Facile quantification of lesions derived from 2′-deoxyguanosine in DNA

Article abstract of DOI:10.1021/ja072174n

OxodG and Fapy?dG are two frequently formed DNA lesions that affect replication in vitro and in cells. They are also potentially important biomarkers for determining the effects of oxidative stress and aging in cells. We report the first method that enabl

Full text of DOI:10.1021/ja072174n

Published on Web 05/11/2007
Facile Quantification of Lesions Derived from 2′-Deoxyguanosine in DNA
Liang Xue and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins UniVersity, 3400 North Charles Street, Baltimore, Maryland 21218
Received March 28, 2007; E-mail: mgreenberg@jhu.edu
Exposure of DNA to free radicals and other electrophiles results
in strand breaks, as well as modified nucleotides (lesions) within
intact strands of the biopolymer. DNA lesions can be genotoxic
and have been implicated in aging and diseases such as cancer.1-3
OxodG and Fapy•dG are examples of lesions produced in significant
yields from dG when DNA is exposed to oxidative stress. It is well-
known that OxodG gives rise to GfT transversions.1 Recent
investigations suggest that Fapy•dG also has significant effects on
slowly were also detected. Reaction of OxodG with spermine and
other diamines under oxidative conditions is well-established.15
repair and replication.4 For instance, OxodG and Fapy•dG result
in high levels of GfT transversions when they are replicated in
simian kidney cells.5 The former has been proposed as a biomarker
when studying the effects of oxidative stress on DNA.2 In addition,
it has also been suggested that the ratio of OxodG:Fapy•dG may
be characteristic of the cancerous state of a cell.6 Currently, these
lesions are detected and quantified via mass spectrometry following
degradation of the DNA.7,8 However, there is some disagreement
regarding how these measurements are made.9-11 We describe a
reagent and accompanying fluorescent detection method that enables
one to selectively quantify OxodG and Fapy•dG.
Confirmation that adducts corresponded to 1:1 adducts between 2
and oxidized OxodG or Fapy•dG was obtained using MALDI-TOF
MS.16 Additional evidence that adduct formation occurred at
Fapy•dG in 4 was obtained by ESI-MS following reaction of 1
and 2 under the same reaction conditions.16
Although the yields of adducts were different for Fapy•dG and
OxodG, for each lesion, they were independent of the flanking
sequence (Figure 2). This was true even when OxodG was present
in sequences that were expected to significantly alter its oxidation
potential from that in 3a-d.16,18 In addition, no adducts were
detected when duplexes containing OxodA or Fapy•dA were reacted
with the strongest oxidant, Na2IrCl6, in the presence of 2 (data not
shown). Moreover, no adducts were observed when DNA containing
5-hydroxy-2′-deoxyuridine or 5-hydroxy-2′-deoxycytidine, which
have more favorable oxidation potentials than OxodG, were reacted
with Na2IrCl6 and 2 (data not shown).19 Their lack of reactivity
under the reaction conditions indicates that oxidation is a necessary
but insufficient requirement for tagging.
In order to quantify the individual amounts of Fapy•dG and
OxodG, other oxidants were explored in the hope of taking
advantage of the lesions’ differing oxidation potentials. Although
both produced adducts with 2 in the presence of Na2IrBr6, only
OxodG was trapped when K3Fe(CN)6 was used as the oxidant
(Figures 1 and 2). The selective biotinylation of DNA containing
the lesions as a function of oxidant indicated that individual amounts
of OxodG and Fapy•dG in randomly damaged DNA could be
determined using 2 as part of a fluorescence assay (Scheme 1).
Amplex Red, which is oxidized to fluorescent resorufin by
horseradish peroxidase in the presence of H2O2, was used to
quantify adducts of 2.20 Experiments were carried out immediately
after tagging by 2 because the adducts decompose with half-lives
on the order of 12-15 h.15,16 The system was calibrated for
application in a microtiter plate using a 287 nt duplex fragment of
M13mp7(L2) prepared by PCR that contained biotin at the
5′-terminus of one strand.16 The fluorescent signal produced by
resorufin depended linearly on the amount of biotinylated DNA as
it varied 10-fold.
Selective DNA lesion detection is attractive because of its
simplicity. One approach exploits a lesion’s reactivity.12-14 The
facile oxidation of OxodG and nucleophilic trapping of its oxidized
product(s) by molecules such as spermine provided inspiration for
developing a system for detecting it and Fapy•dG.15 OxodG (∼0.75
V, NHE) is more readily oxidized than the most readily oxidized
native nucleotide, dG (1.29 V, NHE). The irreversible oxidation
of 1 (1.08 V, NHE) was determined to be more favorable than that
of dG, suggesting that using a similar oxidation/trapping approach
for detecting Fapy•dG was plausible.16
A carboxylated derivative of spermine was used as a precursor
to 2.16,17 The spermine derivative (2) provides a means for tagging
the oxidized species derived from OxodG and Fapy•dG with biotin,
which is used for quantifying the lesions. Slower moving adducts
were detected in good yield when duplex DNA (3 and 4) was
reacted with Na2IrCl6 in the presence of 2 at 25 °C for 1 h (Figures
1 and 2).15 Minor amounts of a product that migrated even more
The ELISA type method was used to measure the amounts of
OxodG and Fapy•dG produced in aqueous solution by γ-radiolysis.
The amount of biotinylated DNA was measured as a function of
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10.1021/ja072174n CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Reaction of 2 with DNA containing (A) OxodG (3b) or (B)
Fapy•dG (4b) under oxidative conditions.
Figure 3. Average adduct yield with 2 in 287 nt duplex DNA (2.5 pmol)
as a function of 137Cs dose. Circles, Na2IrCl6; squares, K3Fe(CN)6.
monocyte cells were subjected to γ-radiolysis.21,22 In summary, we
have developed a simple and sensitive method for quantifying
OxodG and Fapy•dG, two important DNA lesions. This tool will
be widely accessible to researchers studying the effects of oxidative
stress on DNA. In addition, the fluorescence assay should be
generally applicable to the quantification of other lesions that can
be selectively tagged with biotin.
Acknowledgment. We are grateful for financial support from
the National Institutes of Health (CA-074954). We thank Professor
Gerald Meyer, Mr. James Gardner, and Dr. Shanta Dhar for
assistance with the cyclic voltammetry experiment.
Supporting Information Available: Procedures for synthesis and
characterization of all molecules, and all other experiments. ESI-MS
and MALDI-TOF MS from experiments described herein. This material
is available free of charge via the Internet at http://pubs.acs.org.
Figure 2. Average adduct yield with 2 in DNA as a function of sequence.
(A) OxodG (3a-d). (B) Fapy•dG (4a-d). Dark gray, Na2IrCl6; black, Na2-
IrBr6; light gray, K3Fe(CN)6.
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(16) See Supporting Information.
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aqueous solution by 137Cs equals 1.12. This is higher than the ratio
reported when chromatin is irradiated but lower than when
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