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Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides

  • February 2004
  • Nucleosides Nucleotides & Nucleic Acids 22(5-8):1267-72
DOI:10.1081/NCN-120022943
Authors:
Alexandre S Boutorine at Muséum National d'Histoire Naturelle
Alexandre S Boutorine
Vladimir Ryabinin at Institute of Chemical Biology and Fundamental Medicine
Vladimir Ryabinin
Daria Novopashina at Institute of Chemical Biology and Fundamental Medicine
Daria Novopashina
A. G. Ven’yaminova at Russian Academy of Sciences
A. G. Ven’yaminova
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Abstract and Figures

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.
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Stabilization of DNA Double and Triple Helices
by Conjugation of Minor Groove Binders to
Oligonucleotides
A. S. Boutorine a d , V. A. Ryabinin b , D. S. Novopashina c , A. G. Venyaminova c , C. Hélène a
& A. S. Sinyakov b
a Laboratoire de Biophysique , Muséum National d'Histoire Naturelle , INSERM U 565, CNRS
UMR 8646, Paris Cedex, France
b State Research Center for Virology and Biotechnology “Vector” , Novosibirsk Region, Russia
c Institute of Bioorganic Chemistry, Siberian Division , Russian Academy of Sciences ,
Novosibirsk, Russia
d Laboratoire de Biophysique , Museum National d'Histoire Naturelle , 43, rue Cuvier, Paris
Cedex 05, F-75231, France
Published online: 31 Aug 2006.
To cite this article: A. S. Boutorine , V. A. Ryabinin , D. S. Novopashina , A. G. Venyaminova , C. Hélène & A. S. Sinyakov
(2003) Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides,
Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1267-1272, DOI: 10.1081/NCN-120022943
To link to this article: http://dx.doi.org/10.1081/NCN-120022943
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Stabilization of DNA Double and Triple Helices
by Conjugation of Minor Groove Binders
to Oligonucleotides
A. S. Boutorine,
1,
*V. A. Ryabinin,
2
D. S. Novopashina,
3
A. G. Venyaminova,
3
C. He
´le
`ne,
1
and A. S. Sinyakov
2
1
Laboratoire de Biophysique, Muse
´um National d’Histoire Naturelle, INSERM
U 565, CNRS UMR 8646, Paris Cedex, France
2
State Research Center for Virology and Biotechnology ‘‘Vector’’,
Novosibirsk Region, Russia
3
Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of
Sciences, Novosibirsk, Russia
ABSTRACT
New conjugates containing two parallel or antiparallel carboxamide minor
groove binders (MGB) attached to the same terminal phosphate of one oligonu-
cleotide strand were synthesized. The conjugates interact with their target DNA
stronger than the individual components. Effect of conjugated MGB on DNA
duplex and triplex stability and their sequence specificity was demonstrated on
the short oligonucleotide duplexes and on the triplex formed by model 16-mer
oligonucleotide with HIV polypurine tract.
Key Words: Oligonucleotides; Minor groove binders; Oligocarboxamides;
Conjugation; Duplex; Triple helix.
*Correspondence: A. S. Boutorine, Laboratoire de Biophysique, Museum National d’Histoire
Naturelle, 43, rue Cuvier, Paris Cedex 05 F-75231, France; Fax: þ33 1 4079 3705; E-mail:
boutorin@mnhn.fr.
NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1267–1272, 2003
1267
DOI: 10.1081/NCN-120022943 1525-7770 (Print); 1532-2335 (Online)
Copyright #2003 by Marcel Dekker, Inc. www.dekker.com
Downloaded by [Institute of Chemical Biology & Fundamental Medicine] at 01:49 26 February 2015
Two classes of ligands recognizing double-stranded DNA in a sequence-specific
manner are known:N-methylpyrrole=N-methylimidazole oligocarboxamide minor
groove binders (MGB)
[1,2]
and triple helix-forming oligonucleotides (TFO).
[3]
Being
covalently linked together, these ligands are able to bind simultaneously to the major
and the minor grooves of the target dsDNA
[4,5]
and thus potentially interfere with
key cellular processes such as replication and transcription.
The conjugation of oligonucleotides to N-terminal amino group of oligocar-
boxamide minor groove binders was carried out by activation of oligonucleotide
terminal phosphate with triphenylphosphine=dipyridyldisulfide (Fig. 1).
[6,7]
After
incubation with activators in organic media the second activation of the same ter-
minal phosphate group could take place and the second residue of oligocarboxamide
MGB could be attached via prosphorodiamidate formation.
