Fluorophore ATCUN complexes: Combining agent and probe for oxidative DNA cleavage

Article abstract of DOI:10.1039/c5cc04508h

DNA can be oxidatively cleaved by copper complexes of the ATCUN peptide (amino terminal Cu(II)- and Ni(II)-binding motif). In order to investigate the fate of the metal ion throughout this process, we have exploited quenching/dequenching effects of conjugated fluorophores.

Full text of DOI:10.1039/c5cc04508h

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DOI: 10.1039/C5CC04508H
COMMUNICATION
Fluorophore ATCUN Complexes: Combining Agent
and Probe for Oxidative DNA Cleavage
niloCite this: DOI: 10.1039/x0xx00000x
C. Wende,a N. Kulak*a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
fluorophore ATCUN complex conjugates can be used for
monitoring the fate of the metal ion during oxidative DNA
cleavage. This new approach allows to follow the cleavage
process not only by conventional agarose gel electrophoresis
but also by fluorescence, having function (nuclease) and
reporter (fluorophore) combined in one system.
DNA can be oxidatively cleaved by copper complexes of the
ATCUN peptide (amino terminal Cu(II)- and Ni(II)-binding
motif). In order to investigate the fate of the metal ion
throughout
this
process,
we
have
exploited
quenching/dequenching effects of conjugated fluorophors.
There is only one example in the literature where a Cu(II) based
DNA cleaving agent was equipped with a fluorophore. In that
case a ligand exhibiting fluorescence was used for monitoring
cell uptake.14 As to the best of our knowledge, no system has
been described so far where an artificial nuclease was linked to
a reporter molecule for investigating the status of the
nucleolytic metal ion. This could be, however, of interest for
gaining better understanding of such a metal initiated cleavage
reaction. A reporter molecule being part of the agent itself is
supposed to be more reliable than an external reporter due to
spatial proximity.
The peptides 1a – 3a (Figure 1) with the sequence
fluorophore R-2,3-diaminopropionic acid-β-alanine-histidine-
serine-serine-CONH2 (R-Dap-β-Ala-His-Ser-Ser-CONH2) were
synthesized manually on solid support using standard Fmoc
strategy (Supporting Information S-1).15 The yield was
determined by UV/vis spectroscopy exploiting the extinction of
the coupled fluorophores (Rhodamine B in 1a, dansyl chloride
in 2a, and fluorescein isothiocyanate (FITC) for 3a, Supporting
The ATCUN motif has been studied for more than 50 years.1 During
this time the understanding of this binding motif has evolved from a
small metal binding site in natural proteins like albumin – therein
used for the transport of ions in blood plasma – to effective DNA
cleavers with antitumoral activity.2 The latter ones soon found their
way into bioinorganic chemistry due to their simple synthesis and
modification and high affinity towards Cu(II) and Ni(II) ions.3,4
The simplest peptide mimicking the ATCUN motif is the
tripeptide Gly-Gly-His. Several characteristics of it have been
studied: binding properties3, RNA interaction4,5, DNA cleavage
activity6-8, in vitro behaviour9. Variations of the amino acid sequence
allow an increase in binding affinity to Cu(II) ions as well as to
DNA and RNA as targets.7,10-12 Imperiali et al. were able to improve
the selectivity of the peptide for Cu(II) ions by exchanging both
glycines (Gly) by 2,3-diaminopropionic acid (Dap) and β-alanine (β-
Ala). Such, the introduction of an amino group at the N-terminus
rendered the coupling with functional molecules like fluorophores
possible, and lead to the development of a selective Cu(II)
fluorescent chemosensor.13
Information S-2).
†
Based on this work we have demonstrated here that the
quenching of different fluorophores by Cu(II) ions in
Figure 1: Structure of the peptides 1a – 3a and the corresponding Cu(II) complexes 1b – 3b.
