Oxidation of an oligonucleotide-bound CeIII/Multiphosphonate complex for site-selective DNA scission

Article abstract of DOI:10.1002/chem.200902169

Oligodeoxyribonucleotide conjugates of ethylenediamine- N, N, N′, N′-tetrakis(methylenephospho- nic acid) (EDTP) have been used to place a Ce^III/EDTP complex in close proximity to predetermined phospho- diester linkages of a complementary targe

Full text of DOI:10.1002/chem.200902169

FULL PAPER
DOI: 10.1002/chem.200902169
Oxidation of an Oligonucleotide-Bound CeIII/Multiphosphonate Complex for
Site-Selective DNA ACHTNUTGRNEUNGScission
Tuomas Lçnnberg,[a, b] Yuichiro Aiba,[a] Yuya Hamano,[a] Yoshitaka Miyajima,[a]
Jun Sumaoka,[a] and Makoto Komiyama*[a]
Abstract:
conjugates
N,N,N’,N’-tetrakis(methylenephospho-
ACHTUNGTRENNUNGnic acid) (EDTP) have been used to
Oligodeoxyribonucleotide
of ethylenediamine-
linkage. No cleavage occurs at the
other single-stranded regions, which
suggests that the catalytic Ce species is
strictly localized next to the target
phosphodiester linkage. No decrease in
the reaction rate is observed upon in-
troduction of scavengers for hydroxyl
radicals (such as DMSO or MeOH) or
singlet oxygen (such as NaN3) to the
system; this indicates that the reaction
proceeds via a hydrolytic pathway. Any
significant contribution by an oxidative
pathway is further ruled out by the ob-
servation that nucleosides remain
intact after incubation with CeIV/EDTP
complex for extended periods.
place a CeIII/EDTP complex in close
proximity to predetermined phospho-
diester linkages of a complementary
target oligonucleotide. In the presence
of atmospheric oxygen, the CeIII is oxi-
dized into CeIV which, in turn, efficient-
ly cleaves the target phosphodiester
Keywords: cerium · DNA · DNA
cleavage · hydrolysis
Introduction
cleaving agents that are directly compatible with enzymatic
DNA manipulation used in current molecular biology and
biotechnology.
Preparation of man-made cutters for site-selective scission
of DNA has been one of the most attractive and challenging
themes of bioorganic chemistry, mainly because of their po-
tential applications in advanced molecular biology, therapy,
and many other fields.[1,2] Many elegant systems utilizing ox-
idative cleavage of the target DNA have already been de-
veloped and used for various purposes (e.g., structural
probes of DNA/protein or DNA/drug interactions, as well as
inhibition of target gene expression). Artificial hydrolytic
cutters, which cut DNA at a desired site through hydrolysis
of the target phosphodiester linkage, on the other hand,
have been limited in number despite great demand for
Among the metal catalysts reported to hydrolyze the im-
mensely stable phosphodiester linkages in DNA, CeIV ion is
characterized by its high scission activity, being able to hy-
drolyze DNA under physiological conditions.[3] In previous
papers,[4] a single-stranded DNA substrate was selectively
cleaved at the target site by addition of CeIV salts (e.g.,
CeACHTUNTRGENNUG(NH4)2ACHUTNGTREN(NGUN NO3)6) to oligonucleotide–ligand conjugates (or
peptide nucleic acid–ligand conjugates), thus placing the
CeIV complexes at a predetermined site (Figure 1b). In
these artificial cutters, however, large excess of the CeIV salt
to the conjugate (e.g., 100–1000 fold) is usually needed for
efficient cleavage due to competitive formation of the hy-
droxide gel of CeIV around pH 7.[5,6] The large excess of
CeIV, in turn, gives rise to off-target scission at other single-
stranded regions of the target DNA. Clearly, further im-
provements in site selectivity and scission efficiency are re-
quired for more versatile and practical applications of these
tools.
