Peroxynitrite-Induced Reactions of Synthetic Oligonucleotides

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Chem. Res. Toxicol. 1999, 12, 459-466

459

Peroxynitrite-Induced Reactions of Synthetic Oligonucleotides Containing 8-Oxoguanine Natalia Yu. Tretyakova,† Jacquin C. Niles,† Samar Burney,‡ John S. Wishnok,† and Steven R. Tannenbaum*,†,‡ Division of Bioengineering and Environmental Health and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received October 22, 1998

8-Oxoguanine (8-oxo-G) is one of the most common DNA lesions present in normal tissues due to exposure to reactive oxygen species. Studies at this and other laboratories suggest that 8-oxo-G is highly susceptible to secondary oxidation, making it a likely target for endogenous oxidizing agents, such as peroxynitrite (ONOO-). Synthetic oligonucleotides containing 8-oxoguanine were treated with ONOO-, and the reaction products were analyzed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI--MS). CCACAACXCAAA, CCAAAGGXAGCAG, CCAAAXGGAGCAG, and TCCCGAGCGGCCAAAGGXAGCAG (X is 8-oxo-G) were found to readily react with peroxynitrite via the same transformations as those observed for free 8-oxo-2′-deoxyguanosine. The composition of the reaction mixtures was a function of ONOO- concentration and of the storage time after exposure. The oligonucleotide products isolated at low [ONOO-]/[DNA] ratios (10, 2,4,6trioxo[1,3,5]triazinane-1-carboxamidine- and cyanuric acid-containing oligomers were the major products. The exact location of a modified base within a DNA sequence was determined using exonuclease digestion of oligonucleotide products followed by LC/ESI--MS analysis of the fragments. For all 8-oxo-G-containing oligomers, independent of the sequence, the reactions with ONOO- took place at the 8-oxo-G residues. These results suggest that 8-oxo-G, if present in DNA, is rapidly oxidized by peroxynitrite and that oxaluric acid is a likely secondary oxidation product of 8-oxo-G under physiological conditions.

Introduction Peroxynitrite (ONOO-)1 is formed endogenously from a diffusion-limited reaction of nitric oxide with superoxide anion (1). ONOO- is extremely reactive under physiological conditions, and is capable of interacting with various cellular components, including proteins, lipids, and DNA. ONOO- produced by macrophages has been implicated in cell death and an increased risk of cancer in tissues under inflammatory conditions (2, 3). ONOOtreatment of the pSP189 plasmid containing the supF gene induces mutations following replication in bacterial or human cells (4). The majority of mutations are located at G‚C base pairs (98.5% in bacteria and 93.4% in human cells), including G‚C f T‚A transversions (65% mutation level in bacteria and 63% in human cells), G‚C f C‚G transversions, and deletions (4). * To whom correspondence should be addressed: Massachusetts Institute of Technology, 77 Massachusetts Ave., Room 56-731A, Cambridge, MA 02139. Telephone: (617) 253-3729. Fax: (617) 2521787. E-mail: [email protected]. † Division of Bioengineering and Environmental Health. ‡ Department of Chemistry. 1 Abbreviations: CSPDE, calf spleen phosphodiesterase; LC/ESI-MS, liquid chromatography/negative ion electrospray ionization mass spectrometry; NHE, normal hydrogen electrode; 8-nitro-G, 8-nitroguanine; oxazolone, 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentafuranosyl)amino]-5-(2H)-oxazolone; ONOO-, peroxynitrite; 8-oxo-dG, 8-oxo-7,8dihydro-2′-deoxyguanosine; 8-oxo-G, 8-oxo-7,8-dihydroguanine; SVPDE, snake venom phosphodiesterase.