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The sequence of peptide a was developed by Imperiali et al. Supporting Information). An increase in incubation time lead to an
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with the aim of improving metal binding properties when increase in cleavage yield. When the incubation time was doubled,
compared to Gly-Gly-His.13a The exchange of N-terminal the amount of form II DNA decreased dueDtoOIf:o1r0m.10at3i9o/nC5oCfCf0o4rm508IIHI
glycine by 2,3-diaminopropinoic acid was performed in order and fragments (black line in Figure S3.7, Supporting Information),
to allow coupling of a fluorophore. Imperiali et al. have shown indicating a somewhat linear relationship.
that the fluorescence quenching of Cu(II) ions becomes more
Assuming the fluorescence properties of the dyes can be used for
efficient when the linker length of the peptide backbone to the the indirect detection of DNA cleavage by sensing the catalytic Cu
fluorophore decreases. Whereas, the introduction of two serine species, it is important to consider that the fluorescence of
residues at the C-terminus serves the increase of water rhodamine B in TRIS-HCl buffer at pH 7.4 is quenched completely.
solubility of the peptide.13a
In order to be able to exploit fluorescence properties in this case, one
The corresponding Cu(II) complexes 1b – 3b were synthesized has to work at lower pH values.† Thus, the general stability of the
according to a literature procedure by incubation of the peptides copper(II) complexes at different pH values was studied with peptide
1a – 3a with copper(II) chloride in aqueous solution.6 2a (Figure S4.1, Supporting Information). The absorption spectra of
Afterwards their ability to cleave DNA was tested.
peptide 2a in the presence of 1 equivalent Cu(II) at pH 2-4 shows
It is known that Cu(II) complexes of peptides with the ATCUN only a broad band at 800 nm for copper(II) ions in water.17a The
motif are able to cut plasmid DNA in the presence of reducing intensity of d-d transition of the Cu(II)-peptide complex at 525-550
agents like ascorbic acid (Asc).6 Starting from concentration- nm increases with increasing pH value while the band for Cu(II)
dependent gel electrophoresis experiments we could show that decreases. These data suggest that the ATCUN complexes are stable
the complexes 1b – 3b cleave plasmid DNA pBR322 over a wide range of pH.
efficiently (Figure 2 and Supporting Information S-3).
Based on previously published studies6 and based on the fact
that DNA cleavage with the complexes 1b – 3b happens only in
the presence of ascorbic acid as a reductant, the mechanism of
cleavage of DNA is most likely oxidative. In order to
investigate the reaction mechanism and identify potential ROS
that are involved in the cleavage process 1b – 3b were
incubated with plasmid DNA and several literature-known ROS
scavengers (Table 1). The quenching effects were the same for
all complexes including Cu(II)-GGH suggesting that the
mechanism of cleavage is the same for these compounds. The
results are exemplarily shown for 1b in Figure 3.
Form II
Form III / Fragments
100
80
60
40
20
0
1b
2b
1
3b
1b
2b
2
3b
1b
2b
3b
3
Reference
25 µM + Asc
25 µM
Form II
Form III / Fragments
Lane
100
80
60
40
20
0
Figure 2: Cleavage activity of complexes 1b - 3b (0.025 mM) with
respect to pBR322 DNA (0.025 µg/µL) in TRIS-HCl buffer (10 mM,
pH 7.4) in the presence and absence of ascorbic acid (0.25 mM) at
37 °C for 1 h.
As expected all complexes cleave DNA only in the presence of
ascorbic acid (lane 2 in Figure 2 and lanes 2, 4 and 6 in Figures S3.1
– S3.4 in Supporting Information).6,7,12 The degree of cleavage
increased with increasing complex concentration. A concentration as
low as 25 µM was sufficient to cleave supercoiled plasmid DNA
(form I) to at least 30% into the open circular form II (lane 2).
Complex 3b even lead to a double strand break of DNA at this
concentration (form III) (lane 2). At a concentration of 75 µM (lane
6) all complexes cleaved plasmid DNA almost completely into form
III, and even smaller fragments were observed. At a concentration as
low as 1 µM 1b ~10% of the DNA was cleaved (Figure S3.5,
Supporting Information).