[a] Dr. T. Lçnnberg,+ Y. Aiba,+ Y. Hamano,+ Dr. Y. Miyajima,
Dr. J. Sumaoka, Prof. M. Komiyama
Research Center for Advanced Science and Technology
The University of Tokyo, 4-6-1 Komaba, Meguro-ku
Tokyo 153-8904 (Japan)
Fax : (+81)03-5452-5209
E-mail: komiyama@mkomi.rcast.u-tokyo.ac.jp
This paper describes a novel strategy for the preparation
of well-characterized CeIV-based site-selective cutters for
single-stranded DNA scission. As depicted in Figure 1a, an
easily tractable CeIII salt, which is markedly less prone to gel
formation, is used as a precursor of the catalytically active
CeIV species. As a ligand, ethylenediamine-N,N,N’,N’-tetra-
[b] Dr. T. Lçnnberg+
Current address: Department of Chemistry
The University of Turku, Vatselankatu 2, Turku 20014 (Finland)
[+] T.L. started this work, and Y.A. and Y.H. completed it.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902169.
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855

adding CeACTHNUGTRNEUNG(NO3)3 to the mixture and carried out at 508C
and pH 7.0 for 20 h under air. Rapid oxidation of the CeIII
in its EDTP complex to CeIV under these conditions was
confirmed by the decrease in the intensity of luminescence
from the CeIII in the 350–450 nm region (see Figure 2; CeIV
is emission-silent). About 90% of the CeIII was converted to
CeIV in 1.5 h, and ultimately the oxidation was almost com-
plete.[9] No turbidity was observed in the solutions through-
out the oxidation.
Figure 2. Change of the emission spectrum of a mixture of Ce
ACHTUGNRTNE(NUNG NO3)3 and
EDTP upon incubation under air at pH 7.0 and 508C; [Ce(NO3)3]=8 mm,
AHCTUNGTRENNUNG
[EDTP]=2 mm in 5 mm HEPES buffer ([NaCl]=100 mm). Immediately
after the preparation of the sample (the blue line), the spectra were
taken at 10 min intervals (from the top to the bottom).
Figure 1. a) Strategy developed in this paper for the in situ preparation
of site-selective DNA cleavage from CeIII salt. A CeIII salt is first added
to EDTP–ODN conjugate, and the resultant CeIII complex is then oxi-
dized to the corresponding CeIV complex. Minimized unbound catalytic
species gives rise to clear-cut site-selective scission. Selective scission is
achievable even with the use of only one EDTP–ODN conjugate (see
text for details). b) Previously employed methods: A CeIV salt is directly
added to ligand–ODN conjugate. In order to form the active CeIV com-
plex in the presence of competitive formation of CeIV hydroxide gel, a
large excess of CeIV (e.g., 100–1000 fold) is necessary, leading to off-
target scission. c) Structure of EDTP–ODN conjugate used in a).
Figure 3 shows the PAGE patterns for the hydrolysis of
DNA(S1) (85-mer) using various combinations of ODN addi-
tives (20-mers in Figure 3a). In lane 5, 1:1 mixture of
ODN1–EDTP and EDTP–ODN2 was used to place a CeIV/
EDTP complex on both sides of the gap. Notable scission
selectively occurred at the 5-base gap-site (T41–G45), yield-
ing five bands corresponding to the scission of each phos-
phodiester linkage. The scission in the middle of the gap
was especially strong (the total conversion of the scission
was approximately 13%).[10] The DNA scission rate steeply
kis(methylenephosphonic acid) (EDTP) is employed, since
it can strongly bind both CeIII and CeIV to prevent CeIV ions
from aggregating and to recruit them to the cleavage site.[7]
The CeIV/EDTP complex for site-selective DNA hydrolysis
is prepared by oxidizing the CeIII/EDTP complex which is
bound to an oligodeoxyribonucleotide (ODN). The resultant
CeIV/EDTP–ODN complex is free from undesired formation
of CeIV hydroxide gel observed in previously reported CeIV
salt-derived DNA cutters, and shows clear-cut site-selective
scission.
increased with increasing [Ce
when the mole ratio of Ce(NO3)3 to each of the EDTP–
ODN conjugates was around 2–4 (Figure 3c).[11] Increasing
[Ce(NO3)3] beyond this saturation level resulted in off-
target scission, although cleavage of the target site was still
favoured (lane 9, Figure 3c). As expected, no scission oc-
curred when all reactions were carried out under nitrogen,
because the oxidation of the CeIII to CeIV is not possible
(data not presented). When CeCl3 was used in place of
ACHTUNGTREN(NNUG NO3)3], and attained a plateau
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Ce
ACHTUNGTRENNUNG
the use of Ce
N
N
ACHTUNGTRENNUNG
no site-selective scission was observed. Apparently, gel for-
mation of the CeIV ions from the CeIV salt outcompeted the
complex formation with the EDTP. The CeIII salt-derived
DNA cutters are thus far more clear-cut.
