Quantification of abasic sites in DNA and RNA by LC-MS/MS method

Abstract

Apurinic/apyrimidinic (AP) sites are common damage lesions of both DNA and RNA, which are resulted from glycoside bond hydrolysis of normal nucleotides and damage repair mechanisms of the damaged nucleotides with abnormal bases. As an important damage intermediate product, AP sites provides information of the response to physical and chemical factors in DNA and RNA. Quantification of AP sites will reveal the relation to endogenous or exogenous damage sources. However, strictly quantitative research of AP sites is limited by technical problems and the existing analytical methods lack enough sensitivity and selectivity. Since the potential of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and related detecting techniques, we attempted to develop strict quantification method of AP sites in DNA and RNA using these techniques. The method we have built consist of several essential processes: 1) Pretreatment of DNA/RNA samples; 2) enzyme-catalyzed digestion of AP site-containing DNA/RNA; 3) derivatization reaction with pentafluorophenylhydrazine (PFPH); and 4) quantitative studies by LC-MS/MS method combined with isotope dilution technique. The LC-MS/MS detection is performed on a triple quadrupole mass spectrometer that shows high performance in both detection sensitivity and selectivity. The detection limit is as low as 6.5 fmol, equivalent to 4 AP sites per 10⁹ nucleotides in 5μg DNA, and at least 10 times lower than existing quantification methods. The whole processes of the developed method was examined and assessed by using AP site-containing oligonucleotides as reference sample. The quantitative method succeeded in monitoring methyl methanesulfonate (MMS)-induced formation of AP sites in cellular DNA. This method also works in RNA, and allows the comparison of the different response between DNA and RNA under depurinated condition.

Date of Award	2015
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology