Sensing sequence-specific DNA micro-environment with nucleotide-independent nitroxides

In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically stable unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented in this dissertation examine feasibility of probing local DNA structural and dynamic features using a class of nitroxides that are linked to chemically substituted phosphorothioate positions at the DNA backbone (R5-series). Two members of this family, designated as R5 and R5a, were attached to multiple sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label and the identity of a phosphorothioate diastereomer (Rp or Sp). Spectral simulations and molecular modeling have been used to define basic principles for correlating observed variation in EPR spectra with site-specific structural and dynamic features in DNA. Overall, these studies advance our understanding of coupling between DNA and R5/R5a, which may ultimately enable the use of nucleotide-independent probes to obtain quantitative description of sequence-specific properties in large biologically relevant DNA molecules.