Resolution and Quantitation of Ribonucleosides and Deoxyribonucleosides in Digested Genomic DNA by UHPLC-MS/MS

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Deoxyuridine (dUrd) misincorporation into genomic DNA occurs in cancer patients undergoing antimetabolite chemotherapy.
• This process activates DNA repair pathways involving ataxia telangiectasia-mutated and Rad3-related (ATR) and impacts nucleotide metabolism via ribonucleotide reductase (RNR).
• Ceralasertib (AZD6738), a clinical ATR inhibitor (ATRi), may disrupt this pathway, affecting deoxynucleotide triphosphate synthesis.

Purpose of the Study:

• To develop and validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying nucleosides in digested genomic DNA.
• To investigate the effects of ATR inhibition on deoxyuridine misincorporation and other nucleoside levels.
• To provide a tool for understanding the molecular mechanisms of ATRi-induced DNA damage.

Main Methods:

• Genomic DNA was extracted from murine cell lines treated with ceralasertib.
• Nucleosides (deoxyuridine, uridine, guanosine, adenosine, cytidine, thymidine, deoxyguanosine, deoxyadenosine, deoxycytidine) were quantified using LC-MS/MS.
• Chromatographic separation was performed on an Inertsil ODS-3 column with a gradient elution, and detection utilized a SCIEX 6500+ mass spectrometer.

Main Results:

• The developed LC-MS/MS method demonstrated high accuracy (90.8%-114.2%) and precision (<7.73% CV).
• The assay showed acceptable stability under various conditions, including freeze-thaw cycles, long-term storage at -80°C, and short-term storage at room temperature.
• The method was successfully applied to analyze genomic DNA from ceralasertib-treated murine cell lines.

Conclusions:

• A robust and sensitive LC-MS/MS method for quantifying DNA nucleosides has been established.
• This method is suitable for studying the impact of ATR inhibitors on DNA metabolism and integrity.
• The findings contribute to a better understanding of ATR inhibitor mechanisms in cancer therapy.