Using this method, we obtained a series of short DNA duplexes where one oli-
gonucleotide strand was attached to two parallel (N !C) or antiparallel (N !C,
C!N) oligocarboxamide residues (Table 1). The conjugates were purified by HPLC
on reverse phase (C18) and analyzed by denaturing gel electrophoresis and
UV-spectrophotometry.
[7]
Phosphorodiamidate structure of 1:2 conjugates was
confirmed by mass-spectrometry and
31
P-NMR (161.95 MHz).
Sequence-specific stabilization of DNA duplexes by one or two MGB residues
conjugated to one duplex strand has been shown by thermal denaturation method.
The results are shown in Table 1. The most important conclusion is that two
parallel or antiparallel tetrapyrrole ligands attached to the same strand of a duplex
A have similar stabilizing effect on the double-stranded DNA with a complemen-
tary A:T strands (DTm ¼40±4
C) as a hairpin ligand (Py)
4
-g-(Py)
4
described by
Dervan.
[1,8,9]
Figure 1. Synthesis of oligonucleotide conjugates with carboxamide minor groove binders.
1268 Boutorine et al.
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In order to confirm sequence specificity of dsDNA:2(MGB) interaction we have
synthesized a duplex B with alternating G:C pairs. We constructed and attached to
one strand three sequence-specific MGB combinations that must recognize this
sequence:a hairpin octacarboxamide and two linear tetracarboxamides in parallel
or antiparallel orientation (Table 1). According to results reported earlier,
[10,11]
we
replaced one methylpyrrole residue in every tetracarboxamide chain by more flexible
b-alanyl unit. All the three combinations strongly stabilized the target duplex (Table
1, DTm ¼20–30C). This stabilization was observed only when both carboxamide
chains were designed according to Dervan rules
[1,8,9]
and covalently attached to oli-
gonucleotide strand. No stabilizing effect of free MGBs was detected (data not
shown).
These results demonstrate a strong and a sequence specific affinity of the two
linked oligocarboxamide chains for double-stranded DNA that is comparable
(if not better) with classic hairpin MGB conjugates. In order to combine dsDNA-
binding properties of both components in one molecule, we attached one and two
hairpin hexa- or octamethylpyrroles to 16-mer oligopyrimidine DNA or 20-O-methyl
RNA oligonucleotide T
4
CT
4
C
6
T (designated as HIV-T) via triethyleneglycolpho-
sphate linker (Fig. 1). This linker is long enough to circumvent one of the duplex
strands and to connect MGB in the minor groove with TFO in the major groove.
[4]
The oligonucleotide forms a triple helix with a conservative polypurine tract of HIV
proviral DNA which is located in genes pol and nef of the provirus (artificial target in
the form of double-stranded hairpin HIV-Loop is shown on Fig. 2). The remarkable
feature of this sequence is that its 50-adjacent region contains a large A:T tract, an
ideal target for oligopyrrole MGB.
Table 1. Thermal denaturation temperatures of duplexes containing one strand conjugated
to one or two minor groove binders. Conditions of thermal denaturation experiments:
0.01 M phosphate buffer 0.1 M NaCl-0.001 M EDTA, oligonucleotide concentrations 1.3–
3mM, temperature change rate 0.2C=min, detection at 260 and 330 nm.
Expt nDuplex X
1
X
2
T
m
,C
1AO
O
20
2AO
NH(CH
2
)
5
CO(Py)
4
NH(CH
2
)
3
NEt
2
46
3ANH(CH
2
)
5
CO(Py)
4
NH(CH
2
)
3
NEt
2
NH(CH
2
)
5
CO(Py)
4
NH(CH
2
)
3
NEt
2
60
4AO
NH(CH
2
)
5
CO(Py)
4
-
NH(CH
2
)
3
CO(Py)
4
NH(CH
2
)
3
NET
2
58
5ANH(CH
2
)
5
CO(Py)
4
OEt Py(Py)
3
NH(CH
2
)
3
NH56
6BO
O
26
7BO
NH(CH
2
)
5
COImbImPy-
NH(CH
2
)
3
COImbImPyNH(CH
2
)
3
NMe
2
56
8BNH(CH
2
)
5
COImbImPyNH(CH
2
)
3
NMe
2
BocNH(CH
2
)
5
COImbImPyNH(CH
2
)
3
NH52
9BNH(CH
2
)
5
COImbImPyNH(CH
2
)
3
NMe
2
NH(CH
2
)
5
COPyImbImNH(CH
2
)
3
NMe
2
46
: place of attachment to the terminal phosphate (p) of oligonucleotide strand.
