A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (R_p or S_p) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with R_p-R5a spectra effectively reporting on local DNA structural features and S_p-R5a spectra sensing variations in local DNA motions. This establishes R_p- and S_p-R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual R_p and S_p spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of R_p- and S_p-R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.