Results and Discussion
According to the previously described procedure,[8] an
EDTP ligand was attached to either the 5’- or the 3’-end of
an ODN (Figure 1c). By using two of these EDTP–ODN
conjugates, a five-base gap for site-selective scission was
formed in the middle of a single-stranded DNA substrate
(see Figure 1 a).[5] The DNA hydrolysis was started by
In lanes 6 and 7 of Figure 3b, only one CeIV/EDTP com-
plex was placed on either the 5’- or the 3’-side of the gap
(the ODN1–EDTP/H–ODN2 and the ODN1–H/EDTP–
ODN2 combinations, respectively). The site-selective scission
was still successful, although the scission efficiency was
856
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Chem. Eur. J. 2010, 16, 855 – 859

DNA Cleavage
FULL PAPER
Figure 3. Site-selective DNA scission by using CeIII-derived DNA cutters. a) Substrate and ODN additives used in this study. The gap site is underlined.
b) Denaturing 20%-PAGE patterns for site-selective scission of DNA(S1) using various combinations of ODNs: lane 1, marker; lane 2, DNA only; lane 3,
CeIII only (without ODN additives); lane 4, ODN1–H/H–ODN2; lane 5, ODN1–EDTP/EDTP–ODN2; lane 6, ODN1–EDTP/H–ODN2; lane 7, ODN1–H/
EDTP–ODN2; lane 8, ODN1–P/P–ODN2; lane 9, ODN1–P/H-ODN2; lane 10, ODN1–H/P–ODN2. Reaction conditions: [DNA(S1)]=1 mm, [each of ODN
additives]=1 mm, [Ce
tive DNA scission on the concentration of Ce
Ce(NO3)3: [Ce(NO3)3]/[EDTP–ODN]=0 (lane 2), 1 (lane 3), 1.5 (lane 4), 2 (lane 5), 3 (lane 6), 4 (lane 7), 5 (lane 8), and 10 (lane 9). In order to simplify
the system, DNA(S2) (5’-FAM-CAATTAGAATCAGGAATGGCTTATGGTGCAGACTGTCGACCTAAG), which provides no single-stranded over-
hangs, was used as substrate in place of DNA(S1)
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
A
R
ACHTUNGTRENNUNG
.
lower than that in lane 5. It is noteworthy that, in lanes 5–7
in Figure 3b, neither of the overhang regions of DNA(S1)
(from T1–T20 and T66–C85) was cleaved although these
portions were also single-stranded. Apparently, the present
site-selective scission is primarily ascribed to “proximity
effect”: the catalytically active CeIV/EDTP complexes are
strictly localized at the target site, and thus off-target scis-
sion is avoided.
dized to CeIV. Then, the DNA substrate was added to the
mixture. Both the scission efficiency and the selectivity
(lane 3) were essentially the same as without the pre-oxida-
tion procedure (lane 2). Thus, the possibility that free radi-
cals, formed in situ by Fenton-like chemistry, cleave the
DNA via an oxidative pathway was further ruled out (note
that CeIII is required to produce a hydroxyl radical in
Fenton-like chemistry).
In contrast to these successful site-selective DNA scis-
sions, virtually no scission occurred under the same condi-
tions when no EDTP group was bound to the ODN
(ODN1–H/H–ODN2 combination in lane 4 of Figure 3b).