Stabilization of DNA Double and Triple Helices 1269
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Figure 3. Gel retardation experiments at 10C (non-denaturating polyacrylamide gel; 20% in
A) 50 mM MES buffer, 0.05 M NaCl, 0.005 M MgCl
2
, pH 6.0; B) 50 mM HEPES buffer,
0.05 M NaCl, 0.005 M MgCl
2
, pH 7.0; C) 50 mM TBE buffer, 0.05 M NaCl, 0.005 M MgCl
2
,
pH 8.3. 1. Oligonucleotide HIV-Loop (50-
32
P), 2. HIV-Loop with non-modified HIV-T;
3. HIV-Loop with the conjugate HIV-T-(Py)
3
-g-(Py)
3
;4. HIV-Loop with the conjugate
HIV-T-[(Py)
3
-g-(Py)
3
]
2
. Concentration of HIV-Loop is 50 nM, HIV-T-20 mM. Positions of
duplex (D) and triplex (T) are indicated by a
´rrows.
Figure 2. Sequence of the target HIV-Loop DNA and of the triple-helix forming oligo-
nucleotide HIV-T. Original sequence of the HIV proviral polypurine tract fragment from
genes pol and nef is indicated by arrows. For technical reasons, the target sequence was syn-
thesized as a hairpin with two complementary strands connected by tetrathymidylate linker.
Two versions of HIV-T were used:with i) DNA backbone and ii) 20-O-methyl-RNA back-
bone. All the cytosines in DNA version (in italics) were methylated at position 5.
1270 Boutorine et al.
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Formation of the complex between conjugates and the target was demonstrated
by electrophoresis on a non-denaturing polyacrylamide gel with
32
P-labeled double-
stranded target HIV-Loop (gel retardation). As is seen from Fig. 3, all conjugates
form stable triplexes in standard conditions (MES buffer, pH 6 and 7), independently
of the backbone structure (DNA or 20-O-methyl RNA).
TFO with only one attached hairpin hexapyrrole formed slightly more stable tri-
plex (37C compared to 24C for non-modified oligonucleotide) that dissociated at
pH >6. When mismatched oligonucleotide was used, no interaction with a duplex
was detected in the case of 1:1 TFO:MGB conjugate. However, conjugates with
two parallel MGB residues form stable complexes with double-stranded target even
at pH ¼8.3 and at the temperature >55C(Fig. 3). Moreover, the complex of the
target with 1:2 conjugates was formed under these extreme conditions even when
TFO was substituted by a short non-complementary oligonucleotide C
4
T. (data
not shown). It means that, in contrast to 1:1 TFO:MGB conjugates, interaction of
1:2 conjugates with double-stranded target is mainly determined by MGB compo-
nent. Taking into account the size of A:T-rich region of the target DNA and possi-
bility to have different conformations for two attached MGB residues, we proposed a
hypothesis of complex formation mechanism that is shown on Fig. 4.
Strong and sequence-specific dsDNA-binders can serve as the base for potential
therapeutic agents acting on the level of the genomic DNA.
ACKNOWLEDGMENTS
This work was supported by European Community (INTAS Open Project
01-0638) and French Ministry of Foreign Affairs (EGIDE 04542ND). We thank
Mr. Ludovic Halby for excellent technical assistance.
Figure 4. Hypothesis of complex formation and dissociation mechanisms between oligo-
nucleotide-TFO conjugates and target double stranded DNA.
Stabilization of DNA Double and Triple Helices 1271
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... A code for the binding of MGB polyamide hairpin motifs has been proposed wherein Py/Im, Im/Py, Hp (3-hydroxy-1-methylpyrrole)/ Py and Py/Hp combinations recognize C-G, G-C, T-A, and A-T base pairs, respectively [39][40][41][42][43]. MGB polyamide hairpin motifs can recognize many different sequences of dsDNA and bind in the minor groove of dsDNA according to a set of pairing rules. Novopashina et al. and Boutorine et al. have reported that oligonucleotides conjugated with MGB polyamide hairpin motifs to either the 5 ′ -or 3 ′ -end formed stable dsDNA with target DNA by sequencespecific dsDNA stabilization of MGB polyamide hairpin motifs [16][17][18][19][20][21]. ...
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  • Jan 1998
  • Nucleos Nucleot
Oligonucleotide derivatives carrying a side chain of either lysine or histidine at the 3′-end and their complementary oligonucleotides having photoreactive groups a p-azidophenyl-NH(CH2)nNH- (n = 4, 6) residue at the 5′-end were prepared by using new phosphorylating species formed by treatment of oligonucleotides with Ph3P and (PyS)2 or (PyrS)2. in DMF, DMSO or their mixture. Efficient cross-linking of duplexes occurred under UV-irradiation (λ > 300 nm).
