Construction of highly reactive probes for abasic site detection by introduction of an aromatic and a guanidine residue into an aminooxy group

Article abstract of DOI:10.1021/ja904767k

(Chemical Equation Presented) Abasic sites (AP sites) arise from hydrolysis of glycosidic bonds of DNA that is damaged by various external and internal processes; unrepaired AP sites give rise to genetic mutations. We have constructed highly reactive AP-s

Full text of DOI:10.1021/ja904767k

Published on Web 08/28/2009
Construction of Highly Reactive Probes for Abasic Site Detection by Introduction
of an Aromatic and a Guanidine Residue into an Aminooxy Group
Naoshi Kojima, Toshie Takebayashi, Akiko Mikami, Eiko Ohtsuka, and Yasuo Komatsu*
Research Institute of Genome-based Biofactory, National Institute of AdVanced Industrial Science and Technology
(AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
Received June 11, 2009; E-mail: komatsu-yasuo@aist.go.jp
Detection and quantification of DNA lesions plays an important
role in studies on causal relationships between DNA lesions and
mutagenesis.1,2 Chemical probes specific for DNA lesions are
powerful tools for detection3-5 because probe-mediating detection
is convenient and does not require derivatization or digestion of
DNA.6,7 Apurinic/apyrimidinic (AP) sites are a prevalent type
of DNA lesion, arising from hydrolysis of glycosidic bonds of
nucleotides or excision repair of bases modified by radiation and
alkylating agents.8 Oxidized AP lesions, 2-deoxyribonolactone, C4-
AP, or DOB are also produced by the reactive species of antitumor
agent bleomycin9 or by the diradicals of neocarzinostatin chro-
mophore.10 These oxidized AP lesions, as well as normal AP sites,
not only are mutagenic but also cause strand scission of DNA.8,11
Since AP sites exist at equilibrium between cyclic hemiacetal
and open-chain aldehyde forms, an aldehyde-reactive probe (ARP,
Figure 1) is frequently used for detection of AP sites.3,12 Oxidized
AP lesions can also be specifically detected with smartly designed
molecules such as cystein13 and a Tris derivative.14 These probes
make it possible to detect AP sites using a biotin-avidin complex
system after conjugation with the DNA lesion.15,16 Recently, AP
sites are also used as reactive intermediates, provided by deoxy-
uridine and uracil DNA glycosylase (UDG), to obtain labeled DNA
for gene expression profiling.17
naphthalene). The structure of aoN-bio is just like a naphthalene-
inserted construct at the hydrazine of ARP, and aoN-OH bears a
hydroxyl group in place of a biotin residue (Figure 1). Since an
amido bond linking to an aromatic ring is planar,19 we expected
that this arrangement of an aminooxy group close to bis-amido-
naphthalene would act effectively in both conjugation and stacking
in an AP site pocket. We also synthesized aoN-g and ao-g having
a guanidine residue (g denotes guanidino group) to enhance
electrostatic interaction or hydrogen bonding with backbone
phosphate groups of nucleic acids, since a guanidine has a positive
charge at neutral pH (pKa ≈ 12.5).
Although ARP has a simple structure, it is not necessarily
designed for high reactivity toward AP sites.3 We therefore
constructed highly reactive AP site-detecting probes by assembling
a hydrophobic and a hydrophilic residue into an aminooxy group
and report here the construction and the structure-function relation-
ships.
Figure 2. Observed rate constants for each probe for single-stranded
(F-20dU) and double-stranded (F-20dU/PM) ONTs. White bar: F-20dU;
black bar: F-20dU/PM.
In contrast to ARP, aoN-bio was insoluble in aqueous solution
due to the hydrophobic naphthalene residue. Other probes without
a biotin were dissolved, and in particular, aoN-g and ao-g could
be easily dissolved in aqueous solution due to the guanidine
residues. Thus, aoN-OH, aoN-g, and ao-g were subjected to labeling
reactions with AP sites generated by UDG treatment of single-
stranded (ss) (F-20dU) or double-stranded (ds) (F-20dU/PM; Figure
1) oligonucleotides (ONTs) containing a deoxyuridine. Observed
rate constants (kobs) were determined in the presence of excess
amounts of these probes. Rates were slightly higher for aoN-OH
than for ARP, and those for the guanidine-containing probes (aoN-
g, ao-g) were significantly higher than those for aoN-OH (Figure
2 and Figure S2 in Supporting Information (SI)). In particular,
aoN-g showed the fastest reaction rates among all probes, and the
rate for ds-ONT was 9.5-fold higher than that of aoN-OH without
a guanidine.
Figure 1. Structures of aminooxy derivatives and sequences of double-
stranded oligonucleotides (F and underlined U indicate fluoresceine and
deoxyuridine residue, respectively).
These differences in reaction rate are closely related with probe
