A Nucleobase that Releases Reporter Tags upon DNA Oxidation

Article abstract of DOI:10.1021/ja038169a

We have developed a novel nucleosbase, ^edaG, that efficiently releases various reporter units upon one-electron oxidation. The ^edaG-selective degradation of ODNs was achieved by various mild oxidizing agents. The oxidant-dependent mo

Full text of DOI:10.1021/ja038169a

Published on Web 12/18/2003
A Nucleobase that Releases Reporter Tags upon DNA Oxidation
Akimitsu Okamoto, Kazuo Tanaka, and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto UniVersity, and
SORST, Japan Science and Technology Corporation, Kyoto 615-8510, Japan
Received August 27, 2003; E-mail: saito@sbchem.kyoto-u.ac.jp
Scheme 1 a
DNA biosensors offer considerable promise for extracting
information from target genes in a quick and simple manner.
Various DNA probes that give signals in a sequence-specific
fashion, as represented by molecular beacons, have been widely
used.1 However, there are very few DNA probes that can release
useful functional molecules.2 A molecular releasing system that is
triggered by external stimulation such as oxidation or photoirra-
diation would be a useful tool for gene analysis.
Herein, we report a novel nucleoside, ethylenediamine-modified
G (edaG), and a new molecular releasing system controllable by
one-electron oxidation of oligodeoxynucleotides (ODNs). Reporter
units tethered to edaG were easily released from ODNs by mild
oxidation. In a long-range hole transport experiment, DNA duplex
containing edaG efficiently and stoichiometrically released a fluo-
rescent tag.
The synthesis of edaG-containing ODN is outlined in Scheme 1.
8-BromoG (1) protected by a 4,4′-dimethoxytrityl group3 was
converted to 2 by refluxing in ethylenediamine, and the amino
groups were protected to afford 3. edaG phosphoramidite 4 was
incorporated into ODNs by a conventional method. By the
a Reagents and conditions: (a) ethylenediamine, 130 °C, 7 h; (b) ethyl
trifluoroacetate, triethylamine, methanol, 0 °C, 2 h; (c) DMF-dimethylacetal,
DMF, room temperature, 2 h, 63% (three steps); (d) (iPr2N)2PO(CH2)2CN,
tetrazole, acetonitrile, room temperature, 2 h, quant.
postsynthetic modification of edaG-containing ODNs using N-
hydroxysuccinimidyl ester of carboxylic derivatives such as benzoic
acid (Bz) and tetramethylrhodamine (TAMRA), reporter units were
incorporated into the amino side chain of edaG in the ODNs.
We initially examined the photooxidation of single-stranded ODN
containing Bz-edaG, ODN1(Bz-edaG) 5′-d(TATAATXTAATAT)-
3′ (X ) Bz-edaG), in the presence of a photosensitizer riboflavin.4
A sample solution of 10 µM ODN1(Bz-edaG) in 50 mM sodium
cacodylate (pH 7.0) was irradiated at 366 nm in the presence of 50
µM riboflavin at 0 °C. Photoirradiation of ODN1(Bz-edaG) resulted
in a rapid decomposition of ODN (t1/2 ) 6.2 min). The major
isolable photoproducts of the oxidation of ODN1(Bz-edaG) were
identified by mass spectrometric analysis as ODN fragments 5,
cleaved via an abasic site, ([M - H]-, calcd. 1968.32, found
1967.68 by MALDI-TOF), 5′-phosphate end 6 ([M - H]-, calcd.
1870.22, found 1870.11 by MALDI-TOF), and benzamide 7
possessing a guanidinium group (M+, 207 and its fragments 148,
105, 77, and 59 by LC-ESI/MS/MS) (Scheme 2). The identified
products strongly suggest that the rapid decomposition of edaG may
proceed via the G cation radical decomposition mechanism proposed
earlier5 to result in a release of a functional unit as typically
represented by 7.
Scheme 2
hexachloroiridate(IV) (20 µM). Oxidation with Ir(IV) resulted in a
rapid degradation of ODN1(X) at the site of modified edaG (57%
consumption for Bz-edaG and 89% for TAMRA-edaG in 15 min
incubation, as determined by HPLC).
