J. Phys. Chem. B 2010, 114, 10195–10199
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Charge Separation and Photosensitized Damage in DNA Mediated by Naphthalimide, Naphthaldiimide, and Anthraquinone Kiyohiko Kawai,* Yasuko Osakada, Eri Matsutani, and Tetsuro Majima* The Institute of Scientific and Industrial Research (SANKEN), Osaka UniVersity, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan ReceiVed: March 19, 2010; ReVised Manuscript ReceiVed: June 18, 2010
Charge-separation and charge-recombination dynamics and oxidative DNA degradation were investigated for DNA modified with a photosensitizer (Sens) naphthalimide (NI), naphthaldiimide (ND), or anthraquinone (AQ). In all three Sens-modified DNA systems, the formation of long-lived charge-separated states was observed in which the lifetime increased with increasing numbers of A-T base pairs between Sens and the neighboring G-C base pair. The lifetime of the charge-separated state correlated well with the DNA damage yield, indicating that the charge-separated state provides time for the irreversible DNA oxidative damage to occur. The quantum yield of DNA damage was the lowest for ND-modified DNA due to the slow reaction of ND radical anion with molecular oxygen; the process needed to preclude charge recombination. The AQ-modified DNA resulted in the highest charge separation and subsequent DNA damage yield, which would be partly explained by the formation of the spin-forbidden triplet radical ion pairs during charge separation. Introduction Upon exposure of our skin to solar UV radiation, DNA damage takes place through two major pathways: (i) the formation of mutagenic pyrimidine dimers via direct excitation of the pyrimidine bases, and (ii) photoexcitation of a photosensitizer (Sens), which triggers the oxidative damage of nucleobases. Since the nucleobases do not absorb appreciably above 300 nm, the latter pathway become more pronounced when considering UV irradiation with a wavelength longer than 300 nm. The mechanisms of the photosensitized oxidatively generated DNA damage involve oxidation through electron transfer from DNA to the Sens in the singlet or triplet excited state (mechanism type I), as well as oxidation mediated by singlet oxygen (mechanism type II), which is formed through energy transfer from the photoexcited Sens to the molecular oxygen (O2).1,2 Since the lifetimes of Sens in the excited state or of singlet oxygen are usually short in living cells, Sens must be located in the vicinity of DNA for the photosensitized DNA damage to occur. And when the Sens is bound to DNA, not only the type II process but also the type I process can take place because, for many of the well-studied Sens causing DNA damage, electron transfer from DNA to the photoexcited Sens proceeds rapidly.3-9 Hence, it is important to understand the kinetic mechanisms of the photosensitized one-electron oxidation of DNA, the type I process.10,11 The photoirradiation of DNA-bound Sens triggers electron transfer from nucleobases to the excited Sens to produce the radical anion of the Sens (Sens•-) and the radical cation of the nucleobase (hole) in a charge-separated state. Since guanine (G) exhibits the lowest oxidation potential among the four DNA bases,12-14 one-electron oxidation of DNA leads to the generation of a radical cation of G (G•+) and subsequent formation of G oxidative products.15-20 For photosensitized DNA damage * To whom correspondence should be addressed. Tel.: +81-6-6879-8496 (K.K.), +81-6-6879-8495 (T.M.). Fax: +81-6-6879-8499 (K.K., T.M.). E-mail:
[email protected] (K.K.),
[email protected] (T.M.).
by the one-electron-oxidation mechanism to occur, the processes leading to the DNA damage must take place faster than the charge recombination, a process that protects DNA from oxidative degradation.3,7 By using DNA in which naphthalimide (NI) or naphthaldiimide (ND) is covalently attached to A-stretch sequences as a Sens, we have previously reported that, in the initial step of the photosensitized one-electron oxidation of DNA, before a hole is trapped at G, the rapid hole transfer between As helps to separate the Sens•- and a hole causing DNA damage by providing time for G•+ and/or Sens•- to react with water or O2.21-24 Consequently, consecutive A sequences have been demonstrated to serve as good targets in photosensitized DNA damage, or G adjacent to such sequences may be a potential hot spot of oxidatively generated DNA damage.21-24 In this study, we expanded on these studies to investigate the Sens anthraquinone (AQ), which is most widely used to explore DNA damage caused via long-range hole-transfer processes.10,25-32 A series of DNA modified with NI, ND, and AQ were synthesized, and the relation between charge-separation and charge-recombination kinetics and DNA damage processes were investigated by a combination of the transient absorption measurements using the laser flash photolysis and quantitative HPLC analyses of DNA damage. Similar to NI and ND, AQ caused oxidative G degradation via the formation of a longlived charge-separated state in DNA. Among the three examined Sens, AQ-modified DNA showed the highest charge separation and DNA damage yield. This would be partly explained by the rapid intersystem crossing and subsequent charge separation via AQ in the triplet excited state for AQ-modified DNA, forming the spin-forbidden triplet radical ion pairs.33,34 Materials and Methods DNA Synthesis. NI-, ND-, and AQ-modified DNAs were synthesized as previously reported.8,35-37 All other reagents for DNA synthesis were purchased from Glen Research. DNA were synthesized on an Applied Biosystems A3400 DNA synthesizer and purified by reverse phase HPLC and lyophilized. All the NI-, ND-, and AQ-modified DNA studied here were character-
