The CD spectra of different oligonucleotides show that c-MYC and PDGF-A sequences preferentially form the parallel G-quadruplex structure in 50 mm Tris-HCl buffer at pH 7.8, and the maxi
Trang 1hypersensitive elements of human c-myc and PDGF-A
promoters and their ability to form G-quadruplexes
Viktor Viglasky
Department of Biochemistry, Faculty of Sciences, Institute of Chemistry, P J Safarik University, Kosice, Slovakia
G-rich regions appear in several locations in the
human genome, including at the ends of linear
chrom-omes, the immunoglobin switch region, centromeres,
fragile X syndrome repeats, and promoters of some
genes [1] The sequences repeated in tandem, with three
or four adjacent guanines, have been known to form
polymorphic quadruplexes containing G-quartets
stabi-lized by cyclic Hoogsteen hydrogen bondings
Quadru-plex structures are highly stable DNA or RNA
structures formed on G-rich sequences [2] The Na+
and K+ions stabilize the stacking through their
inter-actions with carbonyl oxygens of the eight guanines of
two adjacent quartets [3] Direct evidence for the
pres-ence of G-quadruplex structures in vivo has been
reported both at the telomeres of the ciliate
Stylonychi-a[4] and those of humans [5], and at the promoter of
c-myc [6,7] Moreover, other genomic regions were
shown to be able to adopt quadruplex structures, such
as the promoters of c-kit oncogene [6], HIF-1a [9], Bcl2 [10] and vascular endothelial growth factor [11] The stabilization of the G-quadruplex structure by small molecules is currently emerging as a very promis-ing anti-cancer strategy Therefore, molecules that stabi-lize G-quadruplex structures can be used as potential anti-cancer agents [12] Indeed, recent studies strongly suggest that molecules able to stabilize the quadruplex structure of DNA can lead to an arrest of the prolifera-tion of cancer cells [5,12–14] At each division of somatic cells, telomeres are shortened, a process leading
to senescence and death It has been shown in vitro that G-quadruplex structures of the human sequence (G3T2A)3G3formed in the presence of molecules stabi-lizing the G-quartet stacks, similar to anthraquinones or porphyrins, inhibit the activity of telomerase [13–17] The anti-tumor drug cisplatin (cis-[PtCl2(NH3)2]), known for its high affinity for G-rich sequences, was
Keywords
cisplatin; c-myc; G-quadruplex; PDGF-A
promoter; telomeric sequences
Correspondence
V Viglasky, Department of Biochemistry,
Faculty of Sciences, Institute of Chemistry,
Safarik University, Moyzesova 11, 04011
Kosice, Slovakia
Fax: +421 55 622 21 24
Tel: +421 55 234 12 62
E-mail: viktor.viglasky@upjs.sk
(Received 30 August 2008, revised 5
November 2008, accepted 7 November
2008)
doi:10.1111/j.1742-4658.2008.06782.x
Naturally occurring G-rich DNA sequences that are able to form G-quad-ruplex structures appear as potential targets for anti-cancer chemotherapy, and therefore play an important role in cellular processes, such as cell aging, death and carcinogenesis The telomeric regions of DNA and nucle-ase hypersensitive elements of human c-myc and PDGF-A promoters repre-sent a very appealing target for cisplatin and may interfere with normal DNA function Platinum complexes bind covalently to nucleobases, and especially to the N7 atom of guanines, and the four guanines of a G-quar-tet have their N7 atoms involved in hydrogen bonding Therefore, within a G-quadruplex structure, only the guanines out of the stack of G-quartets should react with electrophilic species such as platinum (II) complexes Platinum complexes have significant influence on the formation of G-quad-ruplexes Results obtained by CD spectroscopy and temperature gradient-gel electrophoresis clearly demonstrate that DNA platination significantly affects G-quadruplex folding for telomeric sequences; the abundance of
un⁄ misfolded DNAs compared to the G-quadruplex is proportional to the platinum concentration
Abbreviation
TGGE, temperature gradient-gel electrophoresis.
Trang 2found to inhibit telomerase activity in testicular cancer
cells [18] and to reduce telomere length in treated cells
[19] The anti-tumor drug cisplatin forms two kinds of
cross-links with DNA: intrastrand and interstrand
Platinum complexes react with cellular DNA, primarily
binding to the N7 positions of guanine bases, forming
60–65% chelates between adjacent guanines named the
1,2-GG adducts, 20% of 1,2-AG adducts and a small
amount of interstrand cross-links [20] It is unclear
how cisplatin induces cytotoxicity but it is widely
attributed to the formation of the major 1,2-GG
adduct because the tumor response correlates with the
levels of 1,2-GG adducts [21] The formation of
inter-strand crosslinks requires partial disruption of the
Watson–Crick base pairing within double strand
DNA, and the cross-linking reaction could therefore
be expected to be rather slow However, by contrast,
kinetic measurements indicate that interstrand
cross-linking is as fast as intrastrand cross-cross-linking, or even
faster [22] However, the four guanines of a G-quartet
have their N7 atoms involved in hydrogen bonding
Therefore, within a G-quadruplex structure, only the
guanines outside the stack of G-quartets should react
with electrophilic species such as platinum complexes
The influence of platination on the telomeric sequences
was described previously by Garnier et al [22], but the
effect of platination on the quadruplex formation has
never been described in detail Guanine-guanine
cross-linking by platinum atoms either welds together
contiguous guanine residues and stabilizes the
G-qua-druplex or the occupancy of N7 hydrogen bonds
desta-bilizes these structural motifs The suggestion that
platinum complexes significantly affect the structure of
G-quadruplexes is shown
Various G-rich sequences prone to form quadruplex
motifs are investigated in the present study
Tempera-ture gradient-gel electrophoresis (TGGE) has been
applied for the first time to study quadruplex
confor-mational stability The results obtained by CD
spec-troscopy and TGGE clearly demonstrate that the
telomeric G-quadruplexes are very sensitive to covalent
platinum modification by platinum, but not nuclease
hypersensitive elements of human c-myc and PDGF-A
promoters; the abundance of unfolded DNA is
propor-tional to the platinum concentration
Results
CD spectrum of G-rich oligonucleotides
CD spectra have been extensively applied to the study
of G-quadruplex structures It is well known that
par-allel G-quadruplex structures, such as propeller forms,
give a positive band at approximately 263 nm and a negative band at approximately 240 nm, whereas anti-parallel G4 structures, such as basket and chair forms, show two positive bands at approximately 295 and
240 nm and a negative band at approximately 260 nm These spectral features are mainly attributed to the specific guanine stacking in various G4 structures Figure 1 shows the CD spectra of c-MYC, PDGF-A, Tel-1 and Tel-2 oligonucleotides in 50 mm Tris–HCl containing 50 mm K+ cations According to the find-ing of multiple conformations, the 293 nm positive CD band associated with a 265 nm positive shoulder of Tel-1 is probably due to the co-existence of both paral-lel and antiparalparal-lel G4 structures in K+ solution [3,23] The CD pattern of Tel-1 is similar to the CD pattern of d(TAGGGTTAGGGT) and NMR analysis has revealed the co-existence of the dimeric antiparallel and parallel G4 structures in K+solution [23,24] The
CD spectra of different oligonucleotides show that c-MYC and PDGF-A sequences preferentially form the parallel G-quadruplex structure in 50 mm Tris-HCl buffer at pH 7.8, and the maximum of ellipticity is observed at 263 nm These results agree with the mea-surements obtained in previous studies [14] However, telomeric sequences Tel-1 and Tel-2 form a preferen-tially antiparallel configuration of G-quadruplex struc-ture in solutions containing the K+ ion, with the maximum being observed at 293 nm The increase of
K+ facilitates the folding of G-quadruplexes, and the peak at 293 nm increases [25] Interestingly, when a
Fig 1 Comparative CD spectra of four known G-quadruplex-forming sequences: Tel-1 (open circle), Tel-2 (solid circle), c-MYC (diamond) and PDGF-A (triangle) in 50 m M Tris–HCl buffer (pH 7.8) with 50 m M KCl Each spectrum corresponds to three averaged scans taken at 20 C and is baseline corrected for signal contribu-tion due to the buffer.