Even when one monophosphate group was attached to
either or both of these two ODN additives (ODN1–P and/or
P–ODN2) to recruit the catalytic Ce species to the cleavage
site, no scission was observed (lanes 8–10). Superiority of
EDTP as the ligand for the site-selective DNA scission by
CeIII-derived CeIV is, hence, evident. In the absence of
EDTP, the CeIV species, formed by in situ oxidation of CeIII,
rapidly aggregates and thus its local concentration at the de-
sired scission site is only marginal.
To assess the possibility of an oxidative cleavage mecha-
nism, the reactions were also carried out in the presence of
various scavengers for oxidation species (Figure 4a). With
100 mm DMSO (a scavenger of hydroxyl radicals), 2.5m
MeOH (also a scavenger of hydroxyl radicals), or 10 mm
NaN3 (a scavenger of singlet oxygen),[12] no measurable re-
tardation was observed. This confirmed that neither hydrox-
yl radical nor singlet oxygen plays an important role in the
The argument against an oxidative cleavage mechanism
was further borne out by experiments involving incubation
of nucleosides with either CeIII-derived CeIV/EDTP (Fig-
ure 5a and b) or FeII/EDTA (Figure 5c). Relatively high cat-
alyst and substrate concentrations were used to compensate
for the fact that the effective local concentration of the
CeIII-derived CeIV/EDTP complex in the present DNA cut-
ters is expected to be large due to the proximity effect.
After 20 h in the CeIV/EDTP solution, 2’-deoxyguanosine,
the most readily oxidized naturally occurring nucleoside, re-
mained completely intact (Figure 5b).[13] In striking contrast,
after only 30 min in the FeII/EDTA solution, a significant
portion of the same nucleoside was oxidized, yielding gua-
nine and other products (Figure 5c).[14]
Conclusions
Unprecedentedly well-characterized site-selective DNA cut-
ters have been obtained by oxidation of CeIII ion bound to
the oligonucleotide–ligand conjugate. It is noteworthy that
the amount of CeIII salt, with respect to the ligand, is far
smaller than reported previously for CeIV salt-derived DNA
cutters. Thus, the amount of unbound catalytic species in the
present DNA scission. In Figure 4b, CeACTHNURGTNEG(UN NO3)3 was first in-
cubated with EDTP–ODN under air at pH 7.0 and 508C for
3 h, during which time almost all (99%) of the CeIII was oxi-
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857

M. Komiyama et al.
reaction mixtures can be minimized and undesired off-target
scission hardly takes place. The present finding should pro-
vide a new approach for the design of novel and highly emi-
nent site-selective DNA cutters.
Experimental Section
Commercially available CeACHTUNTRGENNUG(NO3)3ACHTUNGTREN(NUNG H2O)6 (from Strem Chemicals Inc.)
was used without further purification. EDTP was purchased from Tokyo
Chemical Industry Co. The 5’-FAM-labeled substrate ODN and marker
ODNs were purchased from Sigma Genosys and purified by usual meth-
ods. The oligonucleotide–EDTP conjugates were prepared by the con-
ventional phosphoramidite strategy followed by on-support oxime cou-
pling, as described in Scheme S1 (Supporting Information).[8] The results
of MALDI-TOFMS were as follows: MS; ODN1–EDTP: m/z: 6762.8
(calcd 6759.4), EDTP–ODN2: 6798.5 (calcd 6794.5).
Figure 4. a) Absence of rate-retarding effect of radical or singlet oxy-
gen scavengers on the site-selective scission by CeIII-derived DNA
cutter: lane 1, marker; lane 2, in the absence of scavengers; lane 3, with
100 mm DMSO; lane 4, with 2.5m MeOH; lane 5, with 10 mm NaN3.
Reaction conditions: [DNA(S2)]=1 mm, [each of ODN additives]=1 mm,
For site-selective DNA hydrolysis, the substrate ODN and the additive
ODNs were first heated to 958C for 2 min and then slowly cooled down
to RT. The reaction was initiated by adding a CeIII solution in HEPES
buffer (pH 7.0) to the mixture unless noted otherwise, and carried out at
508C under air. After a predetermined time, EDTP was added to a final
concentration of 4.5 mm, and the mixture was heat-denatured and imme-
diately subjected to denaturing 20% polyacrylamide gel electrophoresis.
The scission fragments were quantified with an imaging analyzer (FLA-
3000G; Fuji Film).
