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. 2026 Mar 20;9:175. doi: 10.1038/s42004-026-01979-8

Singlet oxygen-mediated photocatalytic generation of abasic sites in DNA

Yuuhei Yamano 1,2,✉,#, Kazumitsu Onizuka 1,2,3,✉,#, Okan Altan 1, Madoka Sasaki 1,2, Ahmed Mostafa Abdelhady 1,4, Shigeki Sasaki 5, Fumi Nagatsugi 1,2,✉,#
PMCID: PMC13168487  PMID: 41862674

Abstract

Oxidative stress factors such as light and inflammation are known to damage nucleic acids through various mechanisms. Guanine, among the four canonical nucleobases, is particularly susceptible to oxidation, leading to the formation of oxidatively generated lesions such as 8-oxo-7,8-dihydroguanine (8-oxoG), spiroiminodihydantoin (Sp), and guanidinohydantoin (Gh). In this study, we now report an abasic (AP) site generation from guanine residue oxidation based on photocatalytic reactions. The study used Dickerson–Drew dodecamer DNA as the model oligo DNA for photocatalytic reactions, and the reactions were analyzed using denaturing polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry measurements. It was observed that guanine residues are converted into AP sites by reacting with singlet oxygen generated from the photocatalyst. Guanine residues with high solvent accessibility were found to be particularly reactive with singlet oxygen. Mechanistic investigations revealed that the photocatalytic generation of AP sites proceeds mainly via an 8-oxoG-independent mechanism. These findings are important for the design and development of photocatalyst-modified functional oligo probes and the understanding of their reactions.

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Subject terms: Nucleic acids, DNA, Photocatalysis

Oxidative stress from light and inflammation can damage nucleic acids, with guanine being particularly vulnerable to oxidation. Here, the authors demonstrate that guanine residues in DNA can be converted into abasic sites through photocatalytic reactions with singlet oxygen, advancing our understanding of DNA damage mechanisms and offering insights for designing photocatalyst-modified oligo probes.

Introduction

Chemical modification of oligonucleotides (ONs) has enabled the creation of artificial nucleic acids with diverse properties and functions. Among them, functional ONs conjugated with photoresponsive molecules exhibit intriguing functions such as photoreaction1–4, photo-induced electron transfer5,6, energy transfer7–9, and singlet oxygen generation10–12 in a spatiotemporal-controlled manner. Despite the numerous successful applications of photoresponsive ONs, it is also well-known that nucleic acids can suffer considerable photodamage depending on the wavelength of the irradiated light and the characteristics of the coexisting or modifying functional molecules13–16. These lesions can hinder or impair the functionality of photoresponsive ONs. Therefore, it is essential to gain a thorough understanding of the types of lesions that can occur and the conditions under which they arise.

When focusing on photooxidation lesions among many photo-induced damages, there is no doubt that guanine, which has the lowest redox potential (1.29 V) among the four canonical nucleobases, is one of the most susceptible to oxidative lesion formation17. Photooxidation of guanine, mediated by exogenous or endogenous photosensitizers, has been extensively studied over the past several decades owing to the structural diversity and biological significance of the resulting lesions (Fig. 1a). Representative guanine oxidation lesions include 8-oxo-7, 8-dihydroguanine (8-oxoG)18–21, spiroiminodihydantoin (Sp)22, guanidinohydantoin (Gh)23, dehydroguanidinohydantoin (DGh)24, 2,5-diamino-4H-imidazol-4-one (Iz), and its hydrolysis product 2,2,4-triamino-5-(2H)-oxazolone (Oz)18,25. In Type I oxidation, photoinduced electron transfer between the photosensitizer and nucleic acid generates a guanine radical cation26,27, which can be converted to various lesions, such as 8-oxoG, Sp, Gh, Iz, and Oz. Free radicals, which are highly oxidizing reactive oxygen species (ROS) that can be generated by Type I oxidation, also form 8-oxoG, Iz, and Oz lesions by reacting with guanine. In contrast, Type II oxidation, involving another ROS: singlet oxygen, primarily leads to the formation of Sp and Gh. DGh is predominantly formed via further oxidation of 8-oxoG. Additionally, 8-oxoG can also be the main product of Type II oxidation in the presence of reducing agents such as glutathione21. Lesion formation based on photo-induced electron transfer can also arise when the proximity effect occurs between the photosensitizer and the nucleic acid26,27. Some oxidized nucleobases can form inter- or intra-strand crosslinks through oxidative processes28–30, and similar lesions may also be generated by photooxidation. Although some oxidatively generated lesions have been detected in vivo and possess high mutagenicity, repair enzymes primarily excise and remove these lesions31–33. These repair pathways minimize the risk of diseases associated with the formation of guanine oxidation lesions.

Fig. 1. Oxidatively generated lesions.

Fig. 1

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a Representative oxidatively generated lesions of guanine. b Enzymatic generation of abasic (AP) sites. c Representative crosslink lesions formed from AP sites.

Abasic (AP) sites are a distinct class of lesions owing to their high genotoxicity34,35. They arise from the hydrolysis of N-glycosidic bonds in purine nucleotides under acidic conditions or via enzymatic reactions in the base excision repair (BER) pathway (Fig. 1b)36–38. Notably, AP sites can form crosslinking structures by reacting with exocyclic amino groups of purine bases in complementary DNA strands (Fig. 1c)39,40 or amino acid residues in proteins41,42. Moreover, AP sites serve as scaffolds for introducing functional molecules into nucleic acids43–46 and crosslinking duplexes47,48. Recent research has focused on developing innovative methodologies for recognizing49,50, reacting51, or sequencing52 AP sites containing DNA strands.

