Application of Fluorescent Post-Labeling To Detect DNA Change

Objective: This project was designed to modify and improve a method for analyzing DNA damage, and apply it to the analysis the modified DNA nucleotide, 8-oxodeoxyguanosine-5'-monophosphate (8-oxodGMP). DNA damage can lead to mutagenesis and carcinogenesis, and it would be useful to develop a method that could be used on human buccal mucosa, as it is an easily obtainable source of human DNA.

Methods: 8-oxodGMP is not commercially available, so it had to be prepared. dGMP was treated with H2O2, CuSO4 and ascorbate. Treated dGMP was injected onto an HPLC. One major product was observed, collected, and a small portion enzymatically converted to the corresponding nucleoside, (which is commercially available, but cannot be converted product that can be fluorescently labeled) to confirm its identity. The 8-oxodGMP was converted to a phosphoramidate (PA) derivative by reacting 8-oxodGMP with ethylenediamine (ED), and alkycarbodiimidazole (CDI). The PA has free amino group, and can react with the fluorescent labeling reagent, BODIPY to form a fluorescent derivative of 8-oxodGMP. Without the PA derivitization, nucleotides are not fluorescent and do not reactive with fluorescent labeling reagents. Excess ED was removed by lyophilization at alkaline pH. The fluorescent product was analyzed by HPLC. Also, the ED is available with a 14C-label, allowing detection by accelerator mass spectroscopy, an extremely sensitive technique.

Results: The above reactions were first successfully applied to a normal deoxynucleotide, dGMP, to produce a fluorescent derivative of dGMP, confirming that the labeling was successful. After this, the 8-oxodGMP was successfully labeled.

Conclusion: In conclusion, we can now prepare PA's and fluorescent derivatives of deoxynucleotides, essentially free of interfering products. As DNA can be digested to deoxynucleotides, this method should be very useful in measuring low levels of DNA adducts in human buccal mucosa.