[CeACHTUNGTRENNUNG(NO3)3]=8 mm, [HEPES (pH 7.0)]=5 mm and [NaCl]=100 mm at
508C for 20 h under air. b) Effect of oxidation of CeIII to CeIV prior to
site-selective DNA scission: lane 1, marker; lane 2, in situ oxidation of
CeIII to CeIV (Ce
ACHTUNGTRENNUNG
For monitoring the oxidation of CeIII to CeIV, emission spectra were mea-
to CeIV (Ce
ACHTUNGTRENNUNG(NO3)3 was first incubated with EDTP–ODNs for 3 h under
sured on a JASCO FP-6500 spectrofluorometer (lex =313 nm). The CeIII
/
air for the oxidation, and then the DNA(S2) was added).
EDTP solution was prepared immediately before measurement by
mixing Ce(NO3)3A(H2O)6 in HEPES buffer and EDTP in water (pH 7.0)
and then kept under air at 508C.
A
CHTUNGTRENNUNG
Acknowledgements
This work was partially supported by a Grant-in-Aid for Specially Pro-
moted Research from the Ministry of Education, Science, Sports, Culture
and Technology, Japan (18001001), the Global COE Program for Chemis-
try Innovation, and Research Fellowships of the Japan Society for the
Promotion of Science for Young Scientists (for Y.A.).
[1] Reviews on non-enzymatic scission of DNA: a) E. L. Hegg, J. N.
Burstyn, Coord. Chem. Rev. 1998, 173, 133–165; b) N. H. Williams,
B. Takasaki, M. Wall, J. Chin, Acc. Chem. Res. 1999, 32, 485–493;
c) A. Sreedhara, J. A. Cowan, J. Biol. Inorg. Chem. 2001, 6, 337–
347; d) S. J. Franklin, Curr. Opin. Chem. Biol. 2001, 5, 201–208; e) J.
Suh, Acc. Chem. Res. 2003, 36, 562–570; f) J. R. Morrow, O. Iranzo,
Curr. Opin. Chem. Biol. 2004, 8, 192–200; g) F. Mancin, P. Scrimin,
P. Tecilla, U. Tonellato, Chem. Commun. 2005, 2540–2548; h) J.
Steinreiber, T. R. Ward, Coord. Chem. Rev. 2008, 252, 751–766; i) C.
Liu, L. Wang, Dalton Trans. 2009, 227–239.
[2] DNA scission by using the combination of enzymes and sequence-
recognizing molecules: a) D. H. Pei, D. R. Corey, P. G. Schultz, Proc.
Natl. Acad. Sci. USA 1990, 87, 9858–9862; b) S. A. Strobel, P. B.
Dervan, Nature 1991, 350, 172–174; c) L. J. Ferrin, R. D. Camerinio-
tero, Science 1991, 254, 1494–1497; d) M. H. Porteus, D. Carroll,
Nat. Biotechnol. 2005, 23, 967–973.
Figure 5. Treatment of 2’-deoxyguanosine by CeIII-derived CeIV/EDTP
complex at 508C under air with HPLC analysis of the reaction mixture
a) just after mixing the reagents, and b) 20 h later. The peaks were de-
tected by UV absorbance at 260 nm. No decomposition of 2’-deoxygua-
nosine was observed. Reaction conditions: pH 7.0, [CeACHTNUGTRNEG(UN NO3)3]=5 mm,
[EDTP]=1.25 mm, [2’-deoxyguanosine]=2 mm, and [HEPES]=120 mm.
In c), 2’-deoxyguanosine was treated with FeII/EDTA, which is known to
cleave DNA through an oxidative process, at 378C for 30 min (positive
control). The oxidation products including guanine and others were clear-
ly observed at shorter retention times. Reaction conditions; [FeII/EDTA
complex]=100 mm, [2’-deoxyguanosine]=100 mm, [H2O2]=10 mm, and
[DTT]=50 mm.
[3] a) M. Komiyama, T. Shiiba, T. Kodama, N. Takeda, J. Sumaoka, M.
Yashiro, Chem. Lett. 1994, 1025–1028; b) B. K. Takasaki, J. Chin, J.
