8-Oxo-7,8-dihydro-2'-deoxyguanosine Forms a Relatively Unstable Tetrameric Structure Compared with 2'-Deoxyguanosine.

The hydrogen-bonded guanine tetrad, or G-quartet has been implicated in a variety of biological roles, including the function of chromosome telomeres. Here effect of the hydroxylation of guanosine at the 8 position on the G-quartet formation was examined. Electrospray inonization mass (ESI-MS) spectra of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) were measured in order to know whether or not 8-oxodG forms a tetrameric structure as 2'-deoxyguanosine forms in teromeres. The ESI-MS spectra of dG shows prominent peaks at m/z 290, m/z 557, and m/z 1092, corresponding to [dG + Na](+), [dG2 + Na](+), and [dG(4) + Na](+) in the presence of 0.1 mM NaCl. On the other hand, the ESI-MS spectra of 8-oxodG in the presence of 0.1 mM NaCl shows prominent peaks at m/z 306 and m/z 589, corresponding to [8-oxodG + Na](+) and [8-oxodG(2) + Na](+). The results showed that 8-oxodG forms a relatively unstable tetrameric structure compared with dG.

Introduction

DNA and RNA containing runs of consecutive guanine bases may adopt four-stranded conformations based on the hydrogen-bonded guanine tetrad, or G-quartet (Fig. 1) [1-3]. The hydrogen-bonded guanine tetrad, or G-quartet are stabilized by monovalent ions such as sodium and potassium [4-7]. Such tetraplexes have been implicated in a variety of biological roles, including the function of chromosome telomeres [8], the dimerization of the human immunodeficiency virus RNA genome [9], the site-specific recombination of immunoglobulin genes [10], L1 retropositions [11], promoter regions of DNA such as the triplet repeat sequence that causes fragile-X syndrome [12-14], the retinoblastoma susceptibility gene [15], the chicken β-globulin gene [16], and the insulin gene-linked polymorphic region (ILPR) [17-20]. Their functional importance is supported by the isolation of proteins that bind and promote the formation of tetraplex structure [21, 22].

Fig. 1

The hydrogen-bonded guanine tetrad. R represents 2'-deoxyribose residue.

On the other hand, reactive oxygen species, which are generated in cellular metabolism [23] and ionization radiation [24], produce irreversible modification to DNA. The damage caused by these reactive free radical species has been proposed to contribute to aging, cancer, and other age-related degenerative processes [25, 26]. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was identified in the DNA exposed to oxygen radicals [27], or γ-irradiation [28], or peroxyl radicals [29]. The irradiation of UVA has resulted in the hydroxylation specifically at C-8 of the 5' site of GG and GGG sequence in DNA in the presence of endogeneous photosentizers [30-35]. Site-specific oxidation at GG and GGG sequences in DNA has also been induced by benzoyl peroxide [36]. Increases in the levels of its oxidation product, 8-oxodG have been reported in granulocute exposed to the tumor promoter (tetradeconylphorbolacetate) [37], mitochondorial DNA [38], mononuclear cells from patients with both insulin- and non-insulin-dependent diabetes [39, 40], and the urine [41].

In this paper, effect of the hydroxylation of guanosine at 8 position on the G-quartet formation was examined by using electrospray ionization mass (ESI-MS) spectrometer. ESI-MS was employed in this study because ESI-MS is a very powerful technique for the analysis of binding interactions [7, 42].

Materials and Methods

Materials

2'-Deoxyguanosine (dG) was from NAKARAI CHEMICALS (Kyoto, Japan). 8-oxodG was purchased from Sigma (St. Louis, MO). All other chemicals used were of analytical grade.

HPLC-ESI-MS

The high performance liquid chromatograph-electrospray ionization-mass spectrometer (HPLC-ESI-MS) consisted of a model 7125 injector (Reodyne Cotati, CA) with a 5 ml sample loop, a model L-7100 pump (Hitachi Ltd., Ibaragi, Japan), and a model M-1200AP LC-MS system with an electrospray ionization (ESI) (Hitachi Ltd., Ibaragi, Japan).

The operating conditions of the mass spectrometer were: nebulizer, 180°C; aperture 1, 120°C; N2 controller pressure, 2.0 kgf/cm2; drift voltage, 70 V; multiplier voltage, 1800 V; needle voltage, 3000 V; polarity, positive; resolution, 48.

For the analyses of a mixture of NaCl with dG (or 8-oxodG), the HPLC was performed at flow rate of 50 µl/min without a column. The mobile phase used was water. Three hundred microliter of aqueous solution of 0.1 mM NaCl with 1.0 mM dG (or 1.0 mM 8-oxodG) was injected to the HPLC-ESI-MS.

