Two-color two-laser DNA damaging.

Full text of DOI:10.1002/anie.200353318

Communications
DNA Damage
Two-Color Two-Laser DNA Damaging**
Kiyohiko Kawai,* Xichen Cai, Akira Sugimoto,
Sachiko Tojo, Mamoru Fujitsuka, and Tetsuro Majima*
Photodynamic therapy (PDT) is a promising treatment for
cancer based on a photosensitized oxidative reaction at the
diseased tissues which results in cell death. Membranes,
proteins, and DNA are considered as a potential targets.[1,2] In
contrast to surgery and chemotherapy, the combination of
photosensitizer (Sens) uptake in malignant tissues and
selective light delivery offers the advantage of a selective
method of destroying diseased tissues without damaging
surrounding healthy tissues. Following the absorption of light,
the photosensitizer is activated to the singlet excited state,
1Sens*, which may be converted into the triplet excited state,
3Sens*. The mechanisms of tumor destruction involve oxida-
tion through electron transfer from cellular components to
1Sens* or 3Sens* (type I mechanism), as well as oxidation
[*] Dr. K. Kawai, Dr. X. Cai, Dr. A. Sugimoto, S. Tojo, Dr. M. Fujitsuka,
Prof. Dr. T. Majima
The Institute of Scientific and Industrial Research (SANKEN)
Osaka University
Mihogaoka 8–1, Ibaraki, Osaka 567–0047 (Japan)
Fax: (+81)6-6879-8499
E-mail: kiyohiko@sanken.osaka-u.ac.jp
majima@sanken.osaka-u.ac.jp
[**] This work was partly supported by a Grant-in-Aid for Scientific
Research on Priority Area (417), 21st Century COE Research and by
the Ministry of Education, Culture, Sports, Science, and Technology
(MEXT) of the Japanese Government. We thank the members of the
RadiationLaboratory of ISIR (SANKEN), Osaka University for
running the linear accelerator.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200353318
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Angewandte
Chemie
mediated by singlet oxygen (type II mechanism), which is
formed through energy transfer from 3Sens* to molecular
oxygen.[3] When targeting DNA, the binding of Sens to DNA
will favor the type I process, as the close association of Sens to
DNA is important in the photoinduced one-electron oxida-
tion of DNA.[4]
Excitation of DNA-bound sensitizers produces the SensCÀ/
+
DNAC (ultimately yielding the radical cation of guanine
+
(GC ), the most easily oxidized base, by hole transfer) charge-
separated state through photoinduced electron transfer.
However, the efficiency of producing photosensitized DNA
damage is low because the charge recombination rate is
usually much faster than the process leading to DNA damage,
Figure 2. Electronejectionfrom NDI CÀ promoted by a 532-nm laser
pulse. NDICÀ was generated from electron attachment during the pulse
radiolysis of NDI-ODN (NDI-AAAAAAGTGCGC/TTTTTTCACGCG)
(gray), and photoirradiated with a 532-nm laser pulse at 2 ms after the
electron pulse (black). Inset: Formation and decay of the solvated
electron monitored at 630 nm.
+
such as the reaction of GC with water.[5–8] Thus, the absorption
of a photon by Sens occasionally leads to DNA damage, but
only with the aid of hole transfer, which provides time for
DNAC+ and SensCÀ to react with water or O2.[9–14] Herein, we
report the first study of nanosecond-laser DNA damaging in
which a combination of two-color pulses is used as a
promising new strategy to reach a high DNA-damaging
efficiency. The first laser pulse was applied for the production
caused a decrease in DOD of NDICÀ and an absorption at
630 nm assigned to a solvated electron (eaqÀ) immediately
after the flash (inset), demonstrating the successful ejection of
an electron from NDICÀ to the solvent water.
