Product analysis of photooxidation in isolated quadruplex DNA ; 8-oxo-7 , 8-dihydroguanine and its oxidation product at 3 0G are formed instead of 2 , 5-diamino-4 H-imidazol-4-one †

The formation of quadruplex structure changed the site reactivity and the kinds of guanine photooxidation products of d(TGGGGT). In quadruplex DNA, 8-oxo-7,8-dihydroguanine (8oxoG) and dehydroguanidinohydantoin (Ghox) were mainly formed, although 2,5-diamino-4H-imidazol-4-one (Iz) was mainly formed in singlestranded DNA. In addition, 30-guanine was specifically oxidized in quadruplex DNA compared with single-stranded DNA, which depended on the localization of the HOMO.

DNA bases are the key components for preservation of genetic information.However, DNA nucleobases are frequently oxidized via reactions with environmental agents such as UVAlight.Among the four DNA bases, guanine is the most easily oxidized.Additionally, guanines in contiguous guanine sequences, such as GG and GGG, in double-stranded DNA have lower redox potentials than single guanine sequences. 1Thus, some guanine-rich sequences could be easily oxidized in living organisms.Guanine-rich sequences exist in many important regions, such as telomeres 2 and the promoter element of the proto-oncogene c-myc 3 in the human genome, and these sequences can form quadruplex DNA with eight Hoogsteen hydrogen bonds of four guanines.In recent computational investigations, 4 the quantity of quadruplex-forming sequences in the genome is over 3.5 Â 10 5 .The differences of DNA structure between single-stranded and quadruplex DNA may inuence the process of oxidation.In this study, quadruplex DNA was isolated by HPLC, and the oxidation of the isolated quadruplex DNA was compared with that of the single-stranded DNA.
In particular, we attempt to directly analyze the oligomers containing the oxidation product.This approach enabled us to identify the kinds, the yields, and the locations of the oxidation products at once, unlike a recent report. 5Since the shorter oligomer was appropriate for isolation and identication, we used the 6-mer DNA d(TGGGGT), which is the shortest oligomer among the quadruplex-formative sequences in the presence of several ions. 6This 6-mer oligomer is the truncated telomeric sequence from Tetrahymena or Oxytricha.
The quadruplex structure of d(TGGGGT) was formed in KCl solution in this study, since the intracellular potassium ion concentration is high (approximately 140 mM).Since, d(TGGGGT) can formed in 1 mM KCl.The d(TGGGGT) in 10 and 0.1 mM KCl were heated to 80 C, and incubated at 4 C for 1 min, and the structures were determined by CD spectroscopy.The increasing typical CD spectrum around 260 nm of the quadruplex DNA was detected in 10 mM KCl (Fig. 1a). 7In contrast, the CD spectrum around 260 nm was not observed in 0.1 mM KCl (Fig. 1b), and then the quadruplex DNA was not formed.Next, the d(TGGGGT) in 10 and 0.1 mM KCl were analyzed using HPLC, and the proles are shown in Fig. 1c  and d, respectively.In these proles, two peaks at 8.2 and 18.0 min were detected in 10 mM KCl.The peaks were isolated using HPLC.Furthermore, the isolated products at 8.2 and 18.0 min in Fig. 1c were identied as a single-stranded DNA and a quadruplex DNA containing three potassium ions ([C 240 H 297 N 96 O 144 P 20 K 3 ] m/z 1889.80689,calculated for [M À 4H] 1889.80493) by electrospray ionization-mass spectrometry (ESI-MS) in negative-ion mode (Fig. S1, ESI †).Even though the isolated quadruplex DNA in 10 mM KCl at 4 C was le for at least 3 days, single-stranded DNA was detected in less than 4% (Fig. 2).Although previous reports suggested that singlestranded DNA and quadruplex DNA were separable with HPLC, 8 isolation of quadruplex DNA was not described in the article.To our knowledge, isolation of quadruplex DNA was accomplished by using HPLC for the rst time.Using this isolated quadruplex structures can eliminate the possibility of formation of oxidation products from single-stranded or double-stranded DNA.