Detrimental biological effects of ONOO- are likely due to reactions with DNA that result in cytotoxic and promutagenic lesions. DNA damage induced by ONOOincludes single-strand breaks, nitration, and oxidation of nucleobases (5-11). Guanine (G) is the most reactive nucleobase when exposed to ONOO-; A, C, and T exhibit minimal reactivity (11). Treatment of 2′-deoxyguanosine with ONOO- leads to several products, including 8-oxo7,8-dihydro-2′-deoxyguanosine (8-oxo-dG; 6, 10), 8-nitroguanine (8-nitro-G; 8, 9), 2,2-diamino-4-[(2′-deoxy-βD-erythro-pentafuranosyl)amino]-5-(2H)-oxazolone(oxazolone; 6), 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine (6), and 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-deoxyguanosine (7). 8-Oxoguanine (8-oxo-G), 8-nitro-G, and oxazolone have also been observed following treatment of calf thymus DNA with ONOO- (6, 9, 10). 8-Oxo-G has been found in both rat and human tissues (1-50 per 106 bases), presumably the result of endogenous oxidative DNA damage (12, 13). On the nucleoside level, 8-oxo-dG is approximately 1000-fold more reactive toward ONOO- than 2′-deoxyguanosine itself, suggesting that 8-oxo-G may be a target for reactions with ONOOin vivo (11, 14). Reaction of free 8-oxo-2′-deoxyguanosine and low amounts of ONOO- first produces 3-(2′-deoxyribosyl)-3a-hydroxy-5-imino-3,3a,4,5-tetrahydro-1H-imidazo[4,5d]imidazol-2-one as the major product (15). This unstable intermediate either hydrolyzes (t1/2 ) 4.8 h at pH 7.2) to give 5-iminoimidazolidine-2,4-dione or further

10.1021/tx980235c CCC: $18.00 © 1999 American Chemical Society Published on Web 04/28/1999

460 Chem. Res. Toxicol., Vol. 12, No. 5, 1999

reacts with ONOO- to give 2,4,6-trioxo[1,3,5]triazinane1-carboxamidine and its hydrolytic product, cyanuric acid (15). While the reactions of ONOO- with free 8-oxo-2′deoxyguanosine have been studied in detail (15), analogous transformations of 8-oxo-G when present in DNA have not been elucidated. In this report, we describe the major reactions that occur when 8-oxo-G-containing single-stranded and double-stranded synthetic oligonucleotides are treated with ONOO-. The oligomers studied were either random sequences or sequences representing ONOO--related mutational “hotspot” regions of the supF gene. The reaction mixtures were analyzed by liquid chromatography/electrospray ionization mass spectrometry in the negative ion mode (LC/ ESI--MS), and the locations of the modified bases within DNA were established via partial exonuclease digestions followed by LC/ESI--MS analyses.

Experimental Section Materials and Methods. Synthetic oligonucleotides (CCACAACXCAAA, CCAAAGGXAGCAG, CCAAAXGGAGCAG, and TCCCGAGCGGCCAAAGGXAGCAG, where X is 8-oxo-G) were obtained from Synthetic Genetics (San Diego, CA). The oligonucleotides were purified by HPLC on a 250 mm × 4.6 mm, 5 µm Supelcosil LC-18-DB column with UV detection at 260 nm. The mobile phase was 150 mM aqueous ammonium acetate (A) and 100% acetonitrile (B) with a gradient from 5 to 20% B over the course of 50 min at a flow rate of 1 mL/min. Fractions containing the target oligonucleotide were collected, combined, and thoroughly dried under vacuum to remove most of the ammonium acetate. ONOO- was prepared by ozonation of sodium azide in alkaline solution (0.1 N NaOH) and stored at -80 °C until it was used (5). ONOO- concentrations were determined by spectrophotometry ( ) 1670 M-1 cm-1, λ ) 302 nm in 0.1 N NaOH). Reactions between ONOO- and DNA were carried out in 150 mM potassium phosphate/25 mM sodium bicarbonate buffer at pH 7.2. Oligonucleotide solutions (∼50 µM in 50 µL of buffer) were mixed with ONOO- (final concentration of 50 µM to 5 mM) by vortexing for 1 min. Calf spleen phosphodiesterase (CSPDE) and snake venom phosphodiesterase (SVPDE) were purchased from Boehringer Mannheim (Indianapolis, IN). Oligonucleotides (7-10 µg) were enzymatically digested by incubating in 50 µL of water with 3 × 10-3 unit of CSPDE (5′ f 3′ sequencing) or SVPDE (3′ f 5′ sequencing) at 37 °C for 1-30 min. Aliquots (10 µL) were taken every 5 min and stored in 10 mM EDTA at -20 °C. For LC/ ESI--MS analysis, the samples were warmed to room temperature and then injected directly onto the LC column. Liquid Chromatography/Electrospray Ionization Mass Spectrometry. Reaction mixtures were analyzed by on-line HPLC/ESI--MS system using a Hewlett-Packard 1090 liquid chromatograph interfaced with a Finnigan TSQ 7000 triplequadrupole mass spectrometer. In some experiments, a diode array UV detector was in series with the mass spectrometer. Chromatographic separation was achieved using a 150 mm × 1 mm, 5 µm Jupiter C18 column with one of the following solvent systems. System I consisted of 400 mM aqueous 1,1,1,3,3,3hexafluoro-2-propanol (A) and 400 mM 1,1,1,3,3,3-hexafluoro2-propanol in 70% CH3OH/H2O (B). Both A and B were adjusted to pH 7 with triethylamine (16). The gradient program was from 30 to 100% B over the course of 30 min at a flow rate of 65 µL/min. System II was 50 mM triethylammonium acetate in water (pH 7.2) (A) and 100% acetonitrile (B) with a gradient from 0 to 20% B over the course of 30 min at a flow rate of 50 µL/min. The mass spectrometer was operated in the negative ion mode. Electrospray ionization was typically achieved at a spray voltage of 2.2-3 kV. The temperature of the heated capillary was 180-200 °C. The sheath gas pressure was 60-65 psi. Sheath liquid (80% isopropyl alcohol in water containing 50 mM