Trang 3DNA tract of repetition contains more than three
residues of guanines, then a small amount of DNA
without the presence of K+is folded into the
G-quad-ruplex structure at room temperature Defrosted stock
solution of G-rich oligomer in distilled water contains
a nonzero amount of folded G-quadruplex structure,
which is detectable by CD spectroscopy; this was
observed for Tel-2, c-MYC and PDGF-A [26] This is
most likely due to the residual amount of monovalent
ions that was not removed during desalting process,
but is still sufficient for stabilization of the
G-quadru-plex motif
CD spectra of platinated Tel-1 and Tel-2
Platination of oligonucleotides was performed in
dis-tilled water in the presence of 10 mm KClO4, where
the G-quadruplex should form a stable conformation
In the case where the platination is performed in
dis-tilled water without any univalent ions, a very low
amount of folded G-quadruplex is detected by CD
spectroscopy (data not shown) Figure 2 shows the
influence of platination on the G-quadruplex
destabili-zation for Tel-2 Increasing the number of cisplatin
molecules per oligonucleotide rapidly decreases the
positive and negative elliptic peaks of Tel-2 at 293, 242
and 262 nm, respectively The same effects were
observed for five additional oligonucleotides:
d(G3T3)3G3, d(G4T3)3G4, d(G4T2)3G4, d(G4T2)3G4 and
d(G4T2A)3G4(data not shown) The intersected isosbe-stic points were detected nearby, at 237, 252 and
280 nm at the given conditions for Tel-2 An addi-tional increase of the K+ concentration in solution during the collection of CD spectra has no significant effect on the ellipticity of Tel-2 These multi-isosbestic-point spectra are clear evidence for the formation of intermediates [27] A molar ratio of 1 : 1 platinum complexes incubated with DNA oligomer decreases the ellipticity at 293 nm by approximately 20% An increase in the amount of platinum complexes incu-bated with DNA oligonucleotide (four platinum com-plexes per oligomer) causes an additional decrease of this characteristic peak, by approximately 38% A molar ratio of 16 platinum complexes to oligomer can occupy all guanines of Tel-2 oligonucleotide; this decreases the ellipticity by approximately 73% com-pared to the original nonplatinated oligonucleotides under the same conditions A similar effect had also been observed for trans-platinum complex (data not shown) Interestingly, single platinum molecules per parallel c-MYC and PDGF-A do not show the effect
of a decreasing peak at 263 nm, probably due to the preference of cisplatin to bind with guanines occurring
in the loop of the G-quadruplex motif, and these N7 nitrogens of guanine are not associated with Hoogs-teen pairing of the G-tetrad When the amount of platinum complexes achieves a molar ratio of four molecules per oligonucleotide, then the same effect is observed as for Tel-1 and Tel-2; the decrease of ampli-tude of the characteristic peaks for parallel structures
is observed (263 and 240 nm) The decrease of peaks should correlate with the amount of oligomers correctly folded into the quadruplex structure
Analysis of thermal stability of G-quadruplexes The results shown in Fig 3 clearly demonstrate that the number of G-tetrad in quadruplex is the determining factor for the thermal stability of both parallel and antiparallel G-quadruplexes Thermal stability in the presence of KCl is: Tel-1 < Tel-2 < c-MYC < PDGF-A quadruplexes (Table 1) The results again confirm that the concentration of
K+ is a determining factor for the thermal stability
of G-quadruplexes The data shown in Fig 3A,B,D are normalized
TGGE analysis – influence of platination on thermal stabilization
The original data shown in Fig 3C confirm that platination decreases the amount of correctly folded
Fig 2 Representative CD spectra of Tel-2 in 50 m M Tris–HCl
buffer (pH 7.8) with 50 m M KCl Various amounts of platinum
complexes incubated with DNA were used The molar ratios of
platinum complexes incubated per oligomer were: 0 : 1, 1 : 1, 4 : 1
and 16 : 1 All spectra were collected at 20 C and in a strand
concentration of 15 l M
Trang 4oligomers into the antiparallel G-quadruplex
Platina-tion does not principally affect the melting
tempera-tures of Tel-1 and Tel-2; the normalized data shown in
Fig 3D and Table 1 confirm this claim However, the
transition is widespread for platinated oligonucleotides
An explanation of why the covalent modification of
oligonucleotides does not affect thermal stability is
offered by the TGGE results (Fig 4)
In Fig 4, the thermal mobility profiles of platinated
and nonplatinated Tel-1 are compared Tel-1 mobility
shows a sigmoidal profile, as expected based on CD
measurements, but platination causes an increasing
amount of unfolded conformational states (Fig 4,
