Measuring local conformations and conformational disorder of (Cy3)2 dimer labeled DNA fork junctions using absorbance, circular dichroism and two-dimensional fluorescence spectroscopy

Abstract

The sugar-phosphate backbone of DNA near single-stranded (ss)–double-stranded (ds) junctions likely fluctuates within a broad distribution of conformations to permit the proper binding of genome regulatory proteins that function at these sites. In this work we use absorbance, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS) to study the local conformations and conformational disorder within chromophore-labeled DNA constructs. These constructs employ dimers of the fluorescent chromophore Cy3 that are site-specifically incorporated into the sugar-phosphate backbones of DNA strands at ss–ds DNA fork junctions. We show that these data can be analyzed to determine the local conformations of the (Cy3)₂ dimer, and the degree of conformational disorder. Our analysis employs an essential-state Holstein–Frenkel Hamiltonian model, which takes into account the internal electronic-vibrational motions within each Cy3 chromophore, and the resonant electronic interaction that couples the two chromophores together. Our results suggest that this approach may be applied generally to understand local backbone conformation and conformational disorder at ss–ds DNA fork junctions.