Abstract
DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initio calculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm–1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.
Highlights
Reactive oxygen species (ROS), such as peroxides, superoxides, and hydroxyl radicals, constitute a major source of damage to cellular components as lipids, proteins, and nucleic acids
It has been extensively employed to characterize the chemical structure of DNA nucleotides,[31−34] as well as to reveal changes in the structure of DNA, such as the formation of oxidative products.[35−37] Here, by combining Raman data with ab initio calculations, we demonstrate the quantitative detection of oxidatively damaged deoxyribonucleoside triphosphates (dNTPs) and NTPs at low concentration in standard solutions
We provide evidence that similar analyses can be conducted on samples in very small volumes and/or at very low concentrations, that is, under conditions verified in cellular extracts, by exploiting the high sensitivity of surface-enhanced Raman scattering (SERS),[38−40] combined with properly designed superhydrophobic substrates.[41,42]
Summary
Reactive oxygen species (ROS), such as peroxides, superoxides, and hydroxyl radicals, constitute a major source of damage to cellular components as lipids, proteins, and nucleic acids. The intensity of the two 8-oxo-dGTP bands at νA = 1535 cm−1 and νB = 1607 cm−1, respectively (colored curves in Figure 3B), measures the oxidized base content in the mixture.
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