area highlighted by double arrow) Folded and
unfolded conformations are depicted by solid and
empty arrows, respectively The double arrow
high-lights the ‘smear area’ representing a population of
unfolded and misfolded oligonucleotides The intensity
of this area is increased by the increasing ratio of
cisplatin to DNA
Ridge tracking analysis, as applied for proteins, was
used for this purpose Tm values were the same
(55 ± 2C) for both TGGE and CD measurements
The TGGE results clearly demonstrate that CD spec-troscopy results are a convolution of the misfolded and folded spectra of the G-quadruplexes An identical melting profile of the original (Fig 4, top) and the pla-tinated DNA (Fig 4, bottom) is observed after exclud-ing the smear from the electrophoretic record representing any intermediates (Fig 4) The increased ratio between cisplatin and the oligomer causes an increased abundance of the misfolded population of G-quadruplexes
Discussion
The present study aimed to evaluate whether cisplatin
is able covalently to trap quadruplex structures TGGE and CD spectroscopy were used to characterize the folding of platinated G-quadruplex sequences
This structural motif usually plays an important biological role In particular, the folding of telomeric DNA into the G-quadruplex has been shown to inhi-bit telomerase, an enzyme involved in the maintenance
of the telomere length in cancerous cells [2,14] The human quadruplexes of telomeric sequences have
Fig 3 Normalized elliptic changes of 15 l M c-MYC (A) and PDGF-A (B) oligomers at 260 nm against temperature in 50 m M Tris–HCl buffer (pH 7.8) with 5, 10 and 20 m M KCl Original (C) and normalized elliptic changes (D) of 15 l M Tel-1 and Tel-2 oligomers at 293 nm against temperature in 50 m M Tris–HCl buffer (pH 7.8) with 50 m M KCl 1Pt and 4Pt represent platinated Tel-1; the molar ratios of platinum com-plexes incubated per oligomer were 1 and 4 Melting temperatures of platinated DNAs were not affected by the platinum complex, but less cooperative transitions were observed.
Table 1 Melting temperatures of oligomers at different salt concentration +1 cisplatin represents oligomers platinated by a single molecule
of cisplatin ND, not determined.
Trang 5therefore received attention in the context of the
telomerase inhibition as a potential anti-cancer
ther-apy, using specific small molecules that are able to
stabilize DNA quadruplexes [6,13] Platinum
com-plexes, which are widely used for cancer therapies,
have a high affinity to attack N7 of the guanine
resi-due [20,22] However, N7 nitrogen is associated with
the hydrogen bond stabilization of the G-tetrad The
destabilization of the G-tetrad structure and, finally,
the destabilization of the G-quadruplex structure were
both expected en bloc A proposed model of G-tetrad
destabilization by two molecules of cisplatin is
pro-vided in Fig 5A The variability of the cisplatin mode
to bind with G-rich sequences is vast; in principle,
there is the possibility to bind within each guanine, to
form intrastrand and interstrand bifunctional and
monofunctional adducts, etc Some of the proposed
binding places of studied oligomers possessing a high
affinity for platination are shown in Fig 5B,C for
Tel-1 and c-MYC, respectively The expectation that
folded G-quadruplex would not offer such a wide
spectrum for platinum binding was not observed by
TGGE It is known that platinum related complexes
cause local bending of DNA and steric hindrance for any DNA associated enzymes in the place of their binding [20] Except for these effects, the correct fold-ing of G-quadruplexes is significantly affected by covalent modification due to cisplatin However, for some biological mechanisms, the quadruplexes appear
to be essential
Bertrand et al [28] confirmed that the platination of guanine residues constituting the 5¢ external G-quartet
is feasible by a disruption of Hoogsteen organization, which is particularly favored in the case of the antipar-allel conformation of the quadruplex However, in these studies, the authors had used only human telo-meric sequences forming a hybrid-type intramolecular G-quadruplex structure with mixed parallel⁄ antiparal-lel strands in potassium solution [3] Cation-dependent experiments, which modify the equilibrium between the different quadruplex structures, and molecular modeling both led to the conclusion that the antiparallel and parallel forms exhibit different platination profiles However, it was noted that the identification of the platinable guanines of the mixed-hybrid structure could be problematic [28,29] Human telomeric repeti-tions do not contain any additional residues of guanine
in the connective loop of the G-quadruplex such as that constituting the PDGF-A and c-MYC sequences
