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Complex Sequence Dependence by Excess-Electron Transfer through DNA with Different Strength Electron Acceptors.

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Electron Transfer
DOI: 10.1002/anie.200502551
Complex Sequence Dependence by ExcessElectron Transfer through DNA with Different
Strength Electron Acceptors**
Antonio Manetto, Sascha Breeger,
Chryssostomos Chatgilialoglu, and Thomas Carell*
Numerous studies of the long-distance hole-migration process
through a DNA double strand have revealed detailed insight
into the distance and sequence dependence of charge-movement processes.[1–4] In contrast to the wealth of information
now available on the long-distance hole transfer,[5–9] less is
known about the complementary excess-electron-transfer
process in which an anion, instead of a cation, moves through
the duplex. We,[10–13] and others,[14–19] have recently shown that
excess electrons move through DNA by a hopping-type
mechanism in which the pyrimidine bases dT and dC act as
,stepping stones-.[17]
Although the hopping of excess electrons through DNA is
now a generally accepted model, conflicting data on the
sequence dependence of this process were reported. The
charge-transfer process was investigated by using DNA that
was modified with either arylamines,[14–16] pyrenes, or phenothiazines[18, 19] as the electron donor, or 5-bromouracil as the
electron acceptor. It was shown that GC base pairs, in contrast
to AT base pairs, reduce the efficiency of the excess-electron
transfer through the duplex. Our studies with a reduced flavin
[*] Dipl.-Chem. A. Manetto, Dipl.-Chem. S. Breeger, Prof. Dr. T. Carell
Department of Chemistry and Biochemistry
LMU Munich
Butenandtstrasse 5–13, 81377 Munich (Germany)
Fax: (+ 49) 89-2189-77756
Dr. C. Chatgilialoglu
Consiglio Nazionale delle Ricerche
Via P. Gobetti 101, 40129 Bologna (Italy)
[**] We thank the Deutsche Forschungsgemeinschaft, the Volkswagen
Foundation, and the EU Marie Curie training and mobility program ,
project number MRTN-CT-2003-505086 [CLUSTOXDNA], for
financial support. S.B. thanks the Fonds der Chemischen Industrie
for a predoctoral fellowship.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 318 –321
redox potential of the Br-substituted bases are not all known,
we can assume, based on the reduction potentials of the
unmodified nucleobases, a sequence of BrdU > BrdA BrdG, with BrdU being the easiest to reduce, followed by
BrdA and BrdG.[33] All the hairpins depicted in Figure 1 show
melting points above room temperature. CD measurements
of the hairpins 1–5 are, in all cases, in agreement with a BDNA conformation. All of the hairpins were prepared and
purified in accordance with standard phosphoramidite automated solid-phase-synthesis protocols.
In previous studies the flavin was reduced with sodium
dithionite (Na2S2O4).[10–12] Recently we, and Ito and Rokita,[15]
observed that the dithionite reduction inhibits the excesselectron-transfer process after some time. Therefore, in this
study we used a more stable EDTA-based photoreduction
process.[34] Although the initial yields determined with
dithionite and EDTA are similar, EDTA allowed us to
establish a stable, fully reduced system for more than 1 h.
In a typical irradiation experiment, EDTA (0.02 m) was
added to a solution of DNA (cDNA = 20 mm, 0.01m Tris, pH 7.4,
0.15 m NaCl) and irradiated with white light for 1 min in
fluorescence cuvettes and under anaerobic conditions to
photoreduce the flavin (monitored by fluorescence spectroscopy). After photoreduction, we further irradiated the
samples with a 1000-W Xe lamp, equipped with a cooled
340-nm cut-off filter. For analysis, 10-mL samples were
removed from the assay solution after defined time intervals,
aerated for 30 min, desalted, and analyzed. Analysis of the
data was performed by capillary gel electrophoresis. In the
past, excess-electron transfer
through DNA with 5BrdU,
was evaluated by observation
of strand cleavage that was
induced by the neutral dU
radical after heating of the
DNA with
(CE), in contrast, allowed us
to detect directly the loss of
the Br anion within the intact
19-mer hairpins after electron
capture. An example of the
CE analysis of an irradiation
experiment performed with
the hairpin (1 a) is given in
Figure 2.
The fractions were collected and the corresponding
peaks were analyzed by
MALDI-TOF mass spectrmetry to prove the formation of
the expected product strands.
The yield data obtained for all
15 hairpins are depicted as bar
Figure 1. DNA-hairpin models (1–5) for the excess-electron-transfer study through DNA depending on the
graphs in Figure 3. They show
acceptor strength. A) & = Flavin electron donor 6 in the reduced and deprotonated form; & = electron
the time-dependent formation
acceptor (a = 5BrdU, b = 8BrdA, c = 8BrdG); & = dibrominated electron acceptors (a = dU, b = dA,
of the debrominated 19-mer
c = dG); X = the acceptor counter bases. B) Sequence of hairpins 1–5 for the three acceptors (a, b, and c);
hairpins as average values that
& = flavin 6; & = electron acceptor. Highlighted in gray are the number and the position of GC base pairs.
as the electron donor and a thymine dimer as the electron
acceptor—a system that mimics the repair of UV-induced
thymine dimer DNA lesions by photolyases[20, 21]—showed, in
contrast, no sequence dependence.[13] Based on the likely
reductive opening of the thymine dimer at a rate of
approximately 106 s 1 [22] and under the assumption that
electron-hopping rate could be 108–109 s 1, we speculated
that the dimer opening might be the rate-determining step.
