Interstrand cross-link of DNA by covalently linking a pair of abasic sites

Article abstract of DOI:10.1039/c2cc16785a

A pair of apurinic/apyrimidinic sites formed in DNA has been covalently connected with bis(aminooxy) derivatives. The efficacy of the interstrand cross-link is associated with the structural tethering of two aminooxy groups. The interstrand cross-link con

Full text of DOI:10.1039/c2cc16785a

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COMMUNICATION
Interstrand cross-link of DNA by covalently linking a pair of abasic
sitesw
Kohei Ichikawa,a Naoshi Kojima,b Yu Hirano,b Toshie Takebayashi,b Keiko Kowatab and
Yasuo Komatsu*b
Received 2nd November 2011, Accepted 23rd December 2011
DOI: 10.1039/c2cc16785a
A pair of apurinic/apyrimidinic sites formed in DNA has been
covalently connected with bis(aminooxy) derivatives. The efficacy
of the interstrand cross-link is associated with the structural
tethering of two aminooxy groups. The interstrand cross-link
constructed stable DNA scaffolds for enzyme alignment.
Various chemical and environmental agents generate interstrand
cross-links (ICLs) in double-stranded DNA. Bifunctional alkylating
agents,1 platinum compounds,2 psoralen,3 and unsaturated
aldehydes4 react with the natural bases of double-stranded DNA,
resulting in ICL-duplexes. Site-directed ICLs are also formed by
incorporating chemically modified nucleosides that allow disulfide
bond formation,5 amination,6 radicals formations,7 and photo
activation.8 These ICLs are applicable to some biological studies;
however, most of them lead to the breakage of the connective base
stacking at ICL-sites or protrude bulky linkers outside helices.
At equilibrium, an AP site resulting from the hydrolysis of the
glycosidic bonds of nucleotides exists between a cyclic hemiacetal
form and an open-chain aldehyde form. The latter form having an
electrophile has the potential to conjugate with exocyclic amines of
opposing bases9 and proteins.10 Because an aminooxy group
readily reacts with the aldehyde group, mono-aminooxy-containing
agents are powerful tools for quantification of the AP sites in
DNA.11 We previously reported that an aminooxy compound
having a naphthalene residue improves conjugation efficiency
toward AP sites.12 In addition, an AP site becomes a recognition
site for a number of small ligands which interact with the
nucleobases surrounding the AP site.13
Scheme 1 ICL reaction of an AP pair.
cross-linkers can be tested on a single DNA molecule, resulting in
the production of ICL-duplexes including different cross-linkers.
We synthesized four types of cross-linkers that comprise
naphthalene (aoNao), propyl (aoPao), 1,2- and 1,4-substituted
benzene (aoOBao, aoPBao) at the spacer tethering aminooxy
groups (Fig. 1a). The benzene derivatives were prepared to
examine the distance between the two aminooxy groups needed
to form ICLs. The AP pair was produced from the hydrolysis
of a deoxyuridine pair with uracil DNA glycosylase (UDG)
immediately before the cross-linking reactions (Scheme 1).
We first investigated whether bis(aminooxy) derivatives linked
complementary oligodeoxynucleotides (ODNs) (I/II, Fig. 1b)
after UDG treatment, as shown in Scheme 1 (two-step reactions
of 1 and 2). All reactions were conducted in an aqueous solution
except for that of aoPBao, which was dissolved in 5% dimethyl-
sulfoxide because of its insolubility in aqueous solutions.
Products were analyzed with denaturing polyacrylamide gel.
By exploiting these chemical properties of AP sites, we have
developed a covalent cross-link of a pair of AP sites (AP pair) on
the opposite positions of complementary strands (Scheme 1).
In the reaction, two aldehyde groups of the AP pair are linked
with a bifunctional cross-linker having two aminooxyacetyl
groups. In other words, a single molecule such as an aromatic
ring is covalently introduced in place of a base pair. Thus, various
a Nippon Steel Kankyo Engineering Co. Ltd., 2-1-38 Shiohama,
Kisarazu 292-0838, Japan
b Bioproduction Research Institute, National Institute of Advanced
Industrial Science and Technology (AIST),
2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
E-mail: komatsu-yasuo@aist.go.jp
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc16785a
Fig. 1 (a) Structures of the bifunctional cross-linkers used for the
ICL reactions. (b) Sequences of ODNs. U, f, X and the bold lines
ꢀ
indicate deoxyuridine, the tetrahydrofuran derivative, AP site, and
cross-linker, respectively.
c
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However, similar to aoPao, aNa exhibited few conjugates in
the presence of sodium cyanoborohydride (Fig. S5 in ESIw).