In the present study, we did not localize the binding sites of cisplatin for c-MYC and PDGF-A folded oligonucleotides, although it is suggested that unassociated guanines on the formation of G-terad are more preferred for platination After platination of these more accessible guanines, the excess cisplatin attacks another guanine, similar to that occuring for Tel-1 and Tel-2 [28]
It should not be overlooked that adenine in the con-nective loop can also be platinated, and therefore so too can oligomers where adenine was replaced by thymine (data not shown) However, oligomers d(G3T3)3G3, d(G3T4)3G3 and d(G4T3)G4 behave as uniformly as Tel-1 and Tel-2 oligomers No principal discrepancies were observed, either by CD or TGGE The experimental results obtained clearly demonstrate that platinum derivatives affect the compactness of the G-quadruplex structure In addition, transplatin was used instead of cisplatin in the present study, but the results obtained confirmed that both platinum com-plexes significantly affect the G-quadruplex structure, regardless of whether a parallel or an antiparallel structure is formed However, the experimental data
do not offer a clear answer with respect to whether the monofunctional or bifunctional platinum adduct is mainly responsible for Tel-1 and Tel-2 destabilization Nevertheless, the purification of these adducts and
Fig 4 Representative comparative inverted TGGE records of Tel-1
oligomer (top) and Tel-1-platinum complex (bottom) in 50 m M Tris–
HCl pH 7.8 in the presence of 50 m M KCl One molecule of
cisplatin per Tel-1 was used The gradient of temperature was
per-pendicular to the electric field Folded and unfolded G-quadruplex
structures are indicated by dark and white arrows, respectively.
The intermediate structure represents a ‘smear area’ marked by a
double arrow.
Trang 6subsequent mass spectrometry is required to clearly
indicate the number of platinum complexes bound per
oligonucleotide
A distinguishable G-quadruplex conformation after
platination was expected, but the TGGE experiments
did not confirm this fact An increased population of
misfolded structures characterized by various
thermo-dynamic properties can be observed On the other
hand, the platination of guanine in the loop of the
G-quadruplex could stabilize this form, probably due
to DNA bending [17] However, further investigations
are currently in progress that aim to provide a clear
answer about the structural stability of the
G-quadru-plex containing platinable guanine in the connective
loop
To date, determination of the G-quadruplex folding
in the presence of platinum derivatives in K+solution
remains unresolved
Experimental procedures
DNA oligomers (sequences shown in Table 2) were obtained from Sigma-Aldrich (St Louis, MO, USA) and Biosearch Technologies, Inc (Novato, CA, USA) All DNA oligomers were PAGE-purified and dissolved in dou-ble-distilled water before use Acrylamide : bisacrylamide (19 : 1) solution and ammonium persulfate were purchased from Bio-Rad (Hercules, CA, USA), and N,N,N¢,N¢-tetra-methylethylenediamine was purchased from Fisher Slova-kia (Fisher SlovaSlova-kia, Levocˇa, SlovaSlova-kia) T4 polynucleotide kinase was purchased from Promega (Madison, WI, USA) [c-32P]ATP was purchased from Amersham (Arlington Heights, IL, USA) Cisplatin and transplatin were obtained from Sigma-Aldrich The stock solutions of the platinum complexes at a concentration of 5· 10)4m in
10 mm KClO4were prepared under conditions of darkness
at 25C For platination, it is more suitable to use per-chloride instead of per-chloride salts to avoid per-chloride ions
A
Fig 5 (A) Proposed mechanism of G-tetrad destabilization by cisplatin The first platinum molecule can destabilize any of the Hoogsteen pairings and the second cisplatin destabilizes an additional Hoogsteen pairing Four cisplatin molecules per G-tetrad totally disrupt this struc-ture The propensity of cisplatin binding to Tel-1 and c-MYC oligomers is indicated by arrows The size of an arrow is proportional to the affinity of the platinum complex to attack N7 of the quinines The schemes in (B) and (C) represent Tel-1 and c-MYC oligomers, respectively The c-MYC oligomer contains three guanine residues located in loops of the G-quadruplex structure and five residues (gray arrows) that are not directly associated with the G-quadruplex structure, although these guanines are the most preferred for platination The guanines out of the stack of G-quartets should react with electrophilic species such as platinum complexes This explains why the platination G-quadruplexes formed from Tel-1 and Tel-2 are more sensitive than c-MYC and PDGF oligomers and, in addition, why these oligomers can be stabilized by platinum complexes.
