Combining DNA Nanotechnology and Fluorescence Polarization Microscopy to Determine the Orientation of DNA-bound Fluorophores

We describe a technique to measure the axis of the transition dipole moment of a fluorophore bound to dsDNA and compare results with existing techniques. We use DNA nanotubes to present the dsDNA in a known orientation and query a variety of intercalating (e.g., YO-YO, TO-TO), groove-binding (e.g. DAPI) or covalently linked (e.g., Fluorescein, Cy3, Cy5) dyes. A de S\'{e}narmont prism in front of the camera generates simultaneous images of fluorescence polarized perpendicular and parallel to the DNA nanotube axis, allowing for ratio measurements that are insensitive to bleaching. We suggest the use of technique to detect helical supertwist, and possibly other nanoscale structural features, of DNA nanostructures.