Tretyakova et al.

Figure 1. HPLC/ESI--MS analysis of CCACAACXCAAA (X is 8-oxo-G). HPLC conditions were as follows. System A was 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in water. System B was 400 mM HFIP in 70% CH3OH/H2O. The gradient was from 30 to 100% B over the course of 30 min. Full scan MS from m/z 600 to 1800 over the course of 5 s. imidazole and 50 mM piperidine) was infused at a rate of 10 µL/min with HPLC solvent system II (17). The mass spectrometer was scanned from m/z 600 to 1800 in 5 s. Oligonucleotide spectra were obtained by subtracting the background spectra on either side of an HPLC peak from the averaged data obtained during the elution.

Results ONOO-

Reactions with DNA Containing 8-Oxoguanine. Previous experiments with polyacrylamide gel electrophoresis (PAGE) indicated that single-stranded synthetic oligonucleotide CCACAACXCAAA (X is 8-oxoG) reacted quantitatively with peroxynitrite to give at least two distinct products (11). Since the identity of these products could not be established by PAGE, the reactions were repeated on a larger scale to allow analyses by HPLC/ESI--MS. Analysis of unmodified CCACAACXCAAA (M ) 3608.4), using solvent system I, revealed a single peak in the LC chromatogram at 17.2 min (Figure 1). The negative ion ESI mass spectrum of this peak contained signals corresponding to several charge states of this oligonucleotide, including [M - 6H+]6- at m/z 600.4, [M - 5H+]5- at m/z 720.3, [M - 4H+]4- at m/z 900.8, [M - 3H+]3- at m/z 1202.1, and [M - 2H+]2- at m/z 1803.3 (Figure 1 and Table 1). Treatment of this oligonucleotide with a 5-fold excess of ONOO- led to its complete disappearance, along with the appearance of two major products with retention times shorter than that of the original oligonucleotide (13.7 and 14.2 min, respectively) (Figure 2a). The ESI- mass spectrum of the peak at 13.7 min deconvoluted to a molecular weight of 3596.4 (Figure 2b). The peak at 14.2 min contained two compounds with molecular weights of 3570.4 and 3612.4 (Figure 2c,d). When the molecular weight of CCACAACXCAAA (X is 8-oxo-G, with an M of 3608.4) is compared to the calculated molecular weight of the product eluting at 13.7 min (M ) 3596.4), a mass difference of -12 is observed; this is the same as the difference between 8-oxo-G (M ) 167.1) and 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro-1Himidazo[4,5d]imidazol-2-one (M ) 155.1; 1 in Scheme 1), an unstable intermediate found in mixtures of 8-oxo-2′deoxyguanosine treated with peroxynitrite (Scheme 1; 15). The mass differences between CCACAACXCAAA (X is 8-oxo-G) and the products in the second peak (14.2 min,