10.1021/jp102483k 2010 American Chemical Society Published on Web 07/16/2010
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ized by complete digestion with nuclease P1 and AP to 2′-deoxyribonucleosides. Duplex solutions (20 mM sodium phosphate buffer (pH 7.0)) were prepared by mixing equimolar amounts of the desired DNA complements and gradually annealing with cooling from 80 °C to room temperature. Laser Flash Photolysis. The nanosecond transient absorption measurements were performed by using the laser flash photolysis technique.21-24,38-41 Briefly, the third-harmonic oscillation (355 nm, fwhm of 4 ns, 6 mJ/pulse) from a Q-switched Nd:YAG laser (Continuum, Surelight) was used for the excitation light which was expanded to 1 cm diameter. The light from a xenon flash lamp (Osram, XBO-450) was focused into the sample solution for the transient absorption measurement. Time profiles of the transient absorption in the UV-visible region were measured with a monochromator (Nikon, G250) equipped with a photomultiplier (Hamamatsu Photonics, R928) and digital oscilloscope (Tektronics, TDS-580D). The quantum yield of the formation of the charge-separated state (ΦCS) was determined from the transient absorption of the triplet benzophenone as an actinometer during the 355 nm laser flash photolysis. The time profiles were obtained from the average of 32 laser shots. DNA Damage Quantification. Photoirradiation was carried out in an aqueous solution under air containing 30 µM DNA (strand conc.) and 20 mM pH 7.0 Na phosphate buffer. The solution mixture was photoirradiated with a mercury lamp (Asahi Spectra REX-120) equipped with 350 nm filter. The reaction mixture was subjected to enzymatic digestion with P1 nuclease and alkaline phosphatase. The consumption of G was quantified by reverse-phase HPLC using A as internal standards. For the experiments in Figure 4, DNA damage yield was described as -G %/min which was measured as the average of three measurements. Measurement of the Quantum Yield of DNA Damage (Consumption of G (Φ-G)). Φ-G was measured using NITTTTTCGCGC/AAAAAGCGCG (Φ-G ) 3.3 × 10-3) as a chemical actinometer.23 Photoirradiation was carried out in an aqueous solution containing 40 µM DNA (strand concentration) and 20 mM (pH 7.0) Na phosphate buffer. The solution mixture was photoirradiated with a Q-switched Nd:YAG laser (355 nm, fwhm of 4 ns, 1.5 mJ/pulse, 1 Hz, Continuum, Surelight). NI4, ND4, and AQ4 were photoirradiated for 3 min (0.27 J), 30 min (2.7J), and 9 min (0.81 J), respectively. The reaction mixture was subjected to enzymatic digestion with nuclease P1, phosphodiesterase I, and alkaline phosphatase at 37 °C. The consumption of G was quantified by the reverse phase HPLC on a Nacalai Tesque 5C18MS HPLC column using A as internal standard. Φ-G was measured as the average of three measurements.
Kawai et al.
Figure 1. Chemical structures of Sens NI, ND, and AQ attached at the 5′ end of DNA, sequences of Sens-modified DNA, and kinetic scheme for hole injection, charge separation, and G degradation in Sensmodified DNA.
TABLE 1: Reduction Potentials (Ered),a,b the Singlet (ES)a,c and Triplet (ET)a,c Energies of NI, ND, and AQ, and the Bimolecular Rate Constant between NI•-, ND•-, AQ•-, and O2 (kO2)d Sens
Ered (V vs NHE)
ES (eV)
ET (eV)
NI ND AQ
-1.0a -0.22a -0.34b
3.4a 3.2a 2.9c
2.3a 2.0a 2.7c
kO2 (M-1 s-1)
d
1.3 × 109 8.6 × 106 8.2 × 108
a Derived from ref 35. b Derived from ref 42. c Derived from ref 43. d Measured using NI5, ND5, and AQ5 by comparing the time profile measured under air with that measured under Ar during the 355 nm laser flash photolysis.
Results In order to investigate the relation between charge-separation and charge-recombination kinetics and DNA damage processes, a series of DNA modified with NI, ND, and AQ having varying numbers of A-T base pairs between Sens and neighboring G-C base pair were synthesized (Figure 1). We first performed nanosecond time-resolved transient absorption measurements under Ar to investigate the charge-separation (hole injection) and charge-recombination kinetics of Sens-modified DNA. The reported values of the reduction potentials (Ered)35,42 as well as the singlet (ES) and triplet (ET) energy35,43 of NI, ND, and AQ are summarized in Table 1. NI, ND, and AQ can oxidize G (1.31VvsNHE)andA(1.63VvsNHE)14 uponphotoexcitation.10,21-35 The 355 nm laser irradiation allowed the selective excitation of Sens because the nucleobases do not absorb at 355 nm. The laser excitation triggers electron transfer from the nucleobase
Figure 2. Transient absorption spectrum of NI5, ND5, and AQ5 obtained at 1 µs after the 355 nm laser flash excitation. The sample aqueous solution contained 40 µM DNA in 20 mM Na phosphate buffer (pH 7.0) purged with Ar.
to the excited Sens to produce a charge-separated state in DNA. Transient absorption spectra were measured using NI5, ND5, and AQ5, where transient absorption with a peak maximum at around 400, 495,35,44 and 530 nm,33,34 attributed to the NI•-, ND•-, and AQ•- were observed, respectively (Figure 2). The charge-separation yield (ΦCS) and the lifetime of the chargeseparated state (τ) were measured by monitoring the formation and decay of NI•-, ND•-, and AQ•- (Table 2, Figure 3). The
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TABLE 2: Lifetimes (τ) and Quantum Yields of Formation of the Charge-Separated State (ΦCS) Measured for NIn, NDn, and AQn DNA NI0 NI1 NI2 NI3 NI4 NI5 NI6 NI7
τ (µs)a