Trang 7Single-strand concentrations were determined by measuring
the absorbance (260 nm) at high temperature The
concen-tration of DNA was determined by UV measurements
carried out on Varian Cary 300 Bio UV–visible
spectro-photometer (Amedis, Bratislava, Slovakia) Cells with
opti-cal path lengths of 10 mm were used, and the temperature
of the cell holder was controlled by an external circulating
water bath
CD spectroscopy
CD spectra were recorded on a Jasco J-810
spectropolarime-ter (Jasco Inc., Easton, MD, USA) equipped with a
PTC-423L temperature controller using a quartz cell of
1 mm optical path length and an instrument scanning speed
of 100 nmÆmin)1, with a response time of 2 s, over a
wave-length range of 220–320 nm The scan of the buffer alone
was subtracted from the average scan for each sample CD
spectra were collected in units of millidegrees versus
wave-length and normalized to the total species concentrations
The cell-holding chamber was flushed with a constant stream
of dry nitrogen gas to avoid water condensation on the cell
exterior All DNA samples were dissolved and diluted in
Tris–HCl buffer (50 mm, pH 7.8) and, where appropriate,
the samples contained different concentrations of KCl
(KClO4) The amount of DNA oligomers used in the
experi-ment was maintained at D220–330in the range 0.2–0.8, and the
CD data represent three averaged scans taken at an
experi-mental temperature (25–90C) All CD spectra are
baseline-corrected for signal contributions due to the buffer
Labeling and purification
The DNA oligomers were 5¢-end-labeled with [c32
P]ATP using T4 polynucleotide kinase for 45 min at 37C The
labeling reaction was inactivated by heating the samples at
90C for 5 min after the addition of 1.5 lL of 0.5 m
EDTA The 5¢-end labeled DNA was then purified using a
Bio-Spin 6 chromatography column (Bio-Rad)
Platination of oligonucleotides
For 5¢-end-radiolabeled oligonucleotides, the same
proce-dure was used as described previously [29] Oligomers in
the presence of potassium form a quadruplex structure as confirmed in CD measurements To avoid any multimeric form, the oligomers were heated before platination to 95C and cooled slowly to achieve a final temperature 37C within 1 h Unlabeled oligonucleotides and platinum com-plexes were mixed at ratio 1 : 1, 1 : 4, 1 : 12 and 1 : 16 in
10 mm KClO4 The reaction was performed overnight at
37C in a volume of 10 lL The reaction products were purified on 20% denaturing gel electrophoresis and desalted
on a Sephadex G25 column At least six to nine different bands after denaturing gel electrophoresis were observed, as described previously [29] Only one intensive but ‘smeared’ band was observed under nondenaturing conditions, and this was used for all the CD and TGGE experiments; no additional purification of DNA conformers has been applied
TGGE
TGGE was performed using the same equipment as described previously [30] A temperature gradient was gen-erated in the gel in a direction perpendicular to that of the electrical field The gradient was established on a copper plate placed adjacent to the electrophoretic apparatus by cooling and heating its opposing ends with two indepen-dently circulating water baths DNA samples were run through 15% total polyacrylamide gels [19 : 1 acrylamide: bis(acrylamide)] buffered with 50 mm Tris–HCl (pH 7.8) for 4 h at 6 VÆcm)1 The dried gel was exposed on a phor screen Visualization was performed using a phos-phorimager (Storm 820; Molecular Dynamics, Sunnyvale
CA, USA) and imagetools, version 2.1 (available at: http://ddsdx.uthscsa.edu/dig/itdesc.html) Digital image pro-cessing was used to determine an objective curve consisting
of the darkest points of the electrophoretic records of the deformed electrophoretic band representing the dependence
of DNA mobility on temperature [30]
Acknowledgements
This study was supported by grants from the Slovak Grant Agency (1⁄ 1274 ⁄ 04 and 1 ⁄ 3254 ⁄ 06) and the Science and Technology Assistance Agency (APVT-20-006604) I would like to thank Gavin Cowper and Lenka Sieber for critically reading and correcting the manuscript, my student Lubos Bauer and Professor Vik-tor Brabec for the opportunity to work in his laboraVik-tory and obtain skills with respect to DNA platination
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