In previous work, we developed photocatalyst-tethered ON probes for photochemical labeling of nucleic acids53. Under photoirradiation in the presence of a labeling agent, the desired labeling reaction occurred. However, photoirradiation in the absence of a labeling agent resulted in complex lesions, including AP sites, as a side product. While further analysis was challenging owing to the complexity of our previous system, those findings provided an important clue that AP sites could be generated as significant lesions via photocatalytic oxidation reactions. Crucially, such AP site generation has likely been overlooked in previous studies of oxidative DNA damage. The conventional methodology has relied on high-performance liquid chromatography (HPLC) purification of damaged nucleoside monomers after enzymatic digestion, utilizing UV absorption for detection. However, unlike other oxidative lesions, AP sites lack the aromatic nucleobase required for UV absorption around 260 nm. Consequently, they are effectively invisible in standard HPLC profiles, making them inherently difficult to detect in such workflows.

In this study, to verify the occurrence of this intriguing phenomenon and clarify the details of the mechanism, we decided to investigate it using a much simpler system. We employed an oligomer-based analysis approach to ensure that these lesions were captured without loss. Specifically, we designed a system with a self-complementary oligo DNA duplex. Denaturing polyacrylamide gel electrophoresis (PAGE), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF MS), and ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC/QTOF–MS) were utilized to analyze the lesions. Notably, we found and confirmed that oligo DNA undergoes photooxidative depurination of guanines to generate AP sites as one of the major lesions when irradiated with light in the presence of a photocatalyst such as Rose Bengal.

Results and discussion

Discovery of AP site generation in Dickerson–Drew dodecamer DNA (DD–DNA) under photocatalytic reactions

First, we evaluated DNA damage under photocatalytic reactions using a simplified system. For this purpose, DD–DNA, a self-complementary DNA oligomer whose crystal structure has been elucidated and that forms a typical B-type duplex54, was selected as a model sequence. In the presence of free photocatalysts such as Ruthenium tris(bipyridine) complex (Ru complex), Eosin Y (ESY), or Rose Bengal (RB), DD–DNA was exposed to light with a wavelength close to the absorption maximum of each photocatalyst: 455, 505, or 540 nm (Fig. S1). The irradiated aliquots were then analyzed by denaturing polyacrylamide gel electrophoresis (PAGE). Notably, 120 s of irradiation resulted in the near-complete disappearance of the starting DD–DNA band (Fig. 2). In its place, two groups of bands with different mobilities compared to DD–DNA (oxidatively generated lesions 1 and 2) were generated. Similar gel profiles were obtained regardless of the photocatalyst used. Because RB exhibited the highest reactivity, we decided to use the DD–DNA and RB combination for further analysis of the reaction.

Fig. 2. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of photocatalytic reactions with DD–DNA.

Fig. 2

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Photocatalytic reactions were performed with 100 μM DD–DNA (50 µM duplex) and 50 μM photocatalyst in a 20 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 10% DMSO at 0 °C. Each aliquot was irradiated with LED light corresponding to the absorption wavelength of each photocatalyst (455, 505, or 540 nm) for varying durations (0–120 s). The gel was stained with SYBR gold. After irradiation, two types of slower-mobility bands (oxidatively generated lesions 1 and 2) were observed on the gel, and similar band profiles were observed for all photocatalysts.

To characterize the oxidatively generated lesions in detail, MALDI–TOF MS analysis was performed. After the photocatalytic reaction, aliquots of DD–DNA were purified, desalted, and subjected to analysis. MALDI–TOF MS analysis of an aliquot of DD–DNA after 120 s of irradiation provided valuable insights into the lesions generated (Fig. 3). The peak corresponding to intact DD–DNA (Calcd. [M–H]− 3643.6) was absent from the MS spectrum (Fig. 3b), indicating near-complete consumption, consistent with the results obtained from denaturing PAGE. In its place, peaks corresponding to DD–DNA containing guanine lesions such as Gh and/or Sp, as shown in Fig. 3a, were observed. Additionally, DD–DNA species in which one or two guanine residues were converted to AP sites were detected as major peaks, suggesting that guanine residues in DD–DNA undergo not only established types of oxidations such as Sp and Gh formation but also considerable depurination leading to AP site formation under photocatalytic reaction conditions. These main products likely correspond to the major bands observed in the PAGE analysis (oxidatively generated lesions 2 in Figs. 2 and S2).

Fig. 3. MALDI–TOF MS analysis of photocatalytic reactions with DD–DNA and Rose Bengal (RB).

Fig. 3

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a DD–DNA sequence used in the experiment and representative lesions observed in MALDI–TOF MS analysis. b, c MALDI–TOF MS spectra after 540 nm LED light irradiation for 120 s. DD–DNA containing AP sites and/or guanine lesions such as Gh and Sp were detected as major peaks. d Time course of AP site generation in DD–DNA under photocatalytic reaction conditions. The generation ratio was calculated from the peak areas in the MALDI–TOF MS spectra (see supporting information for details). Photocatalytic reactions were performed with 100 μM DD–DNA (50 µM duplex) and 50 μM RB in a 20 mM phosphate buffer (pH 7.0) containing 100 mM NaCl and 10% DMSO at 0 °C. Aliquots were irradiated with 540 nm LED light for varying durations (0–120 s).

