THESIS
2015
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 m...[
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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 10
9 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.
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