The differences in cleavage efficiency can be explained by the
different bulk of the dyes and their linkers, respectively. Due to the
length and characteristics of the different linkers (1b: amide, 2b:
sulfonic amide, 3b: thiourea) a closer (1b) or more distant (3b)
approach of the dye to the copper centre and thus a change in DNA
interaction is expected. Also the orientation of the fluorophores
might play a role. In comparison to the unfunctionalized copper(II)
peptide complexes b (R=H) and Cu(II)-Gly-Gly-His cleavage
activity of the fluorophore carrying peptides 1b – 3b is somewhat
lower (Figure S3.6, for Cu(II)-Gly-Gly-His see also the literature6).
In order to investigate whether an incubation time longer than
one hour would result in a change in cleavage efficiency, DNA was
incubated with complex 1b also for two and four hours (Figure S3.7,
1
2
3
4
5
6
7
8
II
III
I
1b [µM]
Scavenger
DNA
-
50
-
50
tBuOH
50
DMSO
50
NaN3
50
CAT
50
SOD
DNA
-
Figure 3: Cleavage activity of complex 1b (0.05 mM) with
respect to plasmid DNA pBR322 (0.025 µg/µl) in TRIS-HCl
buffer (10 mM, pH 7.4) in the presence of ascorbic acid
(0.25 mM) and different ROS scavengers at 37 °C for 1 h.
Table 1: Literature-known ROS scavengers and concentrations
applied in this study.
Scavenging agent
ROS
Hydroxyl radicals
Hydroxyl radicals
Concentration
200 mM
200 mM
tert-Butanol18
Dimethylsulfoxide
(DMSO)19
Sodium azide20
Singlet oxygen
Peroxo species
10 mM
2.5 mg/mL
313 u/mL
Catalase (CAT)21
Superoxide dismutase Superoxide
(SOD)20
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Our results show that DNA cleavage with the ATCUN peptide experiments are indications for the essential role of Cu(I) in
is most likely carried out by hydroxyl radicals (lanes 3 and 4) as DNA cleavage.
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DOI: 10.1039/C5CC04508H
well as peroxo species (lane 6) since cleavage is inhibited in the
A
400
200
0
presence of tert-butanol, DMSO and catalase. This is in
accordance with results obtained by Cowan et al.6 Additionally,
sodium azide exhibits a strong quenching effect (lane 5), which
indicates the presence of singlet oxygen. In order to exclude
that this effect is due to a contamination in the azide solution, a
control experiment was carried out. Therein, the complexes
were incubated with DNA as before, however, this time in
deuterated water (Figure S3.8 for 1b, S3.9 for 2b and 3b,
Supporting Information). Singlet oxygen has a lifetime in H2O
of approximately 2 µs.22 In D2O, the lifetime is increased by
factor 10. Therefore reactions depending on singlet oxygen are
more distinctive in D2O.25 This is also true in this case, since
DNA cleavage was 2-3 times stronger regarding the formation
of linear DNA for the compounds tested in D2O, indicating the
presence of singlet oxygen.
550
570
590
610
630
650
670
690
Wavelength [nm]
0.05 mM 1a
0.05 mM 1b
0.05 mM 1b (incubated)
B
500
0
400 450 500 550 600 650 700 750 800
Wavelength [nm]
It is known from the literature that the fluorescence of several dyes
can be quenched by paramagnetic Cu(II) ions.23 Imperiali et al.
showed, that the binding of these ions by the ATCUN motif leads to
quenching of the fluorescence of a dansyl labelled peptide.13a The
effect of Cu(II) complexation on the fluorescence of peptides 1a –
3a was thus studied using fluorescence spectroscopy. A decrease of
fluorescence was observed with increasing Cu(II) concentration with
a rest fluorescence of about 14% at an 1:1 ratio ligand:Cu(II) (Figure
S5.1, Supporting Information, exemplarily 1a).
