crystals with better resolution that can aid in the scaffolding of biological macromolecules for crystallographic structure determination. Applications in many areas of DNA nanotechnology are expected to benefit from a complete analysis of the effects of sticky end length, sequence, and free energy.