In contrast, the product corresponding to the gel band with the lowest mobility (oxidatively generated lesions 1 in Fig. 2) is hypothesized to be cross-linked DD–DNA owing to the considerable band shift observed. This cross-linked DD–DNA may arise from the reaction between a G lesion or AP site and an exocyclic amino group of a guanine residue in the complementary strand39,40. To further validate this assignment, we analyzed the photocatalytic reaction of single-stranded DNAs by denaturing PAGE. Notably, no low-mobility bands corresponding to oxidative lesion 1 were observed in the single-stranded system (Fig. S3), confirming that these species are indeed interstrand crosslinked products that specifically form within the duplex structure.

Additionally, no band shift downstream from DD–DNA was observed on PAGE, and no peaks with a smaller mass-to-charge ratio than AP sites-containing DD–DNA were detected on the MALDI–TOF MS chart (Fig. 3C). This suggests that double-strand breaks, or strand cleavage mediated by AP sites, are not major reactions in this system. To further validate these findings and exclude the possibility that the observed AP sites were artifacts generated during sample preparation (e.g., lyophilization or mixing with matrices) or the MALDI-TOF MS ionization process, we performed analysis using UPLC/QTOF–MS, which employs electrospray ionization (ESI), a softer ionization method. Aliquots containing DD-DNA and RB, taken before and after photoirradiation, were desalted using Amicon® Ultra 3 K centrifugal filters, diluted with water, and analyzed by UPLC/QTOF–MS. In the pre-irradiation samples, the peak corresponding to intact DD-DNA was observed as the sole major peak, with no detectable AP sites (Fig. S4). In contrast, analysis of the aliquots after 120 s of irradiation revealed major peaks corresponding to DD-DNA sequences containing AP sites, Sp (observed as two diastereomers), and Gh. Additionally, a minor peak assigned to a sequence containing both an AP site and a Gh lesion was detected. The observed m/z values for the doubly charged ions ([M-2H]²⁻) of these species were in excellent agreement with the calculated monoisotopic m/z values with high mass accuracy (mass error <6 ppm; see Fig. S4 for detailed values). These UPLC/QTOF–MS results clearly confirm that the AP sites are generated primarily by the photocatalytic reaction.

To quantitatively assess AP site generation under photocatalytic reaction conditions, we calculated the AP site generation ratio from MALDI–TOF MS analysis. The substantial reduction in MS value of DD–DNA upon depurination of guanine enabled us to differentiate between DD–DNA species containing no AP sites, 1 AP site, and 2 AP sites. In negative mode MALDI–TOF MS measurements, many oligo DNAs exhibited peaks corresponding to not only [M-H]− but also adducts with salt, such as [M-2H+Na]− or [M-2H + K]− even after desalting treatment. Based on these points and the classification of the obtained peaks, most of the detectable peaks could be identified (Fig. S5). The ratio of AP site generation in DD-DNA was calculated by dividing the sum of peak areas assigned to sequences containing AP sites by the sum of all detectable peak areas in the m/z value range from 3350 to 3800. The validity of quantifying the AP site generation ratio by peak area was confirmed through MALDI–TOF MS measurements of samples containing specific ratios of DD–DNA and DD–DNA–dS, where the terminal G in the DD–DNA sequence was replaced with an AP site-like spacer (Fig. S6).

The AP site generation ratio for each irradiation time was calculated from three technical replicates of MALDI–TOF MS measurements (including Fig. S7). The average values are shown in Fig. 3d. Before irradiation, intact DD-DNA and its salt adducts were primarily observed in the MS chart. The AP site generation ratio was 4% (Fig. 3d, 0 s), which is likely attributable to the minor depurination that occurs during the laser ionization process of the MALDI-TOF MS analysis55. After 120 s of irradiation, the 1 AP site and 2 AP site generation ratios were 30% and 1%, respectively. The AP site generation ratio increased with increasing irradiation time (Fig. 3d, 10–120 s), confirming that AP sites were indeed generated by the photocatalytic reaction. When DD–DNA was irradiated with 455 or 505 nm light for 120 s in the presence of Ru complex or ESY, respectively, and analyzed by MALDI–TOF MS, similar MS profiles to those obtained with RB were observed (Fig. S8), indicating similar lesion generation with all three photocatalysts.

We also investigated the pH dependence of the AP site and lesion generation (Fig. S9). DD–DNA was irradiated for 120 s in the presence of RB in buffers ranging from pH 6.0–8.0, and the irradiated aliquots were analyzed by MALDI–TOF MS. Experiments at pH 6.0 yielded Gh-containing DD–DNA as a major peak and Sp-containing DD–DNA as a minor peak, whereas experiments at pH 8.0 gave Sp-containing DD–DNA as a major peak. This result is consistent with previous studies demonstrating that Gh lesion formation is favored under acidic conditions, while the Sp lesion predominates at higher pH values56, supporting the validity of our lesion assignments. Notably, the AP site generation ratio remained relatively constant across the pH range studied.