0.05 mM 2a
0.05 mM 2b
0.05 mM 2b (incubated)
C
500
0
450
500
550
600
650
700
When complexes 1b – 3b (Figure 4, red lines) were incubated with
plasmid DNA under gel electrophoresis conditions in the presence of
ascorbate (Figure 4, green lines) a reconstitution of fluorescence was
observed indicating the disappearance of Cu(II) species. For
comparison peptides 1a – 3a are also shown (Figure 4, blue lines).
During the course of these experiments, it is probable that Cu(I) ions
and Cu-”oxo” type species like Cu-OOH and Cu-OH are produced.
Since neither Cu(I) ions (d10 ion) nor Cu-“oxo” species lead to
fluorescence quenching by energy transfer between donor and
acceptor23c,26, fluorescence is reconstituted.
Wavelength [nm]
0.05 mM 3a
0.05 mM 3b
0.05 mM 3b (incubated)
Figure 4: Fluorescence spectra. A) 1a, 1b and 1b (incubated) in
Britton-Robinson buffer (10 mM, pH 5.0). B) 2a, 2b and 2b
(incubated) in TRIS-HCl buffer (10 mM, pH 7.4). C) 3a, 3b and 3b
(incubated) in TRIS-HCl buffer (10 mM, pH 7.4).
Cleavage of DNA is supposed to happen when those Cu(I) ions
are reoxidized by molecular oxygen leading to the generation of
ROS (reactive oxygen species) (Scheme 1).6,11,24 Also the direct
involvement of Cu”oxo” species is feasible.
The incomplete reconstitution of fluorescence (Figure 4, blue lines)
might be due to damage of the fluorescence dyes by ROS, as e.g.
known for rhodamine B and hydroxyl radicals.20 Also, an interaction
of the dyes with ascorbate is conceivable: incubation of 1b - 3b with
an excess of ascorbate as used in the samples for gel electrophoresis
revealed a small quench effect (Figure S5.2). An on/off switching of
the fluorescence of complexes 1b and 2b was observed by titrating
the reducing agent ascorbic acid and the oxidizing agent hydrogen
peroxide alternately in with only a small loss of intensity in the
second cycle (Figure S5.3). Whereas for the dansyl labelled peptide
2b the switching is very distinct and might be considered a proof of
concept, for the rhodamine B labelled peptide 1b the switching (at
pH 5) is less pronounced. The reversibility of the process suggests
the importance of Cu(II) quenching as against dye damaging by
ascorbate, albeit the small loss in intensity throughout the
experiment could be attributed to such a damage. The fluorophores
can thus be used as indicators for the fate of Cu(II) ions responsible
for DNA cleavage.
The three dyes applied in this study exhibit some advantages
but also disadvantages concerning synthesis, fluorescence
properties and stability (S-7). For example, hydroxyl radicals
generated during the redox reactions assumed (Scheme 1),
could damage FITC and rhodamine B after incubation times
longer than 1 h (not shown).27 The experiments should thus be
carried out within less than 1 h. Since short experimental times
are already feasible, this does not pose any disadvantage.
Scheme 1: Mechanism of ROS production by Cu(II) ions in the
presence of ascorbate.24
For Cu(II)-ATCUN complexes it was recently shown by
cyclovoltammetric experiments that a Cu(III)/Cu(II) redox
cycle might be involved in DNA cleavage instead.7,25 For
investigation of the redox cycle in the present case the Cu(II)-
peptide 2b was studied by NMR spectroscopy under an inert
atmosphere in D2O-based TRIS-HCl buffer. The spectrum
reveals typical Cu(II) paramagnetic species. After addition of
ascorbic acid the NMR spectrum showed sharp, diamagnetic
peaks (Figure S6-1). It is thus very likely that Cu(I) is produced
by reduction with ascorbic acid under the conditions applied.
Furthermore, electrophoresis experiments in the presence of the
Cu(I) specific chelator neocuproine showed an inhibition of
cleavage activity by ATCUN complexes (Figure S3-10). Both
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Conclusions
2
3
E. Kimoto, H. Tanaka, J. Gyotoku, F. Morishige, L. Pauling, Cancer
Res., 1983, 43, 824-828. Z. Yu, M. Han, J. A. Cowan, Angew. Chem.