This result confirmed the importance of the combination of
the aminooxy group and aromatic residue in this reaction.
Using aoNao, we examined the effects of the reaction
temperature and the mismatched base pairs adjacent to the
AP pair on the ICL reaction. The ICL duplex was dominantly
obtained at 27 1C whereas the yields of the single stranded
conjugates increased at the higher reaction temperature
(Fig. S4a in ESIw). Similarly, the ICL formation was inhibited
by introduction of the mismatched base pairs adjacent to the
AP pair (Fig. S4b in ESIw). These results indicated that it is
essential for the ICL reaction to form a stable AP pair pocket.
However, it should be noted that the formation of the AP pair
destabilizes duplexes. Thus, we performed UDG reaction in
the presence of aoNao, as shown in Scheme 1 (the reaction
of 1 + 2), to promote a simultaneous reaction. We found that
the ICL-duplex could be obtained with the same efficiency as
in the two-step reactions. It means that the ICL can be
prepared more conveniently from hybridized ODNs.
Fig. 2 Analysis of the ICL reactions. (a) Denaturing polyacrylamide
gel analysis of the reactions using aoNao on single- and double-stranded
ODNs. ssCL and I/UDG indicate the conjugate to the single stranded
ODN-I and UDG-treated ODN-I. (b) Plots of the percentages of ICL
products versus time.
All cross-linkers produced ICL-duplexes (CL1-OB, CL1-PB,
CL1-N, and CL1-P), which showed retarded migration in the
denaturing gel (Fig. 2a). aoNao provided most efficiently
ICL-duplexes and aoPao exhibited the lowest activity (Fig. 2b).
Interestingly, the yields and the migration speeds between
aoOBao and aoPBao were quite different. CL1-OB and CL1-PB
mediated by the benzene cross-linkers were observed as multiple
bands only in the denaturing gel analysis, whereas by treatment
with sodium cyanoborohydride, they were converted to a single
band (Fig. S3 in ESIw). Thus, we concluded that the multiple bands
were derived from the isomers of two oxime linkages.
aoNao could mediate double ICL formations in duplex
III/IV (Fig. 1b). We examined thermal melting curves (Tm)
of the ICL-duplexes containing single (CL1-N, CL1-P) and
double ICL sites (CL2-N). CL2-N did not show a denaturing
profile because both its terminals were constrained by ICLs. In
contrast, CL1-N and CL1-P linked at the center of the sequence
showed standard sigmoidal curves (Fig. S6b in ESIw), and their
Tm values were found to be 75.7 1C (CL1-N) and 70.5 1C (CL1-P),
respectively. These values were much larger than those of I/II
(dU:dU mismatched duplex, 40.5 1C) and I/II-A (dA:dU perfectly
matched duplex, 47.1 1C). Note that CL1-N showed a larger
Tm value than CL1-P. We believe that the increase was due to the
stabilizing effect of the naphthalene residue conjugated in the
inside helix.
The ICL reactions largely depended on the structures of the
cross-linkers. In particular, the hydrophobic residue contributed
to the cross-link of the AP pair as shown in our previous results.12
Because the AP pair provides a space flanked by base pairs, the
stacking interaction between the aromatic ring and the base pairs
might facilitate cross-linker binding into the pocket in the helix.
All cross-linkers efficiently reacted with double strands in
comparison with a single-stranded ODN (ssCL) (Fig. S1 in ESIw).