structures. First, the guanidino group has the capability to increase
the conjugation efficiencies of aminooxy derivatives. The guanidino
group is thought to promote association of these compounds with
ONTs by hydrogen bonding or electrostatic interactions, as previ-
ously reported.20-22 Next, the naphthalene residue also contributes
AP sites in DNA are flanked by bases (or base pairs) at their 5′-
and 3′-sides, and some aromatic hydrocarbons interact with DNA
by stacking interactions.18 To take advantage of the stacking effects
with the adjacent bases, we synthesized naphthalene-containing
aminooxy derivatives (aoN-bio and aoN-OH in Figure 1; N denotes
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13208 J. AM. CHEM. SOC. 2009, 131, 13208–13209
10.1021/ja904767k CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
to fast conjugation, as shown by the different rates for aoN-g and
ao-g. The effect of the naphthalene residue is due to stacking
interactions with bases or base pairs adjacent to the AP site, as
supported by the fact that aoN-g decreased reaction rates for ds-
ONTs containing mismatched base pairs adjacent to the AP site
(Figure S2 in SI). Interestingly, these results indicate that the high
reactivity of aoN-g is due to synergetic effects of guanidine and
aromatic residues and that the hydrophobic interaction of naphtha-
lene might work effectively when the probes are accessible to the
target ONTs with the help of the guanidino group. Although aoN-g
showed high reactivity to AP site, it did not react to 2-deoxyri-
bonolactone (SI).13,23 However, aoN-g should be active for C4-
AP, similar to other aminooxy derivatives.23
aoN-g containing both naphthalene and guanidine is a promising
candidate for a new labeling reagent. Hence, we synthesized its
biotin conjugate by linking a biotin to the guanidine with an
ethyleneglycol linker (aoN-g-bio; Figure 3A). For a cognate probe,
we prepared aoN-dg-bio, which has a guanidino group directly
connected to the naphthalene ring (Figure 3A). Both of the
naphthalene-guanidine-containing probes were still soluble in
aqueous solution, in contrast to aoN-bio, due to the presence of
the guanidino groups. Observed rate constants for these biotin-
tagged probes were obtained from reaction with ONTs (Figure 2).
The rates for aoN-g-bio and aoN-dg-bio were approximately 6- to
8-fold faster than that for ARP. Interestingly, aoN-dg-bio showed
slightly faster reaction rates than did aoN-g-bio in reaction with
either ss- or ds-ONT. Compared with aoN-g, both aoN-g-bio and
aoN-dg-bio decreased the reaction rates with ss- and ds-ONTs, and
the decrease was significant in the reaction with ds-ONT. This
decrease is apparently derived from the biotin attachment, and it is
thought that the biotin insertion obstructed the guanidine-effects
exerted in aoN-g.
thymus DNA samples were incubated in acidic solution for various
times to hydrolyze glycosidic bonds,3 labeling reactions were carried
out in a microwell plate under the same buffer conditions used for
the ONT reactions (SI). AP sites were detected colorimetrically by
an ELISA-like assay using a biotin-streptavidin complex formation
system. For all probes, signal intensity depended on incubation time
(Figure 3B). Both aoN-g-bio and aoN-dg-bio showed higher signal
intensities than did ARP, and aoN-dg-bio showed slightly higher
signal intensity than did aoN-g-bio. These results are consistent
with those obtained for the ONT reactions and also prove the
superiority of the naphthalene-guanidine-containing aminooxy
probes.
In summary, we synthesized aminooxy derivatives containing
either a naphthalene or a guanidine residue and found that probes
with both functional groups can react very efficiently with aldehyde
groups in DNA. Their biotin conjugates enable sensitive detection
of AP sites, compared with conventional detecting probes. We think
that these probes can be useful tools for the detection of DNA
damage or in various genetic assays using AP sites.
Acknowledgment. We thank Dr. Kousuke Sato (Hokkaido
University) for technical assistance and helpful discussions. This
work was supported financially by the National Institute of
Advanced Industrial Science and Technology (AIST).
Supporting Information Available: Experimental procedures for
the synthesis and characterizations of all the compounds, and other
experimental and information. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Figure 3. (A) Structures of biotinylated aminooxy probes. (B) Analysis
of AP sites in calf thymus DNAs by ARP (white bar), aoN-g-bio (gray
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ARPswere used to measure AP sites in genomic DNA. After calf
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