Peroxynitrite Reactions with 8-Oxoguanine in DNA

Chem. Res. Toxicol., Vol. 12, No. 5, 1999 461

Table 1. ESI- Spectra of Oligonucleotides CCACAACXCAAA

M ) 3570.4 and M ) 3612.4) are -38 and 4, respectively, which are equal to those between 8-oxo-G (M ) 167.1) and cyanuric acid (M ) 129.06; 3 in Scheme 1) and 2,4,6trioxo[1,3,5]triazinane-1-carboxamidine (M ) 171.1; 2). In earlier experiments with free 8-oxo-dG, cyanuric acid was found to be a hydrolysis product of 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine (15). In this work, when the analyses were performed under alkaline conditions (pH g9), no oligonucleotide 2 (Scheme 1) was observed, presumably due to its hydrolysis to 3. To test this hypothesis, we isolated oligomer 2 and left it at room temperature at pH 7 for 2 days. HPLC/ESI--MS analysis of the resulting solution confirmed that 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine-containing oligonucleotide 2 was hydrolyzed to the cyanuric acid-containing oligomer 3 (results not shown). The oligomer 1 containing the unstable intermediate 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro-1H-imidazo[4,5d]imidazol-2-one was only observed in reaction mixtures immediately after ONOO- treatment. Storage of 1 for several days at -20 °C resulted in complete hydrolysis to oligonucleotide 4 containing 5-iminoimidazolidine-2,4dione (Scheme 1). If the oligomer 4 was left at room temperature overnight, further hydrolysis took place to give oxaluric acid (5 in Scheme 1). The actual composition of the reaction mixtures depended on the relative concentrations of ONOO- and oligonucleotide. At ONOO-/ DNA molar ratios of greater than 5, 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine- and cyanuric acid-containing oligonucleotides were the main products. At molar ratios of less than 5, the predominant products were the intermediate 1 and its hydrolysis products, 4 and 5 (Scheme 1). The 13-mer CCAAAGGGAGCAG represents a sequence of the supF gene (nucleotides 117-129) that contains the predominant mutational “hotspots” arising from exposure to ONOO- (4). We were therefore interested in the relative reactivity of guanines in this sequence, especially with respect to the effect of preformed 8-oxo-G. To assess this relative reactivity, we replaced guanines 122 and 124 with 8-oxo-G, resulting in sequences CCAAAGGXAGCAG and CCAAAXGGAGCAG (X is 8-oxo-G). The modified oligonucleotides were then treated with ONOO-, and the reaction mixtures were analyzed by LC/ESI--MS. For both oligomers, the products were analogous to those described above for CCACAACXCAAA (X is 8-oxo-G), including those corre-