Position of AP site generation in the DD–DNA based on photocatalytic reactions

Next, the position of AP site generation in DD–DNA was investigated. To identify the positions of oxidized guanine lesions, including AP sites, in DD–DNA, we used hot piperidine treatment. Oxidized guanine lesions undergo depurination upon treatment with hot piperidine, resulting in strand cleavage via the formation of a Schiff base intermediate (Fig. S10)57. The rate of this cleavage reaction is known to vary depending on the type of oxidized guanine lesion, with AP sites being cleaved more rapidly than other lesions. In other words, short-term hot piperidine treatment is expected to provide information primarily on the position of AP sites. Specifically, aqueous piperidine (1 M) and 2-mercaptoethanol (BME) (0.25 M) were directly added to the aliquot after 120 s of light irradiation, followed by incubation at 90 °C and analysis by MALDI–TOF MS. The MS spectrum obtained after 1 min of hot piperidine treatment revealed peaks corresponding to uncleaved AP sites, Gh, and/or Sp-containing DD–DNA, indicating that the incubation time was insufficient (Fig. S11). On the one hand, after 5 min of hot piperidine treatment, only a minor uncleaved peak was observed for the AP site-containing DD–DNA, suggesting that most of it had been cleaved (Fig. S12). On the other hand, the DD–DNA containing Gh or Sp lesion still showed a clear peak because these lesions have been reported to be relatively slow in hot piperidine-mediated cleavage reactions57. Focusing on the cleavage fragments, the major fragment peak corresponded to 5ʹ-CGCGAATTCGCp-3ʹ (calcd. [M–H]− 3394.5). This result indicates that the AP sites are primarily derived from terminal G. A minor peak assignable to 5ʹ-CGCGAATTCp-3ʹ (calcd. [M–H]− 2776.4) or 5ʹ-pCGAATTCGCp-3ʹ (calcd. [M–H]− 2856.4) was also detected, suggesting that extended irradiation time enables the conversion of internal G to AP sites. Additionally, fragments with other guanine lesions, such as 5ʹ-pCGAATTCGC-Gh-3ʹ (calcd. [M–H]− 3112.5) and 5ʹ-pCGAATTCGC-Sp-3ʹ (calcd. [M–H]− 3137.5), were also present among the minor peaks. No fragments containing an intact terminal G were detected. When the incubation time was extended to 10, 30, and 60 min, the peak corresponding to DD–DNA containing Gh lesions nearly disappeared, and the peak of DD–DNA containing Sp lesions became smaller than the main fragment peak, indicating that these lesion-containing DD–DNAs were also cleaved (Figs. S12, 13). The major fragment under these conditions was also 5ʹ-CGCGAATTCGCp-3ʹ. These results underscore that not only AP sites but also Gh and Sp lesions are generated primarily from terminal G.

Identification of reactive oxygen species that mainly contribute to AP site generation in DD–DNA by photocatalytic reactions

To investigate the involvement of oxygen in photocatalytic AP site generation, we performed the reaction under anaerobic conditions via argon (Ar) gas purging, following a previously reported procedure (Fig. S14a)58. Post-reaction aliquots were analyzed by denaturing PAGE and MALDI-TOF MS, and the results were compared with those obtained under aerobic conditions (without Ar gas purging). Denaturing PAGE analysis showed that the intensity of the shifted bands, which were prominent under aerobic conditions, was remarkably diminished in the Ar-purged samples (Fig. S14b). In the MALDI-TOF MS analysis, the peaks corresponding to AP site-containing DD-DNA were not observed as major peaks under anaerobic conditions, even after 120 s of irradiation (Fig. S14c). It was also confirmed that the AP site generation ratio via photocatalytic reaction was markedly decreased under Ar-purged conditions compared to aerobic conditions (Fig. S14d). These findings clearly indicate that the photocatalytic generation of AP sites in DD–DNA is an oxygen-dependent process mediated by reactive oxygen species (ROS).

To identify the major ROS responsible for the photocatalytic AP site generation in DD-DNA, we initially conducted photocatalytic reactions in the presence of sodium azide (NaN3), a known singlet oxygen scavenger59,60. As a result, AP site generation was markedly suppressed in the presence of NaN3, and the AP site generation ratio was comparable to that before irradiation (Figs. 4a and S15). An aliquot of DD–DNA following the photocatalytic reaction in the presence of NaN3 was also analyzed by denaturing PAGE. The results demonstrated that the shifted bands observed in the absence of NaN3 were nearly absent in the presence of NaN3 (Fig. 4a). Moreover, the addition of another singlet oxygen scavenger, 1,4-diazabicyclo[2.2.2]octane (DABCO)61, also reduced the AP site generation ratio to trace levels (Fig. S15). Conversely, the addition of mannitol, catalase or superoxide dismutase (SOD), which scavenge other representative ROS: hydroxyl radical, hydrogen peroxide or superoxide anion60,62, respectively, did not considerably suppress AP site generation (Fig. S15), confirming that these other ROS do not play a major role in the photocatalytic AP site generation observed here.

Fig. 4. Identification of reactive oxygen species that primarily contribute to AP site generation in DD–DNA under photocatalytic conditions.

Fig. 4

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a AP site generation ratio in DD–DNA in the presence of NaN3. The generation ratio was calculated based on peak areas in the MALDI–TOF MS spectra. The p-values were determined by the Welch's t-test. n.d. indicates that there was no significant difference. b Denaturing PAGE analysis of photocatalytic reactions in the presence of NaN3. Photocatalytic reactions were performed with 100 μM DD–DNA (50 µM duplex), 50 mM of NaN3, and 50 μM RB in a 20 mM phosphate buffer (pH 7.0) containing 100 mM NaCl at 0 °C. Aliquots were irradiated with 540 nm LED light for 120 s.

AP sites were also generated when DD–DNA was incubated with an endoperoxide (EP) reagent63, which quantitatively generates singlet oxygen upon heating at 35 °C (Fig. S16). Notably, this reaction was found to be more efficient in a deuterated buffer, which prolongs the lifetime of singlet oxygen10,61. These observations further support the notion that AP sites are primarily generated via the reaction with singlet oxygen (Type II oxidation). Considering the results on the position of AP sites, AP site generation in DD–DNA is thought to occur mainly through the reaction of highly solvent-accessible G residues, such as the terminal G, with singlet oxygen produced by the photocatalyst. Conversion of the terminal G to an AP site, Gh, or Sp lesion by initial irradiation changes the base-pairing stability64 and can increase the solvent accessibility of internal Gs, allowing extended irradiation to generate additional lesions at these internal sites (Fig. S17).

