Nitroxide Sensing of a DNA Microenvironment: Mechanistic Insights from EPR Spectroscopy and Molecular Dynamics Simulations

The behavior of the nitroxide spin labels 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a) and 1-oxyl-2,2,5,5-tetramethylpyrroline (R5) attached at a phosphorothioate-substituted site in a DNA duplex is modulated by the DNA in a site- and stereospecific manner. A better understanding of the mechanisms of R5a/R5 sensing of the DNA microenvironment will enhance our capability to relate information from nitroxide spectra to sequence-dependent properties of DNA. Toward this goal, electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations were used to investigate R5 and R5a attached as R_<i>p</i> and S_<i>p</i> diastereomers at phosphorothioate _pSC₇ of d­(CTACTG_pSC₇Y₈TTAG). d­(CTAAAGCAGTAG) (Y = T or U). X-band continuous-wave EPR spectra revealed that the dT₈ to dU₈ change alters nanosecond rotational motions of R_<i>p</i>-R5a but produces no detectable differences for S_<i>p</i>-R5a, R_<i>p</i>-R5, and S_<i>p</i>-R5. MD simulations were able to qualitatively account for these spectral variations and provide a plausible physical basis for the R5/R5a behavior. The simulations also revealed a correlation between DNA backbone B_I/B_II conformations and R5/R5a rotational diffusion, thus suggesting a direct connection between DNA local backbone dynamics and EPR-detectable R5/R5a motion. These results advance our understanding of how a DNA microenvironment influences nitroxide motion and the observed EPR spectra. This may enable use of R5/R5a for a quantitative description of the sequence-dependent properties of large biologically relevant DNA molecules