To perform quantitative analyses, we determined the observed
rate constants of the reactions to a single reaction site (monoAP
site) in the presence or absence of a nonreactive template (II-A or
II-f, Fig. 1b), which contained an adenosine or tetrahydrofuran
derivative opposite an AP site. The I/II-f duplex is supposed to
offer a space similar to that of an AP pair in the helix. aoNao
efficiently reacted with the monoAP site in the double-stranded
forms, and the rate constants of I/II-f (7.4 ꢁ 10ꢀ2 minꢀ1) and
I/II-A (4.8 ꢁ 10ꢀ2 minꢀ1) were approximately five- and three-fold
higher than that of the single strand (I, 1.4 ꢁ 10ꢀ2 minꢀ1),
respectively. On the other hand, aoPao, aoOBao, and aoPBao
only slightly increased the yields in the double-stranded forms
containing nonreactive templates; however, the rate constants
could not be determined because only few conjugates were
formed. Whereas aoPBao and aoOBao exhibited different
reaction efficiencies in the ICL containing double reaction sites,
they showed equal potential for reacting with the monoAP site.
This might be because the insufficient spatial distance between the
double aminooxy groups of aoOBao did not allow the ICL of the
AP pair.
Because the ICL sites of both CL1-N and CL2-N contained
the naphthalene ring that absorbs ultraviolet light at 290 nm,
their structures were analyzed by complete digestions with
nucleases. Both ICL duplexes showed high nuclease resistance
because of the stabilization by ICLs, and the reaction times of
nuclease digestions were extended for their complete digestions.
As expected, both ICL sites were identified as aoNao-conjugates
with two deoxyriboses (dR2-aoNao). Similar to the standard
sample, the ICL sites exhibited three peaks in the HPLC analyses
(Fig. S7b, d and f in ESIw). They correspond to three structural
isomers based on the two oxime linkages, which could be reduced
to a single bond by sodium cyanoborohydride under the high
acidic conditions (Fig. S7c and e in ESIw).
DNA can offer nanosized platforms for protein arrays14 and
enzyme reactions;15 however, standard duplexes are reversible and
are subject to dissociation. We applied the ICL formation to
construct stable DNA scaffolds for enzyme reactions. 50-Biotin- or
50-fluorescein-modified ODN of 20 bases was linked with each
complementary strand by using aoNao to anchor horseradish
peroxidase-streptavidin (HRP-SA)16 or alkaline phosphatase-
anti-fluorescein antibody (ALP-Ab)17 on DNA,16 resulting
in two-stranded ICL-duplexes (HRP-CL1-N, ALP-CL1-N,
Fig. S8 in ESIw). In addition, we constructed a three-stranded
duplex, HRP/ALP-CL2-N, that involved both 50-biotin- and
50-fluorescein-modified ODNs arranged in tandem on the
The aoNao showed the potent activity for the ICL, and we
synthesized an alternative naphthalene derivative (aNa)
containing bis-primary amine instead of aminooxy groups.
c
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implanting various molecules in the DNA helix. Hence, this
site-specific ICL reaction is applicable for the construction of
stable DNAs with different functionalities.
We thank Dr Eiko Ohtsuka and Dr Kousuke Sato (Hokkaido
University) for their helpful discussions and technical assistance.
This work was supported by KAKENHI (21510239).
Notes and references
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Fig. 3 (a) Schematic drawing of enzyme reactions on ICL-duplexes
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HRP oxidizes hydroquinone (H2Q) to benzoquinone (BQ) and
ALP produces p-aminophenol (PAP) from p-aminophenol
phosphate (PAPP). Both catalytic actions were evaluated by
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scanning electrochemical microscope (SECM) (Fig. 3a).16,18
As shown in Fig. 3b and c, these enzymes were found to
actually promote reactions at exact DNA spots. In particular,
HRP/ALP-CL2-N succeeded in accommodating both the enzyme
binding sites on the single molecule. On the other hand, the
enzymatic productions from the PM-bio and PM-F were signifi-
cantly decreased to 67% and 38% of the corresponding ICL
duplexes (Fig. S10 in ESIw). This result suggests that a part of
standard duplexes was subject to dissociation into single strands
during the chip preparation or enzyme reactions. The cross-linked
duplexes provided stable DNA scaffolds for enzyme reactions.
In conclusion, a pair of AP sites formed in double-stranded
DNAs can be linked with a single bis(aminooxy) molecule.
This ICL distinctly stabilizes double strands by covalently
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c
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