Possible mechanisms for AP site generation under the photocatalytic reaction

To investigate the mechanism of AP site generation in greater detail, we endeavored to elucidate the mechanism at the molecular level. To our knowledge, there is no detailed report on the mechanism of AP site generation via photocatalytic reactions. However, G lesions such as Gh and Sp have been extensively studied using G monomers and oligomers, and the generation mechanism of these lesions based on the reaction with singlet oxygen has been well-established19,56,65. According to previous studies, G primarily undergoes a Diels–Alder reaction with singlet oxygen to produce intermediate 1. The cleavage of the C–O bond in intermediate 1 leads to the formation of 8-peroxy intermediate 256. Owing to its high reactivity, intermediate 2 is dehydrated to form intermediate 3 (Fig. 5 pathway a), which then interacts with water to yield intermediate 456. The subsequent addition of water and decarboxylation of intermediate 4 result in the production of Gh, while acyl transition leads to Sp generation. Alternatively, intermediate 2 can be directly reduced to form 8-oxoG (Fig. 5 pathway b). Furthermore, the reaction of 8-oxoG with another singlet oxygen molecule also yields 4 through highly reactive intermediates 5 and 6, resulting in the formation of Sp and Gh19,65. Intermediate 6 could also lead to Iz, Oz or DGh lesion. In the photocatalytic reaction involving DD–DNA, there was no significant generation of AP sites based on the elimination of nucleobases other than G. Therefore, it is highly likely that the pathway for AP site generation shares partial commonality with the generation of G lesions and branches from any of the Type II oxidation intermediates 1–6.

Fig. 5. Lesions and AP site generation via the reaction with singlet oxygen.

Fig. 5

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The pathways for Gh and Sp generation from G have been previously reported, which involve intermediates 1–6.

While it is extremely challenging to fully determine the specific intermediate from which the AP site is generated, examining the role of 8-oxoG in the primary pathway for photocatalytic AP site generation in DD–DNA could yield important mechanistic insights. The MALDI–TOF MS chart in Fig. 3 shows no peaks corresponding to 8-oxoG-containing DD–DNA after 120 s of photoirradiation with 540 nm light. However, after irradiating DD–DNA for just 10 s in the presence of 5 μM RB, a peak associated with 8-oxoG-containing DD–DNA was detected (Fig. S18). Additionally, this peak vanished after 300 s of irradiation, and in its place, peaks corresponding to DD–DNA containing Gh, Sp, or AP sites emerged. These findings suggest that 8-oxoG contributes to the formation of at least one of these lesions in this system.

Motivated by this result, we designed the DD–DNA-8-oxoG (Fig. 6a), wherein the terminal G residue of the DD–DNA sequence is replaced by 8-oxoG. If AP sites are primarily produced from intermediates 5 and 6 in pathway b, then the AP site generation ratio for DD–DNA-8-oxoG, which bypasses the initial reaction step with singlet oxygen, should be considerably higher than that of DD–DNA. Conversely, if AP sites arise from initial intermediates 1, 2, or 3, substituting 8-oxoG for terminal G should lead to a substantial decrease in the AP site generation ratio. In the presence of RB, DD–DNA-8-oxoG was irradiated with 540 nm light for either 10 or 120 s, desalted, purified, and analyzed using MALDI–TOF MS. The results indicated that the intact DD–DNA-8-oxoG peak nearly vanished after 10 s of irradiation; however, peaks associated with AP sites were not detected as main peaks, and substantial AP site generation could not be confirmed. Instead, the terminal 8-oxoG was quickly transformed into lesions such as Gh and Sp (Fig. 6b). Previous studies have noted that intermediate 6 can also transform into the DGh lesion, and its mass spectrum is very similar to that of Gh24, suggesting that some main peaks may contain DGh rather than Gh. Alongside these major peaks, several minor peaks were also identified. These may represent damaged DD–DNA-8-oxoG related to the imidazolone (Iz) lesion and its hydrolysate, which have similarly been documented in the reactions of 8-oxoG with singlet oxygen66. This result clearly demonstrates that 8-oxoG and the downstream intermediates 4, 5, or 6 are not part of the main pathway for AP site generation. Furthermore, while the multi-step hydrolysis of Iz lesions can also lead to the formation of AP sites67, the considerable reduction in AP site generation rates following 8-oxoG substitution implies that Iz-derived AP site generation is not the primary mechanism in this system either. Extended irradiation time resulted in considerable AP site generation; however, the main peaks in the mass spectrometry chart included both Gh/Sp lesions and AP sites (Figs. 6c and S19). It is likely that the destabilization of the terminal base pairing owing to the transformation of the terminal 8-oxoG lesion allows singlet oxygen to access the internal G, leading to further AP site generation (Fig. 6d). These findings suggest that the generation of AP sites primarily occurs through the reaction of initial intermediates 1 or 2 with singlet oxygen during Type II oxidation. In other words, it is likely that AP sites are formed via direct depurination of intermediates 1 or 2 or their overoxidized derivatives (Fig. S20). To further refine this hypothesis, we compared our findings with the reported reactions of 1O2 analogues, 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), with G derivatives68. These dienophiles react with guanine to form transient [4 + 2] cycloaddition adducts equivalent to intermediate 1, which subsequently rearrange into stable 8-adducts. Importantly, no glycosidic bond cleavage or AP site formation has been reported in these reactions with 1O2 analogues. If depurination primarily originated from intermediate 1, similar glycosidic bond cleavage would be expected in the MTAD/PTAD systems as well. The absence of such side products suggests that the depurination pathway in our system likely branches from intermediate 2—a highly reactive hydroperoxide species—or its subsequent overoxidized species. Although the precise chemical details of this mechanism require further investigation, which is currently underway, this mechanistic analogy provides supportive evidence for our proposed pathway.

