All oxidations were conducted in 20 mM MPi (pH 7.4) 120 mM MCl (M = Na or K) at 37 °C at a 10 µM ODN concentration. Oxidation sites were determined on reactions doped with 20,000 cpm of 5’-32P labeled strand in a 50-µL reaction utilizing the following oxidant conditions: (1) Riboflavin (Type I photooxidant) was added to give a 50 µM final concentration and solutions were exposed to 350-nm light for 5–20 min, (2) CO3•− was produced when SIN-1 generated ONOO− (<3 mM) was allowed to react with 25 mM MHCO3 (M = Na or K) present, and the reaction progressed through the thermal decomposition of SIN-1 for 0.5–3 h, (3) Rose Bengal (Type II photooxidant) was added to a final concentration of 50 µM and solutions were exposed to 350-nm light for 5–20 min, and (4) Cu-mediated oxidations were conducted with 10 µM Cu(OAc)2 that was preincubated with the ODNs for 30 min prior to the addition of 10–1000 µM H2O2, and allowed to react for 30 min prior to reaction termination with 5 mM EDTA. Following the oxidation, samples were dialyzed overnight then worked up with 1 M piperidine at 90 °C for 2 h, and then the piperidine was removed by lyophilization. Next, the samples were resuspended in 12 µL of loading buffer (30% glycerol, 0.25% bromophenol blue, and 0.25% xylene cyanol) and 6 µL was loaded on a 20% denaturing PAGE gel, and electrophoresed at 75 W for 2.5 h. A Maxam-Gilbert G-lane was run alongside each reaction to determine the G oxidation sites.74 (link) The cleavage sites were observed and quantified by storage-phosphor autoradiography on a phosphorimager (see Supporting Information ).
Oxidation products were determined by nuclease digestion followed by HPLC analysis. To achieve the analysis, reactions were conducted similarly to those described above with the exception of not adding the 5’-32P labeled strand. For each oxidant studied, 20 reactions were conducted and then combined to have 10 nmoles of oxidized ODN. Next, the samples were dialyzed overnight to remove the reaction buffer. Rose Bengal was removed with a NAP-25 column (GE Healthcare) following the manufacturer’s protocol. After concentrating the samples by lyophilization, they were resuspended in 50 µL of digestion buffer and digested with nuclease P1, snake venom phosphodiesterase and calf intestinal phosphatase, as previously described.32 (link) Next, the products were quantified by HPLC following a previously described protocol32 (link) for which the complete details of the entire process are described in theSupporting Information .
Oxidation products were determined by nuclease digestion followed by HPLC analysis. To achieve the analysis, reactions were conducted similarly to those described above with the exception of not adding the 5’-32P labeled strand. For each oxidant studied, 20 reactions were conducted and then combined to have 10 nmoles of oxidized ODN. Next, the samples were dialyzed overnight to remove the reaction buffer. Rose Bengal was removed with a NAP-25 column (GE Healthcare) following the manufacturer’s protocol. After concentrating the samples by lyophilization, they were resuspended in 50 µL of digestion buffer and digested with nuclease P1, snake venom phosphodiesterase and calf intestinal phosphatase, as previously described.32 (link) Next, the products were quantified by HPLC following a previously described protocol32 (link) for which the complete details of the entire process are described in the
