Measuring Structure and Disorder of (Cy3)₂ Dimer Labeled DNA Fork-Junctions Using Two-Dimensional Fluorescence Spectroscopy (2DFS)

DNA is commonly represented as a static B-form double helix. Biologically relevant forms of DNA, however, include single (ss) and double strands (ds), as well as various types of ss-ds junctions. DNA is not a single static conformation, but rather fluctuates over a range of conformations that deviate significantly from canonical structures. This is necessary for proteins to access functionally required structural motifs. In order to characterize such heterogeneity, we have performed absorption, circular dichroism, and two-dimensional fluorescence spectroscopic experiments on DNA constructs labelled with the fluorescent chromophore cyanine-3 (Cy3). These DNA constructs form strongly-coupled dimers (Cy3)₂ which are incorporated into the backbone near the ss-ds regions of model DNA replication forks. The vibronic nature of the monomer transitions and the excitonic coupling between monomers yield rich spectra which are sensitive to the range of conformations present. Detailed analyses of the data permit the extraction of conformational parameters and characterization of the conformational disorder.