Fig. 6. The photocatalytic reaction involving 8-oxoG-modified DD–DNA (DD–DNA-8-oxoG) and Rose Bengal.

Fig. 6

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a The sequence of DD–DNA-8-oxoG used in the experiment. b MALDI–TOF MS chart obtained after 10 s of irradiation with a 540 nm LED light. The photocatalytic reactions were performed with 100 μM DD–DNA-8-oxoG (50 µM duplex) and 50 μM photocatalyst in a 20 mM phosphate buffer (pH 7.0) containing 100 mM NaCl at 0 °C. DD–DNA-8-oxoG featuring Gh and Sp was identified as the main peaks, while AP site generation was minimal. c The time course of AP site generation in DD–DNA-8-oxoG during the photocatalytic reaction. The generation ratio was determined from the peaks in the MALDI–TOF MS chart. The ratio and rate of AP site formation in DD–DNA-8-oxoG were considerably lower than those in unmodified DD–DNA. d Proposed mechanism for oxidatively generated lesions and AP site formation in DD–DNA-8-oxoG. The conversion of the terminal 8-oxoG to an oxidative lesion may enhance the solvent accessibility of the internal G, facilitating the generation of additional AP sites and G lesion formation.

AP site generation in various duplexes, single strands, and G4 DNA by photocatalytic reaction

To explore the sequence dependence of AP site generation through photocatalytic reactions, we first designed a sequence where the 5ʹ-CGCG segment of DD–DNA was changed to either CGGC or GGGG and subsequently examined the AP site generation ratio for each sequence (Figs. 7, S21, and S22). The results indicated that all sequences exhibited similar generation ratios to DD–DNA, suggesting that rearranging the sequences near the termini does not considerably impact the AP site generation ratio. Conversely, when a photoreaction was performed using a non-terminal G-C sequence—where TTT and AAA were added to both ends of DD–DNA—the AP site generation rate remained largely unchanged following photoirradiation (Figs. 7 and S23). Additionally, when G and C were added to both ends of the non-terminal G sequence, forming a terminal G–C sequence, the AP site generation ratio increased after the photoirradiation (Figs. 7 and S24). These findings further indicate that the terminal G, with its high solvent accessibility, plays a key role not only in the efficient formation of 8-oxoG–derived lesions69 but also in the effective generation of AP sites in duplex DNA. On the other hand, the non-terminal G-C sequence has a highly solvent-accessible terminal adenine (A); however, no sequences containing A-derived lesions were observed. This result is in good agreement with previous studies showing that A is inactive against 1O220,70, emphasizing that lesion formation, including the AP site in this system, is G-specific.

Fig. 7. AP site generation ratio in various duplexes via photocatalytic reaction.

Fig. 7

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The reactions were performed using a 100 μM DNA or RNA strand (50 µM duplex) and 50 μM RB in 20 mM phosphate buffer (pH 7.0) with 100 mM NaCl and 10% DMSO at 0 °C. Aliquots were irradiated with 540 nm LED light. The p-values were determined by the Welch's t-test. n.d. indicate that there was no significant difference.

In addition, the photocatalytic reaction of DD-DNA-11: 5’-CGCGAATTCGC-3’, DD-DNA lacking the terminal G, was tested (Fig. S25). Consequently, AP sites were generated after 120 s of irradiation; however, the generation ratio of DD-DNA-11 (12%) was significantly lower than that of DD-DNA (31%). This result emphasizes the importance of the terminal G for efficient AP site generation by photocatalytic reactions and suggests that the internal G near the terminal can also generate AP sites. Furthermore, it is worth considering whether specific functional groups at the termini, such as the free 5’-hydroxyl group of the 2-deoxyribose moiety, might facilitate the N-glycosidic bond cleavage via neighboring group participation. However, our results demonstrate that AP sites are generated efficiently regardless of whether the guanine residue is located at the 5’- or 3’-terminus. This indicates that the specific chemical involvement of terminal hydroxyl groups is not the primary driving force. Instead, this observation strongly reinforces the conclusion that the high solvent accessibility and structural flexibility inherent to the terminal positions are the dominant factors facilitating the efficient reaction with singlet oxygen.

Given that the glycosidic bond in RNA is stronger than that in DNA, it is assumed that depurination occurs less frequently in RNA than in DNA71. Consequently, research on AP sites in RNA is notably limited compared to DNA72, leaving this area largely unexplored. We investigated whether AP sites can also form in RNA under photocatalytic oxidation conditions. When we used DD–RNA, which has an RNA backbone in place of DD–DNA, the generation ratio was lower than that observed in DD–DNA; however, significant AP site generation was confirmed following light irradiation (Figs. 7 and S26). This finding suggests that AP sites can also represent a major type of lesion in RNA under photocatalytic oxidation conditions.

We also examined photocatalytic AP site generation in single-stranded DNA. The DNA sequences C11-G: 5’-CCCCCCCCCCCG-3’ with one terminal G and C6-G-C5: 5’-CCCCCCGCCCCC-3’ with an internal G were designed. The photocatalytic reaction of each single-stranded DNA was performed under the same conditions as those for the duplex DNA. Consequently, C11-G (22%) generated AP sites more efficiently than C6-G-C5 (8%) after 120 s of irradiation (Fig. S27), suggesting that the terminal G is a hot spot for AP site generation even in single-stranded DNA.