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Aliphatic/Aromatic Amino Acid Pairings for Polyamide Recognition in the Minor Groove of DNA
Article
  • Jun 1998
Selective placement of an aliphatic β-alanine (β) residue paired side-by-side with either a pyrrole (Py) or imidazole (Im) aromatic amino acid is found to compensate for sequence composition effects for recognition of the minor groove of DNA by hairpin pyrrole−imidazole polyamides. A series of polyamides were prepared which contain pyrrole and imidazole aromatic amino acids, as well as γ-aminobutyric acid (γ) “turn” and β-alanine “spring” aliphatic amino acid residues. The binding affinities and specificities of these polyamides are regulated by the placement of paired β/β, Py/β, and Im/β residues. Quantitative footprint titrations demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-γ-6) with two Py/β pairings affords 10-fold enhanced affinity and similar sequence specificity for an 8-bp target sequence. The 6-γ-6 hairpin ImPyImPyPyPy-γ-ImPyPyPyPyPy-β-Dp, which contains six consecutive amino acid pairings, is unable to discriminate a single-base-pair mismatch site 5‘-TGTTAACA-3‘ from a 5‘-TGTGAACA-3‘ match site. The hairpin polyamide Im-β-ImPyPyPy-γ-ImPyPyPy-β-Py-β-Dp binds to the 8-bp match sequence 5‘-TGTGAACA-3‘ with an equilibrium association constant of Ka = 2.4 × 1010 M-1 and ≥48-fold specificity versus the 5‘-TGTTAACA-3‘ single-base-pair mismatch site. Modeling indicates that the β-alanine residue relaxes ligand curvature, providing for optimal hydrogen bond formation between the floor of the minor groove and both Im residues within the Im-β-Im polyamide subunit. This observation provided the basis for design of a hairpin polyamide, Im-β-ImPy-γ-Im-β-ImPy-β-Dp, which incorporates Im/β pairings to recognize a “problematic” 5‘-GCGC-3‘ sequence at subnanomolar concentrations. These results identify Im/β and β/Im pairings that respectively discriminate G·C and C·G from A·T/T·A as well as Py/β and β/Py pairings that discriminate A·T/T·A from G·C/C·G. These aliphatic/aromatic amino acid pairings will facilitate the design of hairpin polyamides which recognize both a larger binding site size as well as a more diverse sequence repertoire.
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Discrimination of 5'-GGGG-3', 5'-GCGC-3', and 5'-GGCC-3' sequences in the minor groove of DNA by eight-ring hairpin polyamides
Article
  • Jul 1997
Eight-ring hairpin polyamides which differ only by the linear arrangement of pyrrole (Py) and imidazole (Im) amino acids were designed for recognition of six base pair DNA sequences containing four contiguous G,C base pairs. The respective DNA binding properties of three polyamides, ImImPyPy-γ-ImImPyPy-β-Dp, ImPyImPy-γ-ImPyImPy-β-Dp, and ImImImIm-γ-PyPyPyPy-β-Dp, were analyzed by footprinting and affinity cleavage on a DNA fragment containing the respective match sites 5‘-TGGCCA-3‘, 5‘-TGCGCA-3‘, and 5‘-TGGGGA-3‘. Quantitative footprint titrations demonstrate that ImImPyPy-γ-ImImPyPy-β-Dp binds the designed match site 5‘-TGGCCA-3‘ with an equilibrium association constant of Ka = 1 × 1010 M-1 and >250-fold specificity versus the mismatch sequences, 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘. The polyamides ImPyImPy-γ-ImPyImPy-β-Dp and ImImImIm-γ-PyPyPyPy-β-Dp recognize their respective 5‘-TGCGCA-3‘ and 5‘-TGGGGA-3‘ match sites with reduced affinity relative to ImImPyPy-γ-ImImPyPy-β-Dp, but again with high specificity with regard to mismatch sites. These results expand the DNA sequence repertoire targeted by pyrrole-imidazole polyamides and identify sequence composition effects which will guide further second-generation polyamide design for DNA recognition.