Isolated quadruplex DNA (700 mM, DNA concentration was expressed as single-stranded molarity) in 10 mM KCl solution or the single-stranded DNA in 0.1 mM KCl were photooxidized with 75 mM riboavin (RF, i.e., vitamin B 2 ), and these products were analyzed using HPLC. 9To determine the oxidation products of single-stranded and quadruplex at almost the same conversion rate (approximately 20%), 10 we determined the rates of the oxidation in single-stranded and quadruplex DNA.The reaction of quadruplex DNA was analyzed aer UVA irradiation for 0, 10, 20, and 30 min, and the reaction of single-stranded DNA for 0, 1, 2, and 3 min.The conversions of the singlestranded DNA and the quadruplex DNA aer UVA irradiation are shown in Fig. S2 (ESI †).Those conversions were determined with HPLC.As a result, the quadruplex DNA was oxidized more slowly than the single-stranded DNA.Some groups reported that quadruplex of guanine-rich DNA strands reduced the rates of reactions, 11 which agreed with our results.
HPLC chromatograms following oxidation of singleand quadruplex DNA were shown in Fig. 3.The singlestranded and quadruplex DNA were irradiated for 2 min and 20 min, respectively, and both conversion rates were approximately 20%.Those photooxidized solutions were analyzed by HPLC.In irradiated single-stranded DNA, three major peaks were detected at 20.5, 20.7, and 21.1 min, as shown in Fig. 3b.These products were isolated and identied as oligomers containing 2,5-diamino-4H-imidazol-4-one (Iz) ([C 58 H 74 N 23 O 36 P 5 ] m/z 910.66052, calculated for [M À 2H] 910.65937) by ESI-MS in negative-ion mode (Fig. 3a and S3 †).The location of Iz in the oligomer was identied by piperidine treatment as previously reported. 12The peaks at 20.5 and 20.7 min were identied as d(TGGGIzT) and d(TIzGGGT), respectively (Fig. S4a-c, ESI †).The peak at 21.1 min was composed of two products: d(TGIzGGT) and d(TGGIzGT).
The HPLC proles of oxidized guanine in quadruplex DNA signicantly differed from those in single-stranded DNA. 13In irradiated quadruplex DNA, two major peaks were detected at 18.6 and 20.4 min (Fig. 3c).The peaks at 18.6 and 20.4 min in Fig. 3c were isolated and identied as the oligomer containing dehydroguanidinohydantoin (Ghox) ([C 59 H 75 N 24 O 37 P 5 ] m/z     3a and S5, ESI †).The unoxidized single-stranded DNA was detected at 21.7 min (Fig. 3c).Although photooxidation in single-stranded DNA mainly produced the oligomers containing Iz, they were not detected in quadruplex DNA.The location of Ghox was then identied by piperidine treatment. 14s a result, d(TGGGGhoxT) was detected (Fig. S4d, ESI †).Meanwhile, 8oxoG was stable under conditions of piperidine treatment. 15Therefore, four synthetic oligomers containing 8oxoG and its oxidation products were analyzed by HPLC, and the peak at 20.4 min in Fig. 3c was conrmed by those HPLC proles (Fig. S6, ESI †).As a result, the peak at 20.4 was identied as d(TGGG8oxoGT).Surprisingly, the experiments shown in Fig. 3 revealed that the 3 0 -guanine of d(TGGGGT) was selectively oxidized in quadruplex DNA.In double-stranded DNA, 3 0 -guanine was hardly oxidized by the photooxidation of 5 0 -GG-3 0 , 1 5 0 -GGG-3 0 , 16 and 5 0 -GGGG-3 0 . 17To the best of our knowledge, specic oxidation of 3 0 -guanine in guanine-rich sequences has not previously been reported.
Thus, the guanine oxidation products from quadruplex DNA differ from those from single-stranded DNA, with differences in the structure possibly leading to alterations in the oxidation pathway of guanine.The one-electron oxidation 18 and the oxidation with singlet oxygen 19 were mediated by RF under UVA irradiation.To determine whether singlet oxygen was involved in the oxidation of guanine in singlestranded or quadruplex DNA, single-stranded or quadruplex DNA was photooxidized in 80% D 2 O, the solvent in which singlet oxygen is known to have a longer half-life.As a result, the amounts of these oxidation products were little changed by 80% D 2 O (Fig. 4).Thus, the oxidations were likely to undergo one-electron oxidation in single-stranded and quadruplex DNA, and singlet oxygen was not involved in the reactions.