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DOI: 10.1039/C5CC04508H
Investigation of DNA cleavage by agarose gel electrophoresis
showed that fluorophore-carrying peptides 1a – 3a coordinated
to Cu(II) (1b - 3b) are efficient DNA cleaving agents in the
presence of a reducing agent. Plasmid DNA is cleaved even at
very low concentrations. Due to the linked sterically bulky
dyes, and thus the hindered approach of the complexes to DNA,
our system is, however, less efficient under very similar
conditions than the copper complexes of GGH, KGHK and the
unfunctionalised peptide a (in the case of KGHK the positive
charge of the lysine side chains supports interaction with
DNA6). Still, the cleavage activity of 1b - 3b which is in the
low micromolar range (cf. Fig. S3.5), is close to the one of
Cu(phen)2, the first reported and one of the most efficient
oxidative chemical nucleases.28 Experiments regarding the
mechanism of DNA cleavage lead to the conclusion that DNA
is cleaved oxidatively by hydroxyl radicals, peroxo species and
singlet oxygen.
By spectroscopic means (fluorescence, NMR) we demonstrated
that Cu(II) is reduced to Cu(I) which is required to reduce O2 to
superoxide and subsequently produces H2O2. Hydrogen
peroxide can then interact with Cu(I) to form metal-oxo species
and hydroxyl radicals that induce DNA cleavage (Scheme 1).24
Quenched fluorescence of the Cu(II)-coordinated peptides 1b -
3b is regained when the Cu(II) ions react during this process.
These new complexes not only initiate cleavage of DNA, but at
the same time display changes of the oxidation state of the
involved Cu(II) ions. Thus, our system simultaneously
comprises a DNA cleaving agent and a redox-sensitive probe.
After this proof of concept application of the herein described
Cu(II) fluorescent peptides in cell experiments is conceivable
(except for 1b which requires working at pH 5).
Int. Ed., 2015, 54, 1901-1905.
S.-J. Lau, T. P. A. Kruck, B. Sarkar, J. Biol. Chem., 1974, 249, 5878-
5884.
4
5
6
7
E. C. Long, Acc. Chem. Res., 1999, 32, 827-836.
Y. Jin, J. A. Cowan, J. Biol. Inorg. Chem., 2007, 12, 637-644.
Y. Jin, J. A. Cowan, J. Am. Chem. Soc., 2005, 127, 8408-8415.
Y. Jin, M. A. Lewis, N. H. Gokhale, E. C. Long, J. A. Cowan, J. Am.
Chem. Soc., 2007, 129, 8353-8361.
8
9
J. C. Joyner, J. Reichfield, J. A. Cowan, J. Am. Chem. Soc., 2011,
133, 15613-15626.
A. C. Matias, N. Villa dos Santos, R. Chelegão, C. S. Nomura, P. A.
Fiorito, G. Cerchiaro, J. Inorg. Biochem., 2012, 116, 172-179.
10 J. C. Joyner, W. F. Hodnick, A. S. Cowan, D. Tamuly, R. Boyd, J. A.
Cowan, Chem. Commun., 2013, 49, 2118-2120; S. Bradford, Y.
Kawarasaki, J. A. Cowan, J. Inorg. Biochem., 2009, 103, 871-875.
11 C. Harford, B. Sarkar, Acc. Chem. Res., 1997, 30, 123-130.
12 D. F. Shullenberger, P. D. Eason, E. C. Long, J. Am. Chem. Soc.,
1993, 115, 11038-11039.
13 A. Torrado, G. K. Walkup, B. Imperiali, J. Am. Chem. Soc., 1998,
120, 609-610; T. R. Young, C. J. K. Wijekoon, B. Spyrou, P. S.
Donnelly, A. G. Wedd, Z. Xiao, Metallomics, 2015, 7, 567-578.
