Spectroscopic approaches for studies of site-specific DNA base and backbone 'breathing' using exciton-coupled dimer-labeled DNA.

DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites.

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of 'internally-labeled' (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.