sponding to intermediate 1 (M ) 4021.7) and compounds 2 (M ) 4037.7), 3 (M ) 3995.7), 4 (M ) 3979.7), and 5 (M ) 3998.7) (Scheme 1, Figure 3, and Table 2). Similar reactivity was seen for a longer sequence of the supF gene, 23-mer TCCCGAGCGGCCAAAGGXAGCAG (X is 8-oxo-G; see Table 3 and Scheme 1). To simplify the analysis of these relatively long (g13 bases) oligonucleotides, a new LC/MS method was developed as follows. The molecular weight of these molecules may be too large for detection of mass differences between various products resulting from reactions at a single base. This problem can be partially solved if the charge state distributions for related oligomers are shifted toward lower charge states (17). In the case of TCCCGACGGCCAAAGGXAGCAG, for example, the [M - 8H+]8- ions for when X is 8-oxo-G and when X is 2,4,6trioxo[1,3,5]triazinane-1-carboxamidine 2 differ by less than 0.5 m/z unit (889.6 vs 890.1; Table 3). This difference is increased to more than 1 m/z unit for the [M - 4H+]4ions (1780.1 vs 1781.2) (Table 3). Therefore, identification of the oligonucleotide products is simplified by using ions with lower charge states. Solvent system I, containing aqueous 1,1,1,3,3,3-hexafluoro-2-propanol in methanol/ water, favors multiple charge states ([M - 7H+]7- to [M - 9H+]9-) for the 23-mers. Spectra of the same oligomers obtained in solvent system II (triethylammonium acetate in acetonitrile/water), on the other hand, were dominated by a single charge state ([M - 4H+]4-). The ESI- spectra of 13-mers obtained in solvent system II contain single peaks with a z of -3 (Figure 4), whereas various charge states between -3 and -6 are seen in solvent system I (results not shown). Molecular weights determined by this approach are based on a single m/z value and are somewhat less accurate than those determined from spectra containing multiple charge states. This technique, however, facilitates identification of the reaction products of longer oligomers (g13 base pairs) that differ in weight by less than 10 units. In addition, the simplified spectra, containing a single peak for each oligomer, are helpful when analyzing complex mixtures such as those arising from phosphodiesterase digestion for sequencing (see below). Determining the Position of Lesions within DNA Sequences Using Enzymatic Digestion and LC/MS. The methods described above are suitable for detecting DNA modifications in oligonucleotides, but they do not allow the determination of the position of the lesion

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Figure 2. HPLC trace (a) and ESI- mass spectra (b-d) of the products of CCACAACXCAAA (X is 8-oxo-G) treatment with 0.2 mM ONOO-: (b) X is 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro1H-imidazo[4,5d]imidazol-2-one (1), (c) X is cyanuric acid (3), and (d) X is 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine (2). See Figure 1 for the HPLC/MS conditions.

within the sequence. This information is important since reactions can potentially take place at multiple positions within an oligonucleotide. Sequence information for oligonucleotides up to 20 bases in length can be obtained directly from the collision-induced dissociation spectra of their molecular ions using the mass differences between adjacent fragments in characteristic ion series (18, 19). This method requires instrumentation capable of MS/ MS, and in many cases, a prior knowledge of the sequence is needed. Alternatively, enzymatic digestion of modified oligonucleotides followed by LC separation and ESI--MS analysis of the resulting fragments can be

Tretyakova et al.

used. Treatment with nuclease P1, for example, allows 5′-, 3′-, and internal DNA modifications to be distinguished, although this method does not provide a complete sequence (20). Partial digestion with 3′- and 5′phosphodiesterases generates DNA ladders that can be used to obtain a complete oligonucleotide sequence (21). In this work, stepwise digestions with 3′ f 5′ and 5′ f 3′ exonucleases followed by on-line HPLC/ESI--MS were applied to determine the position of DNA modifications. First, the method was applied to synthetic oligonucleotides with the modified base at a known position. CCACAACXCAAA, CCAAAGGXAGCAG, and CCAAAXGGAGCAG (X is 8-oxo-G) were digested with SVPDE for 3′ f 5′ sequencing and CSPDE for 5′ f 3′ sequencing (Tables 4 and 5). This approach is illustrated in Figure 5 for SVPDE digestion of CCACAACXCAAA. At early time points, oligomers missing several nucleotides from the 3′-terminus of the starting DNA are found in the digest (Figure 5, 1 min digest). As the incubation time is increased to 5-30 min, the size of the remaining sequence decreased, until a complete ladder of oligomers resulting from the stepwise removal of all nucleotides was obtained (Figure 5 and Table 5). The resulting mixtures of DNA fragments could not be fully resolved by HPLC, but this was not necessary, since the ESI spectra of the mixtures of oligomers of various lengths provided sufficient information to deduce the oligonucleotide sequence (Tables 4 and 5). These experiments were carried out with LC solvent system II (triethylammonium acetate/ acetonitrile); under these conditions, each oligomer exhibited only one low charge state, thus simplifying interpretation of the MS data. Complete digestion of the oligomers was achieved with SVPDE (Table 5), while the CSPDE digestion stopped at the 8-oxo-G (Table 4). In the case of SVPDE digestion, the spectrum was dominated by signals corresponding to the fragments resulting from removal of oligonucleotides up to the 8-oxo-G site (Figure 5), suggesting that the digestion is slowed by DNA modification. Since the two enzymes work in opposite directions, the information from the two digestions was combined to obtain a complete sequence. These experiments confirmed the sequence of synthetic oligomers CCACAACXCAAA, CCAAAXGGAGCAG, and CCAAAGGXAGCAG (X is 8-oxo-G), and suggested that this method could be applied to oligonucleotides containing nucleobase modifications at unknown locations. Sequencing the Oligonucleotide Products Resulting from ONOO- Reactions with CCAAAGGXAGCAG and CCAAAXGGAGCAG. DNA sequences containing runs of guanines are known to have an increased susceptibility to the action of oxidizing agents, with the guanine at the 5′-end of the G run being the most reactive (22-24). Theoretical studies have attributed this effect to electrostatic and orbital interactions that lead to a reduced oxidation potential for the 5′-guanine (22, 23). Similar interactions have been reported for 8-oxo-Gcontaining DNA (23, 24). Synthetic oligonucleotides CCAAAGGXAGCAG (II) and CCAAAXGGAGCAG (III) used in our experiments contain sequences 5′-GGX-3′ and 5′-XGG-3′, respectively, where X is 8-oxo-G. 8-Oxo-G is typically 1000-fold more reactive than guanine (11, 14). However, sequence effects such as guanine stacking may shift the reaction of oligomer II with ONOO- toward the 5′-guanine, affecting the regioselectivity of oxidation. Since in oligomer III, 8-oxo-G is located 5′ from two