Through the use of DNA duplexes that contain two thymine
dimers in a row, we recently provided evidence that the
electron is able to hop over a thymine dimer without cleaving
it. This lends support to the assumption that dimer cleavage is
slower than electron hopping.[23]
To investigate in more detail how the rate of reaction of an
acceptor with excess electrons influences the observed
sequence dependence of charge movement in DNA, we
prepared a series of 15 DNA hairpins (1–5 depicted in
Figure 1). Within these hairpins, we have placed the light* -red = 2.6 V against
dependent flavin electron injector 6 (Eflav
NHE) in the loop region of the hairpin, and three electron
acceptors (5-bromouracil[14–16] = series a, 8-bromoadenine =
series b, and 8-bromoguanine[25] = series c) positioned in the
stem region at a distance of about 17.5 ? from the donor.
The bimolecular rates of reaction of a solvated electron,
with all three electron acceptors (BrdA, BrdG and BrdU), are
known but they are in the diffusion controlled environment[26]
with approximately 1 @ 1010–2 @ 1010 m 1 s 1.[27–29] The primary
reduction is followed by a release of Br .[27, 30–32] Although the
Angew. Chem. Int. Ed. 2006, 45, 318 –321
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Irradiation of hairpin (1 a). Time-dependent debromination of
the acceptor within hairpin 1 a. The signals for the parent and the
debrominated strand are highlighted in blue and red, respectively.
Their identity was confirmed after collection of the fractions by MALDIMS analysis. The inset shows the debromination rate (10.5 % min 1)
as calculated by peak-area integration and linear approximation of the
data (Beckman Coulter P/ACE-MDQ DNA system, UV detection at
254 nm, 30-cm fused-silica capillary filled with 6 % polyacrylamide gel
in 0.1 m tris-borate, 2 mm EDTA, pH 8.4, electrokinetic injection by
applying 10 kV for 2–10 s, separation at 9 kV for 45 min).[36]
Figure 3. Debromination rates u for the hairpins 1–5. The calculated
repair rates are given as % Br-anion loss per minute. Analysis was
performed by CE of at least three independent measurements per
strand. Calculation was carried out on the data of up to 5 minutes of
irradiation time by peak area integration followed by linear approximation (for an example, see the inset in Figure 2).
were obtained from at least three independent experiments.
We believe that the hydrogen atom needed for the reduction
of the nucleobase radicals may be efficiently donated from
the EDTA in our experiments.
We first investigated how the type of acceptor influences
the debromination yield within the hairpin series 1 a–c, which
have only AT base pairs between the flavin donor and
acceptor. These data show that the yield of debromination
depends strongly on the kind of acceptor. The fastest
debromination was observed with BrdU (1 a), followed by
BrdA (1 b), and BrdG (1 c), which reflects the ease of
reduction of these acceptors. This shows that in the process
of electron migration through DNA, which involves electron
injection, migration, and capture, the reduction of the
acceptor can indeed be the rate-determining step! Next, we
studied 2 a–c and 3 a–c and how the system reacts if we replace
one or two AT base pairs by GC base pairs. For the BrdU
acceptor strands 2 a and 3 a, we observe that the yield drops by
50 % with every GC base pair. For the weaker purine
acceptors, BrdA and BrdG, a much smaller influence is
observed, which indicates that in these systems the acceptor is
not strong enough to fully report the sequence effect. In our
explanation, reduction of the acceptor is the rate-determining
step with BrdA and BrdG. More important, however, is the
observation that the position of the GC base pair, relative to
the acceptor, is more important than the number of GC base
pairs. If we shift either one or both GC base pairs so that they
stack directly on top of the acceptor, dramatic decreases in
the yields are observed. In the 5BrdU series (a), the yield
drops from 10.5 % min 1 to about 1.4 % min 1 with one GC
base pair (4 a) and again to 0.6 % min 1 with two GC base
pairs (5 a). The same was observed when the BrdA or BrdG
acceptors were used, although the yield reductions were in
these cases much less pronounced (series b and c). This is
again in agreement with our idea that reduction of the
acceptor is rate determining if the weaker Br-purine acceptors
are used.
From these studies it becomes clear, that a GC base pair
between the donor and acceptor reduces the excess-electrontransfer efficiency by a factor of approximately 2. This is,
however, only observable with very efficient acceptors such as
BrdU. Weaker electron acceptors provide systems with ratedetermining electron-capture steps. More important, however, is our observation that the position of the GC base pairs
between the donor and the acceptor plays a larger role than
the number of GC base pairs. Although this is difficult to
explain, one reason could be modulation of the redox
potential of the acceptors by the stacking of the GC base
pairs on top of the acceptor. This situation is similar to that
observed in the hole-transfer process, in which the stacking of
guanine makes the oxidation of nucleobases more efficient.
Received: July 21, 2005
Published online: November 29, 2005
Keywords: DNA damage · electron transfer · nucleobases ·
redox potential
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complex, excess, sequence, acceptor, strength, transfer, dna, different, electro, dependence
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