Am. Chem. Soc. 1994, 116, 1121–1122; c) K. Bracken, R. A. Moss,
K. G. Ragunathan, J. Am. Chem. Soc. 1997, 119, 9323–9324; d) M.
Komiyama, N. Takeda, H. Shigekawa, Chem. Commun. 1999, 1443–
1451; e) T. Igawa, J. Sumaoka, M. Komiyama, Chem. Lett. 2000,
356–357; f) H. B. Shen, J. F. Xia, H. F. Yang, Y. M. Luo, Sci. China
858
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DNA Cleavage
FULL PAPER
Ser. B: Chem. 2001, 44, 169–174; g) A. L. Maldonado, A. K. Yatsi-
mirsky, Org. Biomol. Chem. 2005, 3, 2859–2867.
[9] Molecular oxygen tends to oxidize CeIII to CeIV species at neutral
pH: a) S. A. Hayes, P. Yu, T. J. OꢁKeefe, M. J. OꢁKeefe, J. O. Stoffer,
J. Electrochem. Soc. 2002, 149, C623–C630; b) P. Yu, S. A. Hayes,
T. J. OꢁKeefe, M. J. OꢁKeefe, J. O. Stoffer, J. Electrochem. Soc. 2006,
153, C74–C79.
[10] When one-base gap system was formed by using two EDTP-
attached oligonucleotides and treated with CeIV/EDTA, only one
phosphodiester linkage at the gap site was selectively hydrolyzed
(see Figure S1 in the Supporting Information). Apparently, the
strong CeIV binding by EDTP is very effective to hydrolyze smaller
gaps.
[4] a) M. Komiyama, T. Shiiba, Y. Takahashi, N. Takeda, K. Matsumura,
T. Kodama, Supramol. Chem. 1994, 4, 31–34; b) H. B. Shen, W. J.
Zhou, Y. T. Yang, F. Zhang, Sci. China Ser. B: Chem. 2004, 47, 75–
79; c) H. B. Shen, F. Wang, Y. T. Yang, Chin. Chem. Lett. 2005, 16,
1663–1666.
[5] Alternatively, homogeneous solutions of CeIV and EDTA were first
prepared from CeACHTUNGTRENNUNG(NH4)2AHCTUNGTREN(NUGN NO3)6 and EDTA, and used for selective
scission of single-stranded DNA at gap-sites. The scission was great-
ly promoted by attaching monophosphate groups to the ODNs and
placing them near the gap site; W. Chen, Y. Kitamura, J. M. Zhou, J.
Sumaoka, M. Komiyama, J. Am. Chem. Soc. 2004, 126, 10285–
10291.
[11] Attempts to determine the composition of the catalytically active
species of the present DNA cutters through the analysis of the rate–
[CeACTHNUTGRNEGNU(NO3)3] dependence, have so far been unsuccessful.
[6] The CeIV solutions in reference [5] were also used for site-selective
scission of double-stranded DNA in the presence of two peptide nu-
cleic acid strands: M. Komiyama, Y. Aiba, Y. Yamamoto, J. Sumao-
ka, Nat. Protoc. 2008, 3, 655–662.
[7] a) S. T. Frey, W. D. Horrocks, Inorg. Chem. 1991, 30, 1073–1079;
b) J. X. Meng, H. J. Wu, D. X. Feng, Spectrochim. Acta Part A 2000,
56, 1925–1928.
[12] a) Y. Jin, J. A. Cowan, J. Am. Chem. Soc. 2005, 127, 8408–8415;
b) E. L. Hegg, J. N. Burstyn, Inorg. Chem. 1996, 35, 7474–7481.
[13] Consistent with this result, no oxidation product was observed when
2’-deoxyadenosine, 2’-deoxcytidine, or thymidine was used instead.
[14] C. E. Grey, P. Adlercreutz, Nucleosides Nucleotides Nucleic Acids
2006, 25, 259–278.
[8] T. Lçnnberg, Y. Suzuki, M. Komiyama, Org. Biomol. Chem. 2008, 6,
3580–3587.
Received: August 4, 2009
Published online: November 24, 2009
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