Peroxynitrite Reactions with 8-Oxoguanine in DNA

Chem. Res. Toxicol., Vol. 12, No. 5, 1999 463

Scheme 1. Peroxynitrite-Induced Reactions of 8-OxoG-Containing Oligonucleotides CCACAACXCAAA (I), CCAAAGGXAGCAG (II), CCAAAXGGAGCAG (III), and TCCCGAGCGGCCAAAGGXAGCAG (IV)

at the neighboring guanine bases. Table 7 contains the sequencing results for a product of ONOO- reaction with oligonucleotide III (M ) 3998.7). The latter product was identified as CCAAAXGGAGCAG, where X is oxaluric acid, confirming that, as expected, the reaction took place at 8-oxo-G.

Discussion

Figure 3. HPLC/ESI--MS analysis of the products of the treatment of 180 µM CCAAAGGXAGCAG (X is 8-oxo-G) with ONOO- (50 µM): (a) X is 8-oxo-G (unreacted, m/z 1343.6) and (b) X is 5-iminoimidazoline-2,4-dione (m/z 1325.6) (4 in Scheme 1). System A was 50 mM triethylammonium acetate in water, and system B was 100% acetonitrile. The gradient was from 0 to 20% B over the course of 30 min.

guanines, the same factors would increase the reactivity of 8-oxo-G in this sequence. Although the products isolated from the reaction of ONOO- with both oligonucleotides II and III had molecular weights consistent with oxidation at 8-oxo-G (see Table 2 and Scheme 1), the predominant oligonucleotide products were sequenced to unequivocally determine the site of reaction. Oligonucleotides II and III in single-stranded or doublestranded form were treated with peroxynitrite, and the reaction products were separated by HPLC. Fractions containing the products were collected and concentrated under vacuum, and sequencing was performed by exonuclease digestion followed by LC/ESI--MS as described above. The results obtained from SVPDE digestion and LC/ ESI--MS analysis of the oligomer with a molecular weight of 4037.7 resulting from ONOO- reaction of oligonucleotide II are summarized in Table 6. These data are consistent with the sequence CCAAAGGXAGCAG, where X is 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine (2 in Scheme 1), and show that, despite the sequence effects, the reaction is taking place at 8-oxo-G and not

