Measuring oxidized DNA and RNA precursors by micro-Raman spectroscopy

DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring during oxidative stress. 8-oxo-dGTP and 8-oxo-rGTP are the prevalent oxidized nucleotides which can be incorporated into DNA or RNA and lead to mutations and cell death. The MTH1 enzyme protects against these effects by hydrolysing oxidized nucleotides. Since MTH1 inhibition is currently under development as novel target in cancer therapy, measurements of cellular 8-oxo-dGTP or 8-oxo-rGTP concentration provide an important strategy to monitor the enzyme function. Here we present a novel method based on Micro-Raman spectroscopy to reveal oxidative damage in the nucleotide pool. The analysis of d/rGTP and 8-oxo-d/rGTP Raman spectra with the support of ab initio calculations allowed us to identify specific and highly sensitive spectroscopic markers of oxidation. We developed a procedure to determine the concentration of 8-oxo-dGTP in dGTP, and their ribo counterpart, down to very low concentration. Present experiments pave the way for employing this procedure to identify the composition and quantitatively determine the oxidatively damaged nucleotide pool present in the cell.