+
of the SensCÀ and DNAC , and the second laser pulse for the
ejection of an electron from SensCÀ, making the reaction
irreversible.[15]
To test the efficiency of two-color two-laser irradiation for
DNA damaging, the consumption of G upon irradiation of
ODN-G and ODN-GG bound to added N,N’-bis-[3-(N-
dimethyl)propyl]-1,4,5,8-naphthaldiimide dichloridite (NDI-
HCl) and NDI-ODN was compared with that of single-laser
experiments. Interestingly, a higher consumption of G was
observed with the combination of the first and second laser
irradiations than with irradiation by the 355-nm pulses alone
(Table 1).[23] If only 532-nm pulses were used, no DNA
damage would occur because this would require a non-
resonant two-photon excitation, as neither nonreduced NDI
nor DNA absorbs above 450 nm.[24,25] Hence, the second laser
alone cannot damage DNA, but does provide enough addi-
tional energy to cross the ionization threshold of NDICÀ,
which results in the transfer of the electron to the solvent and
The proposed two-color two-laser DNA damaging is
represented as a two-step process in a simplified scheme
(Figure 1). In this study, naphthaldiimide (NDI) was selected
Table 1: Consumption of G in the photosensitized damaging of ODNs.[a]
ODN
ÀG [%][a]
355 nm 532 nm 355+532 nm
Figure 1. Schematic representation of two-color two-laser DNA
damaging.
ODN-G[b]
(TG)6T 0.6
/(AC)6A
(TTGG)3T 2.5
/(AACC)3A
<0.3
<0.2
0
2.6
16.7
14.2
ODN-GG[b]
NDI-ODN[c] NDI-T3CGCGCT2 0.8
/A3GCGCGA2
as a photosensitizer that can be excited with a first laser at a
wavelength of 355 nm.[16–20] First, to assess the feasibility of
electron ejection from SensCÀ bound to DNA, a pulse
radiolysis–laser flash photolysis of NDI-conjugated oligo-
deoxynucleotide (NDI-ODN) was performed (Figure 2).[21,22]
NDICÀ with a maximum absorption peak at 495 nm[16] was
generated from electron attachment during the pulse radiol-
ysis of NDI-ODN. Since SensCÀ often absorbs light at a longer
wavelength than it does in its non-reduced form, laser pulses
with a longer wavelength can be used for the excitation of
SensCÀ, and a 532-nm laser was applied as the second laser.
Irradiation of NDICÀ in NDI-ODN with a 532-nm laser pulse
[a] Laser flash photolysis was carried out inanaqueous solution
containing ODN (strand conc.=40 mm) and Na phosphate buffer
(20 mm, pH 7.0). Inthe case of ODN-G and ODN-GG, NDI-HCl (40 mm)
was added. For the two-wavelength irradiation, the 532-nm laser pulse
(20 mJ/pulse) was synchronized with the 355 nm laser pulse (1.6 mJ/
pulse) with a 10-ns delay. Photoirradiated ODNs were digested with
snake venom phosphodiesterase/nuclease P1/alkaline phosphatase to
2’-deoxyribonucleosides, and the consumption of G was quantified by
HPLC with A as an internal standard. [b] Photoirradiated for 20 min
(355 nm: 19 J; 532 nm: 240 J). [c] Photoirradiated for 5 min (355 nm:
4.8 J; 532 nm: 60 J).
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Communications
makes the reaction irreversible.[15] A higher consumption of G
focal point of the lasers.[29–33] The photosensitizer NDI used in
this preliminary experiment is not adequate for practical as
since the charge-separation yield is small (about 2%) owing
to the fast charge recombination from the contact radical ion
pair.[27,34] Therefore, a second laser exerts its effect only on
NDICÀ generated by occasional escape from the charge
recombination by hole transfer. Thus, for efficient oxidative
DNA damage to occur, it is necessary to increase the charge-
separation yield by decreasing the rate constant for charge
recombination. This can be achieved by the use of triplet
sensitizers,[4] and by hole generation on adenine to promote
fast hole transfer by an adenine-hopping mechanism that
helps to separate the hole and SensCÀ.[14,21]
was observed for ODN-GG than for ODN-G; the ionization
potential of G in the former is lowered by a stacking
interaction between G units,[26] demonstrating that the con-
sumption of G is based on the photoinduced electron transfer
by the first laser. The acceleration of DNA damaging by the
second laser was the highest for ODNcovalently bonded with
NDI, in which case the sequence was designed to generate a
hole selectively on adenine and to have a lifetime of charge-
separated state in the order of 100 ns.[14,27] Figure 3 shows the
Received: November 13, 2003 [Z53318]
Keywords: charge transfer · DNA damage · laser chemistry ·
.
oligonucleotides · photooxidation
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