Finally, we investigated the potential for generating AP sites in DNA structures other than duplex and single-strand under photocatalytic oxidation conditions. For this purpose, we used the human telomere (HT) G4 DNA sequence, known to form a hybrid G4 structure in the presence of K+ and an anti-parallel G4 structure with Na+. We performed photocatalytic reactions with each salt (Figs. S28 and S29). Notably, the generation ratio of AP sites in HT G4 DNA following the photocatalytic reaction with K+ was found to be higher than that observed with Na+ (Fig. S30a). Conversely, the photocatalytic generation ratio of AP sites in DD–DNA in K+ phosphate buffer for hybrid G4 formation showed no considerable difference compared to that in the standard phosphate buffer used in this study (Fig. S31 and 3). These findings show that the increased AP site generation in HT G4 when K+ is present is attributable to changes in the solvent accessibility of G residues owing to differences in topology, rather than the influence of the cation itself. Furthermore, this AP site generation ratio surpasses that of all duplex DNA examined to date, with a higher proportion of sequences containing multiple AP sites observed after irradiation. This indicates that certain G quartet-forming G residues, which are highly solvent-accessible, can be effectively converted to AP sites by singlet oxygen (Fig. S30b).

Conclusions

In this study, we discovered the phenomenon of AP site generation when DD–DNA was irradiated with light in the presence of a photocatalyst such as RB. The evaluation of AP site generation was performed by analyzing the peaks in the MALDI–TOF MS chart, allowing for a quantitative calculation of the AP site generation ratio in oligo DNA. It is important to note that not only AP sites but also other oxidatively generated lesions, such as Gh and Sp, can be observed and easily analyzed without the need for enzymatic digestion. It was surprising to discover that the AP site was generated at a ratio comparable to that of the well-known Gh and Sp. This comparison was primarily enabled by the MALDI–TOF MS analysis. In previous studies, oxidized DNA was typically examined only after enzymatic digestion and isolation via high-performance liquid chromatography. Consequently, this kind of AP site generation may have been overlooked in previous studies.

Treatment with hot piperidine following the photocatalytic reaction, along with photocatalytic reactions using various duplexes, revealed that Gs with high solvent accessibility are hotspots for AP site generation. Verification with various reactive oxygen species scavengers indicated that this photocatalytic AP site generation was primarily mediated by singlet oxygen derived from the photocatalyst. Experiments involving 8-oxoG suggested that AP sites are formed not via 8-oxoG, but rather from initial intermediates 1 or 2, or their overoxidized species. However, the proposed mechanism is still speculative, and further studies are required to determine a more precise mechanism. Additionally, AP sites were efficiently generated in the hybrid HT G4 DNA during the photocatalytic reaction. Furthermore, AP site formation occurs not only in DNA but also in RNA. Given that RNA can adopt various higher-order structures, it is likely that further hotspots may be identified in the future. When using photo-crosslinking or photo-labeling techniques on RNA, precautions should be taken to minimize the generation of AP sites owing to unintended oxidation.

Our findings shed light on the specific locations and mechanisms through which AP sites are generated via photocatalytic reactions. This knowledge will greatly assist in the design and development of photocatalyst-modified functional oligo probes that exhibit fewer side reactions, as well as enhance our understanding of their functions and interactions.

Materials and methods

General

General chemicals were purchased from Fujifilm Wako Pure Chemical, Tokyo Chemical Industry, Kanto Chemical, Aldrich, or Nacalai Tesque. Target DNA and RNA sequences were obtained from JBioS (Japan). Lyophilization was performed using LABCONCO FreeZone 2.5.

Experimental procedures

Photoreaction for denaturing polyacrylamide gel electrophoresis analysis

Aliquots (2 μL) of DNA duplex (50 μM), photocatalyst (RB, ESY, or Ru complex) (50 µM), phosphate buffer (20 mM, pH 7.0), NaCl (100 mM), and 10% DMSO were prepared. For the photoreaction, these aliquots in 0.5 mL tubes were irradiated with 540, 505, or 455 nm light by Twin LED Light (Relyon, for RB at 540 nm 37.0 ± 7.0 mW cm−2) at 0 °C. The distance between the lamp and the sample was 30 mm. After irradiation for varying durations (0–5 min), loading buffer (2.0 μL, 95% formamide, 50 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0) was added to the mixtures. PAGE was performed using a 16% acrylamide gel containing 6.0 M urea, 20% formamide, and 1× TBE at 250 V for 120 min. The gel was stained with SYBR gold, and the ODNs were visualized using an FLA-5100 (Fujifilm Co.) and a ChemiDoc™ Touch Imaging System (Bio-Rad).

Photoreaction for MALDI–TOF MS analysis

Aliquots (20 μL) of DNA duplexes or G4 DNA (50 μM), photocatalyst (RB, ESY, or Ru complex) (50 µM), phosphate buffer (20 mM, pH 5.8–7.0), NaCl or KCl (100 mM), and 10% DMSO were prepared. For the photoreaction, the aliquot was divided into 10 tubes, with 2 μL of solution added to each tube. The tubes were then irradiated with 540, 505, or 455 nm light by Twin LED Light (Relyon) at 0 °C. The distance between the lamp and the sample was 30 mm. The irradiated aliquots in the 10 tubes were combined, and double-distilled water (dd-H2O) (80 µL) was added. Prior to MALDI–TOF MS measurements, this 100 μL aliquot was purified using a NAPTM-5 column (Cytiva) and desalted with a Sep-Pak C18 Plus short cartridge (WatersTM) followed by lyophilization. The dry compound was dissolved in dd-H2O (10 µL) and passed through a Zip-Tip® pipette tip (Millipore) to prepare the DNA sample. The MALDI–TOF MS spectrometry matrix was prepared by mixing di-ammonium hydrogen citrate (5 mg/50 µL) in H2O and 3-hydroxypicolinic acid (25 mg/500 µL) in 1:1 H2O:CH3CN (v/v). A matrix (1 µL) was spotted on a MALDI–TOF MS sample plate, and a DNA sample (1 µL) was applied on top of the matrix. The samples were dried at room temperature and analyzed by MALDI–TOF MS. MALDI–TOF MS analysis was performed using a Bruker Autoflex Speed instrument. The AP site generation ratio was calculated from three technical replicates of MALDI–TOF MS measurements.

