Characterizing Absolute Orientations in DNA Self-Assembly of Single Molecules.

DNA self-assembly of single molecules (i.e., dyes) with deterministic orientations is a powerful approach for engineering molecular excitons. However, current determination methods of the dye orientation relative to DNA do not account for the orientation of the dye plane, which is a missing degree of freedom needed to define absolute three-dimensional orientations. In this work, we combine fluorescence-detected linear dichroism, defocused dipole imaging, and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy to determine the absolute three-dimensional orientations of single Cy5 dyes relative to host DNA duplexes, which includes the dye plane orientation. The data revealed that the absorption and emission dipoles are perpendicular to the DNA duplex, and the mean dye plane is parallel to the DNA bases, which supports the notion that Cy5 dyes intercalate between DNA base pairs. The presented methodology will inspire investigation of the dye plane orientation for controlling dye arrangement configurations beyond spontaneous π-stacking between dyes as well as achieving novel dye-DNA arrangements.