The chemical reactivity of free nucleosides often differs from that of nucleobases within DNA (25). Differences in the electron density distribution, along with steric factors in the polynucleotide chain, contribute to this effect. In earlier work in this laboratory, we studied the ONOO--induced transformations of 8-oxo-2′-deoxyguanosine (15), but the applicability of these findings to 8-oxo-G-containing DNA remained uncertain. The results presented here demonstrate that reactions analogous to those found for free 8-oxo-2′-deoxyguanosine also take place in oligonucleotides containing 8-oxo-G. In both cases, the composition of the reaction mixtures depends on the [ONOO-]/[8-oxo-G] molar ratio and on the length of the postexposure storage. 8-Oxo-G reaction with ONOO- proceeds through the formation of an unstable intermediate, tentatively assigned as 3a-hydroxy-5-imino3,3a,4,5-tetrahydro-1H-imidazo[4,5d]imidazol-2-one (1 in Scheme 1). At the low ONOO- concentrations ([ONOO-]/ [8-oxo-G] < 5), 1 is predominantly hydrolyzed into 5-iminoimidazolidine-2,4-dione-containing oligomers 4. The latter undergo further hydrolysis to oxaluric acidcontaining oligonucleotides 5 (Scheme 1). With high ONOO- treatments ([ONOO-]/[8-oxo-G] g 5), oligonucleotides 1 are converted to 2,4,6-trioxo[1,3,5]triazinane-1carboxamidine-containing oligomers 2 and their hydrolytic products, cyanuric acid-containing oligonucleotides 3. These results suggest that 8-oxo-G will probably not accumulate in DNA under conditions where it can further react with peroxynitrite to form more stable secondary products. At the low ONOO- concentrations expected to be encountered in vivo, oxaluric acid is a likely final product of peroxynitrite-induced oxidation. These findings should help in the elucidation of the mechanisms of ONOO--induced DNA damage.

464 Chem. Res. Toxicol., Vol. 12, No. 5, 1999

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Table 2. ESI- Spectra of Oligonucleotides CCAAAGGXAGCAG and CCAAAXGGAGCAG

Table 3. ESI- Spectra of Oligonucleotides TCCCGAGCGGCCAAAGGXAGCAG

Table 4. Sequencing (5′ f 3′) of CCACAACXCAAA (X Is 8-Oxoguanine) Using CSPDE Digestion and LC/ESI--MS sequence

M

CCACAACXCAAA CACAACXCAAA ACAACXCAAA CAACXCAAA AACXCAAA ACXCAAA CXCAAA XCAAA

3608.4 3319.2 3030.0 2716.8 2427.6 2114.4 1801.2 1512.0

observed ions (m/z) charge state (z) 1201.8 1658.3 1515.0 1357.3 1213.1 1056.3 899.9 1512.1

-3 -2 -2 -2 -2 -2 -2 -1

From a methodological point of view, our results demonstrate the applicability of LC/ESI--MS in investigations of the transformations of synthetic oligonucleotides. The advantages of using oligonucleotides rather than free nucleosides in these studies include the application of a more realistic DNA-based system and the ability to assess the effects of DNA secondary structure on nucleobase reactivity. Since short oligonucleotides are amenable to direct LC/MS analyses, product identification can be performed immediately after exposure to detect unstable intermediates. One potential problem in applying these methods to larger oligonucleotides (>20 bp) is the small mass change associated with reactions at a single base, which complicates the identification of the lesions. However, careful selection of the HPLC conditions and the use of a charge-reducing sheath liquid

Table 5. Sequencing (3′ f 5′) of CCACAACXCAAA (X Is 8-Oxoguanine) Using SVPDE Digestion and LC/ESI--MS sequence

M

CCACAACXCAAA 3608.4 CCACAACXCAA 3295.2 CCACAACXCA

2982.0

CCACAACXC

2668.8

CCACAACX CCACAAC CCACAA CCACA CCAC CCA

2379.6 2034.4 1745.2 1432.0 1118.8 829.6

observed ions (m/z) charge state (z) 1201.2 1646.6 1097.0 1490.3 992.5 1333.4 889.0 1188.6 1016.2 871.6 715.2 558.2 829.3

-3 -2 -3 -2 -3 -2 -3 -2 -2 -2 -2 -2 -1

for ESI (17) make it possible to shift the charge state envelope toward lower charge states and thus to identify the reaction products with greater confidence. Enzymatic digestion of modified oligonucleotides with 3′ f 5′ and 5′ f 3′ exonuclease followed by LC/ESIanalysis allows full sequence analysis for locating the ONOO--induced DNA lesions. In this work, we demonstrate the applicability of this method both to synthetic oligonucleotides with known modifications and to oligonucleotide products of ONOO- treatment. Sequencing of the 13-mer oligonucleotide products of ONOO- reaction with CCAAAGGXAGCAG (X is 8-oxo-G) enabled us to