For the analyses of a mixture of KCl with dG (or 8-oxodG), the HPLC was performed with a column (150 mm long × 4.6 mm i.d.) packed with TSKgel ODS-120T (TOSOH Co., Tokyo, Japan) at flow rate of 50 µl/min. The mobile phase used was 0.1 mM KCl aqueous solution. One milliliter of a mixture of 0.1 mM KCl with 1 mM dG (or 1 mM 8-oxodG) was injected to the HPLC-ESI-MS. The HPLC fraction of dG (or 8-oxodG) was introduced to the HPLC-ESI-MS. Thus, Na+ ions contaminated in a mixture of KCl with dG (or 8-oxodG) were replaced by K+ ions.

For the analyses of 1 mM 2'-deoxyguanosine (or 8-oxodG) with various concentrations of NaCl, the HPLC was performed at flow rate of 50 µl/min without a column. One milliliter of acetonitrile solutions of 1 mM dG (or 1 mM 8-oxodG) with various concentration of NaCl were injected to the HPLC-ESI-MS. The one milliliter acetonitrile solutions contained 50 µl of water.

Results and Discussion

ESI-MS spectra of the solutions of 8-oxodG with NaCl or dG with NaCl (or KCl) were measured in order to know whether or not 8-oxodG forms a tetrameric structure as dG forms in teromeres.

The ESI-MS spectra of the mixture of dG with NaCl showed prominent peaks at m/z 290, m/z 557, and m/z 1092 (Fig. 2B), corresponding to [dG + Na]+, [dG2 + Na]+, and [dG4 + Na]+. On the other hand, ESI-MS spectra of the mixture of 8-oxodG with NaCl showed prominent peaks at m/z 306 and m/z 589 (Fig. 2A), corresponding to [8-oxodG + Na]+ and [8-oxodG2 + Na]+.

Fig. 2

Electrospray inonization mass (ESI-MS) spectra of mixtures of 8-oxodG [or 2'-deoxyguanosine (dG)] with NaCl. HPLC-ESI-MS conditions are as described in Materials and Methods. A, an ESI-MS spectrum of a mixture of 8-oxodG with NaCl; B, an ESI-MS spectrum of a mixture of dG with NaCl.

The ESI-MS spectra of the mixture of dG with KCl showed prominent peaks at m/z 306, m/z 573, and m/z 1108 (Fig. 3B), corresponding to [dG + K]+, [dG2 + K]+, and [dG4 + K]+. On the other hand, ESI-MS spectra of the mixture of 8-oxodG with KCl showed prominent peaks at m/z 322 and m/z 605 (Fig. 3A), corresponding to [8-oxodG + K]+ and [8-oxodG2 + K]+.

Fig. 3

Electrospray inonization mass (ESI-MS) spectra of a mixture of 8-oxodG [or 2'-deoxyguanosine (dG)] with KCl]. HPLC-ESI-MS conditions are as described in Materials and Methods. A, an ESI-MS spectrum of a mixture of 8-oxodG with KCl; B, an ESI-MS spectrum of a mixture of dG with KCl.

The analyses of 1 mM dG (or 8-oxodG) with various concentrations of NaCl were performed (Fig. 4). Relative peak heights of m/z 1092, [dG4 + Na]+ were much larger than those of m/z 1156, [8-oxodG4+Na]+ throughout this experiments.

Fig. 4

Relative intensities of m/z 1092, [dG4 + Na]+ or m/z 1156, [8-oxodG4 + Na]+ observed in the mixtures of 1 mM dG or 1 mM 8-oxodG with various concentration of NaCl. HPLC-ESI-MS conditions are as described in Materials and Methods. (closed circle), m/z 1092, [dG4+Na]+; (open circle), m/z 1156, [8-oxodG4 + Na]+.

8-OxodG seems to be difficult to form a tetrameric structure as dG does. The equilibrium of 8-oxodG lies so far to the 8-keto form (Fig. 5) [43]. The N (7) nitrogen atom of 8-oxodG may be difficult to participate in the hydrogen bonds.

Fig. 5

Equilibrium between enol form and keto form of 8-oxodG.

Since the site-specific hydroxylation at GG and GGG sequences in DNA has been induced by various oxygen stresses [30-36], the hydrogen-bonded guanine tetrad, or G quartet, which is related to a variety of biological roles, is possibly disintegrated by the oxygen stresses in the biological systems.