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Recognition of the Minor Groove of DNA by Hairpin Polyamides Containing α-Substituted-β-Amino Acids
Article
  • Jun 2000
Incorporation of the flexible amino acid β-alanine (β) into hairpin polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids is required for binding to DNA sequences longer than seven base pairs with high affinity and sequence selectivity. Pairing the α-substituted-β-amino acids (S)-isoserine (SIs), (R)-isoserine (RIs), β-aminoalanine (Aa), and α-fluoro-β-alanine (Fb) side-by-side with β in hairpin polyamides alters DNA binding affinity and selectivity relative to the parent polyamide containing a β/β pairing. Quantitative DNase I footprinting titration studies on a restriction fragment containing the sequences 5‘-TGCNGTA-3‘ (N = A, T, G, and C) show that the polyamide ImPySIsImPy-γ-PyPyβImPy-β-Dp (SIs/β pairing) binds to N = T (Ka = 4.5 × 109 M-1) in preference to N = A (Ka = 6.2 × 108 M-1). This result stands in contrast to the essentially degenerate binding of the parent ImPyβImPy-γ-PyPyβImPy-β-Dp (β/β pairing) to N = T and N = A, and to the slight preference of ImPyβImPy-γ-PyPySIsImPy-β-Dp (β/SIs pairing) to N = A over N = T. Additionally, this study reveals that incorporation of RIs, Aa, and Fb into polyamides significantly reduces binding affinity. Therefore, DNA binding in the minor groove is sensitive to the stereochemistry, steric bulk, and electronics of the substituent at the α-position of β-amino acids in hairpin polyamides containing β/β pairs.
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Trauger JW, Baird EE, Dervan PBRecognition of DNA by designed ligands at subnanomolar concentrations. Nature 382:559-561
Article
  • Sep 1996
Small molecules that specifically bind with high affinity to any predetermined DNA sequence in the human genome would be useful tools in molecular biology and potentially in human medicine. Simple rules have been developed to control rationally the sequence specificity of minor-groove-binding polyamides containing N-methylimidazole and N-methylpyrrole amino acids. Two eight-ring pyrrole-imidazole polyamides differing in sequence by a single amino acid bind specifically to respective six-base-pair target sites which differ in sequence by a single base pair. Binding is observed at subnanomolar concentrations of ligand. The replacement of a single nitrogen atom with a C-H regulates affinity and specificity by two orders of magnitude. The broad range of sequences that can be specifically targeted with pyrrole-imidazole polyamides, coupled with an efficient solid-phase synthesis methodology, identify a powerful class of small molecules for sequence-specific recognition of double-helical DNA.
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Sequence-specific DNA recognition by polyamides
Article
  • Jan 2000
  • CURR OPIN CHEM BIOL
Sequence-specific DNA-binding small molecules that can permeate cells could potentially regulate transcription of specific genes. Simple pairing rules for the minor groove of the double helix have been developed that allow the design of ligands for predetermined DNA sequences. Some of these polyamides have been shown to inhibit specific gene expression in mammalian cell culture.
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Triple helix formation and the antigene strategy for sequence-specific control of gene expression
Article
  • Jan 2000
  • Biochim Biophys Acta
Specific gene expression involves the binding of natural ligands to the DNA base pairs. Among the compounds rationally designed for artificial regulation of gene expression, oligonucleotides can bind with a high specificity of recognition to the major groove of double helical DNA by forming Hoogsteen type bonds with purine bases of the Watson-Crick base pairs, resulting in triple helix formation. Although the potential target sequences were originally restricted to polypurine-polypyrimidine sequences, considerable efforts were devoted to the extension of the repertoire by rational conception of appropriate derivatives. Efficient tools based on triple helices were developed for various biochemical applications such as the development of highly specific artificial nucleases. The antigene strategy remains one of the most fascinating fields of triplex application to selectively control gene expression. Targeting of genomic sequences is now proved to be a valuable concept on a still limited number of studies; local mutagenesis is in this respect an interesting application of triplex-forming oligonucleotides on cell cultures. Oligonucleotide penetration and compartmentalization in cells, stability to intracellular nucleases, accessibility of the target sequences in the chromatin context, the residence time on the specific target are all limiting steps that require further optimization. The existence and the role of three-stranded DNA in vivo, its interaction with intracellular proteins is worth investigating, especially relative to the regulation of gene transcription, recombination and repair processes.
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Designed Sequence-Specific Minor Groove Ligands
Article
  • Feb 2000
  • ANNU REV BIOPH BIOM
In the past decade, a general design for sequence-specific minor groove ligands has evolved, based on the natural products distamycin and netropsin. By utilizing a basic set of design rules for connecting pyrrole, imidazole, and hydroxypyrrole modules, new ligands can be prepared to target almost any sequence of interest with both high affinity and specificity. In this review we present the design rules with a brief history of how they evolved. The structural basis for sequence-specific recognition is explained, together with developments that allow linking of recognition modules that enable targeting of long DNA sequences. Examples of the affinity and specificity that can be achieved with a number of variations on the basic design are given. Recently these molecules have been used to compete with proteins both in vitro and in vivo, and a brief description of the experimental results are given.
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