One-electron oxidation of guanine generates a guanine radical cation (G_ + ), 20 and G_ + undergoes two competitive pathways (Scheme 1).In pathway 1, G_ + deprotonates at the N1 position to produce the neutral guanine radical [G(-H)_], and the addition of superoxide (O 2 _ À ) 21 to G(-H)_ produces a transient peroxyl radical.Subsequently, nucleophilic addition of water induces the subsequent rearrangement and release of formamide, 21a and leads to the formation of Iz.In pathway 2, hydration of G_ + is caused.Following the generation of 8-hydroxy-7,8-dihydroguanyl radical, 22 the radical is oxidized to 8oxoG (see ESI †).Since the major oxidation product of 8oxoG by one-electron oxidation was Ghox, 23 the d(TGGGGhoxT) should be formed by d(TGGG8oxoGT).In quadruplex DNA, hydrogen bond between the N1 proton of G_ + and the O6 of its neighbor guanine is formed (Scheme 2).Therefore, N1 proton in quadruplex DNA is more strongly retained than N1 proton in singlestranded DNA (see ESI †).Pathway 1 was blocked by the inhibition of deprotonation in the quadruplex, and 8oxoG was considered to be mainly formed in quadruplex DNA.
The 3 0 -guanine of d(TGGGGT) sequences was selectively oxidized in quadruplex DNA (Fig. 3c).Then, we calculated localization of the highest occupied molecular orbital (HOMO) of the quadruplex structure (see ESI †).As a result, the estimated HOMO was localized on the 3 0 -guanine of d(TGGGGT) (Fig. 3d), and the calculated result was matched with the experimental results (Fig. 3b and c).We concluded that the specic oxidation of 3 0 -guanine was induced by the localization of the HOMO.In summary, isolation and identication of quadruplex DNA were accomplished by using HPLC and ESI-MS (Fig. 2 and S1 †).We attempted to identify the oxidation products of this isolated quadruplex and single-stranded DNA.In quadruplex DNA, major photooxidation products of guanine were identied as 8oxoG and its oxidation product, Ghox (Fig. 3c).The singlestranded DNA and quadruplex DNA signicantly differed in the kinds of major oxidation products.Furthermore, 3 0 -guanine was mainly oxidized in quadruplex.We show that the specic oxidation of 3 0 -guanine depended on the localization of the HOMO (Fig. 3d).We found that the kinds and the locations of guanine oxidation products depended on the structures of quadruplex or single-stranded DNA.
Many important guanine-rich oligonucleotides can form quadruplex structure, and the quadruplex stability inuences the telomere elongation by telomerase 24 and the transcription activity of c-myc. 258oxoG affects the of quadruplex, 26 but it is not investigated whether the other oxidation products except for 8oxoG affect the quadruplex stability.Therefore, we will investigate the relation between this specic oxidation and the stability of quadruplex DNA.
This work was supported by research grants from Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Nakatomi Foundation.

Fig. 1
Fig. 1 The CD and HPLC profiles of d(TGGGGT) in KCl.CD spectroscopy was performed in (a) 10 mM or (b) 0.1 mM KCl. HPLC analyses were performed in (c) 10 mM KCl or (d) 0.1 mM KCl.Samples were analyzed by HPLC with a COSMOSIL 5C 18 -MS column (Nacalai Tesque, 5 mm, 150 Â 4.6 mm, elution with a solvent mixture of 50 mM TEAA (pH 7), 5-30% CH 3 CN/30 min at a flow rate of 1.0 ml min À1 ) and monitored at 260 nm absorbance in panel c and d.

Fig. 4 Scheme 2
Fig. 4 The oxidations with SOD or D 2 O. Single-stranded d(TGGGGT) (700 mM) with 75 mM RF in 0.1 mM KCl and 5 mM cacodylate buffer (pH 7) was irradiated at 365 nm for 2 min.Quadruplex d(TGGGGT) (700 mM) with 75 mM RF in 10 mM KCl and 5 mM cacodylate buffer (pH 7) was irradiated at 365 nm for 20 min.The reactions were initially performed in 80% D 2 O or with 20 U ml À1 SOD.The amounts of oxidation products were calculated using HPLC.The mean values and errors were calculated using data from two independent experiments, and the error for Iz was not detected in the reaction with SOD.(a) Amounts of oligomers containing Iz in irradiated single-stranded DNA.(b) Amounts of oligomers containing 8oxoG or Ghox in irradiated quadruplex DNA.Scheme 1 The proposed pathways for the oxidation of guanine in quadruplex DNA.