A thiol-reactive nitroxide derivative, 3-iodomethyl-1-oxy-2,2,5,5-tetramethylpyrroline, was reacted with individual DNA strands that contain one phosphorothioate modification at a specific site. This reagent was prepared freshly from a precursor, 1-oxyl-2,2,5,5-tetramethyl-3-methane-sulfonyloxymethylpyrroline, following a published procedure (36 (link),38 ,39 (link)) (Supplementary Data). In the DNA labeling reactions, up to 0.1 mM of phosphorothioate modified DNA was mixed with 60–100 mM of 3-iodomethyl-1-oxy-2,2,5,5-tetramethylpyrroline in a mixture containing 100 mM of MES [2-(N-Morpholino)ethanesulfonic Acid (pH 5.8)] and 50% (v/v) formamide. After incubation in dark for 24 h at room temperature, excess nitroxide was removed using anion-exchange HPLC. The labeled DNA was desalted, lyophilized and stored as described above.