The method using edaG constitutes a facile strategy for detecting
long-range hole transport through DNA without complicated and
unwieldy analyzing processes such as quantification of oxidative
guanine damage of labeled DNA11 or the analysis of photodynam-
ics.12 We examined the detection of TAMRA released from ODN2
containing a TAMRA-tethered edaG via long-range hole transport
through DNA (Figure 1). The reaction sample containing the
ODN2/ODN2′(U*) duplex possessing a cyanobenzophenone-modi-
fied uridine (U*)13 as a hole injector was irradiated at 312 nm
(Figure 1a).14 The photolyzed ODN was then removed from the
sample solution by passing through a centrifugal filter (Microcon
YM-3). A strong fluorescence at 576 nm was observed from the
filtrate of the ODN2/ODN2′(U*) sample after photoirradiation, and
the fluorescence after 60 min of irradiation was 7 times stronger
The site-selective oxidation of edaG-containing ODN was also
observed with other one-electron oxidants. Ir(IV) is a highly
selective oxidant that reacts exclusively with oxidized nucleo-
bases such as 8-oxoG and 8-oxoA.6 The oxidation potential (E1/2
)
of Bz-edaG is 0.59 V (vs NHE),7 which is close to that of 8-oxoG
(0.56 V,8 0.60 V9). The edaG-containing ODN ODN1(X) (X ) Bz-
and TAMRA-edaG) was mixed with Ir(IV).6,10 A solution of single-
stranded ODN1(X) (10 µM) in sodium cacodylate buffer (pH 7.0)
was incubated at room temperature in the presence of sodium
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C O M M U N I C A T I O N S
(U*) sample, whereas the emission from the filtrate of photoirra-
diated ODN2/ODN2′(T), a control sample, was negligible. The
incorporation of TAMRA-edaG into the duplex makes it possible
to detect hole transport through DNA without PAGE analysis.
In conclusion, we have developed a novel nucleosbase, edaG, that
efficiently releases reporter tags upon one-electron oxidation. The
edaG-selective degradation of ODNs can be achieved by various
mild oxidizing agents. This oxidant-dependent molecular releasing
technique is useful not only for drug releasing systems but also for
the release of a fluorescent tag after gene analysis.
Supporting Information Available: Detailed experimental data
of edaG and the related ODNs (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 1. Release of TAMRA from TAMRA-edaG via long-range DNA
oxidation. (a) Sequences of duplex ODNs. (b) Fluorescence intensity of
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than that of the control ODN2/ODN2′(T) without U* (Figure 1b).
The fluorescence intensity of the ODN2/ODN2′(U*) sample
increased in proportion to the irradiation time. The change of
fluorescence intensity showed a good correlation with the strand
cleavage at edaG site, which was independently quantified by PAGE
for the experiment using the ODN2/ODN2′(U*) (Figure 1c).15 In
addition, in the PAGE analysis for the photoirradiated duplex, it
was observed that lesions at the GGG sites, located between U*
and edaG, were strongly suppressed. Thus, edaG acts as a very
efficient hole trap, and the hole generated in the duplex by U* is
selectively trapped at the edaG site via a long-range hole transport
to result in the release of TAMRA from the duplex.
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(14) The decrease of the fluorescence intensity of TAMRA by photobleaching
was less than 5% after 312 nm photoirradiation for 90 min.
(15) The 18% of TAMRA-edaG in intact ODN was also damaged by hot
piperidine treatment (90 °C, 20 min).
The fluorescence from the photoirradiated sample was visually
detectable. As shown in Figure 1d, a strong visible emission was
observable with the filtrate of the photoirradiated ODN2/ODN2′-
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