14 S. S. Bhat, A. A. Kumbhar, H. Heptullah, A. A. Khan, V. V. Gobre,
S. P. Gejji, V. G. Puranik, Inorg. Chem., 2011, 50, 545-558.
15 S. I. Kirin, F. Noor, N. Metzler-Nolte, W. Mier, J. Chem. Educ.,
2007, 84, 108-111.
16 L. Fülöp, B. Penke, M. Zarándi, J. Peptide Sci., 2001, 7, 397-401.
17 H. Korpi, P. J. Figiel, E. Lankinen, P. Ryan, M. Leskelä, T. Repo,
Eur. J. Inorg. Chem., 2007, 2465-2471; K. P. Neupane, A. R. Aldous,
J. A. Kritzer, Inorg. Chem., 2013, 52, 2729-2735.
18 M. Von Piechowski, M.-A. Thelen, J. Hoigné, R. E. Bühler, Ber.
Bunsenges. Phys. Chem., 1992, 96, 1448-1454.
19 E. L. Hegg, J. N. Burstyn, Inorg. Chem., 1996, 35, 7474-7481.
20 A. Sreedhara, J. D. Freed, J. A. Cowan; J. Am. Chem. Soc., 2000,
122, 8814-8824.
Acknowledgements
The authors would like to thank Biprajit Sarkar for helpful
discussions.
21 S.-H. Chiou, J. Biochem., 1983, 94, 1259-1267.
22 J. G. Parker, W. D. Stanbro, J. Photochem., 1984, 25, 545-547.
23 G. K. Rollefson, R. W. Stoughton, J. Am. Chem. Soc., 1941, 63,
1517-1520; W.-Y. Lin, H. E. van Wart, J. Inorg. Biochem., 1988, 32,
21-38; J. Brunner, R. Krämer, J. Am. Chem. Soc., 2004, 126, 13626-
13627.
Notes and references
Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstr.
a
34/36, D-14195 Berlin, Germany. E-mail: nora.kulak@fu-berlin.de; Fax:
+49 30 838 454697; Tel: +49 30 838 54697.
†
There is indication from the literature that the thiourea bond (as in
24 L. Guilloreau, S. Combalbert, A. Sournia-Saquet, H. Mazarguil, P.
Faller, ChemBioChem, 2007, 8, 1317-1325.
peptide 3a) is not stable during cleavage from the solid support with
TFA (trifluoroacetic acid).16 Under the conditions chosen, however, the
coupling of FITC on solid support was possible, the yield though, was
low.
25 C. Hureau, H. Eury, R. Guillot, C. Bijani, S. Sayen, P.-L. Solari, E.
Guillon, P. Faller, P. Dorlet, Chem. Eur. J., 2011, 17, 10151-10160.
26 L. Fabbrizzi, M. Licchelli, P. Pallavicini, A. Perotti, A. Taglietti, D.
Sacchi, Chem. Eur. J., 1996, 2, 75-82; G. De Santis, L. Fabbrizzi, M.
Licchelli, C. Mangano, D. Sacchi, Inorg. Chem., 1995, 34, 3581-
3582.
Whereas the basic ATCUN sequence does not bind Cu(II) at pH
values lower than 6,11,17 at pH 5 the rhodamine B labelled peptide 2a
shows Cu(II) binding as well as fluorescence emission. This thus allows
application of the presented concept also at non-physiological pH values.
Electronic Supplementary Information (ESI) available: experimental
section, determination of peptide yields, DNA cleavage, UV/vis,
fluorescence and NMR experiments. See DOI: 10.1039/c000000x/
27 S. Feng, X. Chen, J. Fan, G. Zhang, J. Jiang, X. Wei, Anal. Lett.,
1998, 31, 463-474; J. Fan, H.-Q. Guo, S.-L. Feng, J. Fluoresc., 2007,
17, 257-264.
28 J. M. Veal, K. Merchant, R. L. Rill, Nucleic Acids Res., 1991, 19,
3383-3388.
1
T. Peters Jr., Biochim. Biophys. Acta 1960, 39, 546-547.
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