A tantalizing question in lupus research has been how anti-dsDNA antibodies bind DNA, and whether there are physiological mimetopes by which B cells expressing these antibodies might be selected. Because no crystals could be generated, there is no available X-ray structure of anti-dsDNA antibodies or Fab fragments bound to DNA. With cryo-electron microscopy, we now have a tool comparable—and potentially superior—to traditional X-ray crystallography techniques. In this way, we compare the structure of antibodies that differ by a single or a few amino acids and are dsDNA-binding and nonbinding, respectively. However, to obtain structures of antibodies bound to DNA fragments, we have to constrain the flexibility of DNA—the part of it that is not bound to antibody. In vivo, DNA is constrained in complexes such as chromatin or retroviral pre-integration complex. Similarly, for our structural study we constrain the dsDNA in synthetic nucleosome or DNA origami structures.
During the last decade, DNA origami has progressed past an art form and is being tried for a number of applications. The specificity of the interactions between complementary base pairs makes DNA an exciting construction material for molecular origamis—creating scaffolds that hold other molecules in place or structures all on its own. For our research, we use two examples of antibody complexes with constrained dsDNA. In one example, synthetic DNA is folded into a 6-helix honeycomb structure, which forms a tube. In the other example, the antibodies are complexed with synthetic nucleosomes.
The figure shows electron micrographs (EM) of DNA-antibody complexes. In the upper panel, an antibody is bound to a 6-helical honeycomb tubular structure made of DNA. To the left of the panel is an interpretation of the first EM picture; antibody is in blue, DNA in grey. It appears that antibodies mostly bind to the ends of the origami structure, where the full DNA double helices are more accessible. In the lower panel, an antibody is bound to a synthetic nucleosome. To the left, interpretation of the first EM picture. To the right, enlarged schematic of the nucleosome structure; DNA (gray) in a ring outside, and histones (blue) in the center. (In collaboration with Eugene Palovcak, Yifan Cheng, Lea Tröster, Maryam Shansab and Shawn Douglas).