Photoreaction for UPLC/QTOF–MS analysis

Aliquots (20 μL) of DD-DNA duplex (50 μM), Rose Bengal (50 µM), phosphate buffer (20 mM, pH 7.0), NaCl (100 mM), and 10% DMSO were prepared. For the photoreaction, the aliquot was divided into 10 tubes, with 2 μL of solution added to each tube. The tubes were then irradiated with 540 nm light by Twin LED Light (Relyon) at 0 °C. The distance between the lamp and the sample was 30 mm. The irradiated aliquots in the 10 tubes were combined, and dd-H2O (480 µL) was added. Prior to UPLC/QTOF–MS analysis, this 500 μL aliquot was desalted using an Amicon® Ultra 3 K centrifugal filter (Millipore). The sample was centrifuged at 14,000 × g for 25 min at 25 °C, followed by the addition of 400 μL of dd-H2O and further centrifugation (14,000 × g, 25 min, 25 °C). This washing cycle was repeated twice. The final DNA concentration was adjusted to 0.5 μM for UPLC injection. UPLC analysis was performed on an ACQUITY UPLC system (WatersTM) using an Oligonucleotide BEH C18 Column (130 Å, 1.7 µm, 2.1 × 50 mm) with a 5 µL injection volume. The mobile phase consisted of solvent A (15 mM TEA and 400 mM HFIP, pH 7.9) and solvent B (solvent A /MeOH = 1/1, v/v). A linear gradient of 35% to 70% solvent B was applied over 10 min at a flow rate of 0.2 mL/min and a column temperature of 40 °C. MS measurements were conducted using a Xevo G2-XS Qtof mass spectrometer in ESI negative resolution and continuum mode. The mass range was set from 450 to 3000 Da, with Leucine-enkephalin (LE) used as the internal standard. The source parameters were as follows: cone voltage, 40 V; capillary voltage, 4 kV; desolvation temperature, 600 °C; and desolvation gas flow, 700 L/h.

Hot piperidine treatment and purification of photoirradiated aliquots

The irradiated and collected duplex DNA aliquot (2 μL × 10 tubes) was prepared using the same procedure as described above. Aqueous solution of piperidine (2 M) and β-mercaptoethanol (BME, 0.5 M) (20 μL) was added to the aliquot. This mixed solution was incubated at 90 °C for 5–30 min, and dd-H2O (60 µL) was added. This 100 μL solution was purified and desalted using a NAPTM-5 column, Sep-Pak C18 Plus short cartridge, and Zip-Tip® pipette tip in the same manner as above. The purified sample was subsequently analyzed by MALDI–TOF MS.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Transparent Peer Review File (717.8KB, pdf)
Supplementary Information (5.5MB, pdf)
Reporting Summary (7.6MB, pdf)

Acknowledgements

We thank Miyuki Aihara for assistance with sample preparation for MALDI-TOF MS analysis, Yuri Tagawa for UPLC/QTOF-MS measurements, and Rika Kimura for administrative support. We thank the Tagen Central Analytical Facility and the support program for young researchers from the Tohoku University Technical Support Center for supporting MALDI-TOF MS measurement. This work was supported in part by the Japan Science and Technology Agency (JST) FOREST program (No. JPMJFR2002 to K.O.); Grant-in-Aid for Scientific Research (B) (No. JP24K01641 to K.O. and No. JP23H02076 to F.N.), Scientific Research on Transformative Research Areas (A) “Biophysical Chemistry for Material Symbiosis” (No. JP23H04051 to K.O.), JSPS Fellows (No. JP22J01195 to Y.Y.), and Early-Career Scientists (No. JP22K14792 to Y.Y.) from the Japan Society for the Promotion of Science (JSPS); and the research program of the Crossover Alliance to Create the Future with People, Intelligence and Materials from MEXT, Japan.

Author contributions

K.O. and Y.Y. designed the experiments. Y.Y., O.A., M.S., A.M.A., and S.S. performed experiments. Y.Y. and K.O. mainly wrote the manuscript. K.O. and F.N. mentored the research. All authors discussed the results and provided feedback on the study and manuscript.

Peer review

Peer review information

Communications Chemistry thanks Jialong Jie, Jean-Luc Ravanat and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The raw data that support the findings of this study are available in the Figshare repository at 10.6084/m9.figshare.31436068.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yuuhei Yamano, Kazumitsu Onizuka, Fumi Nagatsugi.

Contributor Information

Yuuhei Yamano, Email: yuuhei.yamano.c4@tohoku.ac.jp.

Kazumitsu Onizuka, Email: onizuka@tohoku.ac.jp.

Fumi Nagatsugi, Email: nagatugi@tohoku.ac.jp.

Supplementary information

The online version contains supplementary material available at 10.1038/s42004-026-01979-8.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Transparent Peer Review File (717.8KB, pdf)
Supplementary Information (5.5MB, pdf)
Reporting Summary (7.6MB, pdf)

Data Availability Statement

The raw data that support the findings of this study are available in the Figshare repository at 10.6084/m9.figshare.31436068.

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