Peroxynitrite Reactions with 8-Oxoguanine in DNA

Chem. Res. Toxicol., Vol. 12, No. 5, 1999 465 Table 7. Sequencing (3′ f 5′) of CCAAAXGGAGCAG (X Is Oxaluric Acid) Using SVPDE Digestion and LC/ESI--MS sequence

M

CCAAAGGXAGCAG 3998.7

Figure 4. ESI- mass spectra of the oligonucleotides present in the reaction mixture after treatment of 180 µM CCAAAGGXAGCAG (X is 8-oxo-G) with a low concentration of ONOO(50 µM): (a) X is 8-oxo-G (unreacted) and (b) X is 5-iminoimidazolidine-2,4-dione (4 in Scheme 1). Full scan MS from m/z 500 to 1800 over the course of 5 s.

CCAAAXGGAGCA CCAAAXGGAGC CCAAAXGGAG

3669.5 3356.3 3067.1

CCAAAXGGA

2737.9

CCAAAGGX

2424.7

CCAAAXG

2095.5

CCAAAX CCAAA CCAA

1766.3 1456.1 1142.9

observed ions (m/z) charge state (z) 1331.6 998.6 1221.8 1117.2 1531.9 1020.9 1367.8 911.0 1211.0 806.9 1046.0 697.3 881.9 1454.9 1141.8

-3 -4 -3 -3 -2 -3 -2 -3 -2 -3 -2 -3 -2 -1 -1

digestion of the oligomer past the lesion was possible using SVPDE. The information about the site of DNA modification is valuable, since the spectrum of chemical damage can then be compared to mutational spectra to assess the biological importance of various lesions and to provide insight into the mechanism of mutagenesis.

Acknowledgment. We thank the National Institutes of Health for continuing support (Grant 5-P01-CA2673119) and the U.S. Department of Energy for the mass spectrometer (Grant DE-FG02-957E00056). J.C.N. is supported by NIH Fellowship HG00144-04.

References

Figure 5. ESI- mass spectra of 3′ f 5′ digests of CCACAACXCAAA with snake venom phosphodiesterase as a function of incubation time: m/z 1201.2 is CCACAACXCAAA (z ) -3); m/z 1646.6 and 1097.0 are CCACAACXCAA (z ) -2 and -3); m/z 1333.4 is CCACAACXC (z ) -2); m/z 1188.6 is CCACAACX (z ) -2); m/z 871.6 is CCACAA (z ) -2); m/z 715.2 is CCACA (z ) -2); m/z 558.2 is CCAC (z ) -2); and m/z 829.3 is CCA (z ) -1). Table 6. Sequencing (3′ f 5′) of CCAAAGGXAGCAG (X Is 2,4,6-Trioxo[1,3,5]triazinane-1-carboxamidine) Using SVPDE Digestion and LC/ESI--MS sequence

M

CCAAAGGXAGCAG CCAAAGGXAGCA CCAAAGGXAGC CCAAAGGXAG

4037.7 3708.5 3395.3 3106.1

CCAAAGGXA CCAAAGGX

2776.9 2463.7

CCAAAGG CCAAAG CCAAA CCAA

2114.5 1785.3 1456.1 1142.9

observed ions (m/z) charge state (z) 1344.1 1235.0 1130.6 1551.8 1034.5 924.2 1230.1 820.1 1056.4 891.5 727.4 1142.0

-3 -3 -3 -2 -3 -3 -2 -3 -2 -2 -2 -1

locate the site of reaction at the 8-oxo-G position, despite the presence of two guanines 5′ from 8-oxo-G. Similar sequencing methods can be applied to DNA that has been modified with other agents, although there are probably limitations to the size of the adduct. For example, the phosphodiesterases have been reported to stop one or two bases before biotin-labeled bases (20). For the products of oxidative damage studied in this work, complete

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