A metal-ion-releasing probe for DNA detection by catalytic signal amplification

Article abstract of DOI:10.1002/anie.200504319

(Figure Presented) Seeing the light: Upon binding to a complementary nucleic acid, a metal-complex-DNA conjugate releases copper(II) ions that activate the precatalyst 1,10-phenanthroline (phen) and amplify the primary signal through catalytic production

Full text of DOI:10.1002/anie.200504319

Angewandte
Chemie
Allosteric Signal Transduction
a cofactor and activates a chemical redox catalyst, which
converts a nonfluorescent substrate into a fluorescent prod-
uct. This approach is more flexible in so far as a structural
similarity between the primary and transduced signals is no
longer essential. The PCR-independent, amplified catalytic
detection of nucleic acid sequences has recently gained
considerable interest, with a focus on enzyme- and ribo-
DOI: 10.1002/anie.200504319
A Metal-Ion-Releasing Probe for DNA Detection
by Catalytic Signal Amplification**
[
5]
zyme-based techniques.
Nora Graf, Mareike Göritz, and Roland Krämer*
The primary receptor in Scheme 1 is the 20 mer DNA 1
with 2,2’:6’,2’’-terpyridine (tpy) attached to both termini
(Scheme 2). The synthesis of 1 and 3’-mono(tpy)-DNA 2 is
Chemical and physical signals are very efficiently enhanced in
biological systems and rapidly converted into a biochemical
response by signal cascades which combine several signal
transduction and catalytic amplification steps. Signal amplifi-
cation by catalysis has been used in the development of highly
sensitive analytical detection schemes. Many powerful assays
rely on enzymatic amplification, for example, the widely
applied heterogeneous ELISA test (enzyme-linked immuno-
sorbent assay). The design of bioinspired amplification
strategies involving chemical catalysts is an emerging research
[
6]
described elsewhere.
[
1]
field. An example is a chloride sensor in which the analyte is
[
2]
[3]
an allosteric regulator and activates a synthetic catalyst. In
[
4]
a sensing assay developed by Anslyn and co-workers,
a
metal-ion analyte (the primary signal) interacts with a
macrocyclic receptor by replacing another, bound-metal ion.
The latter (the transduced signal) assembles as a cofactor with
a precatalyst into the active, signal-amplifying organometallic
catalyst.
Our new sensing concept relies on a unique allosteric
transduction of the primary signal (a DNA oligonucleotide)
into a metal-ion signal (Scheme 1). The metal ion functions as
Scheme 2. Structures of DCFH, DCF, conjugated tpy, and phen.
Sequences of oligonucleotide-terpyridine conjugates 1 and 2, of
complementary DNA 3 and mismatch DNA 4.
II
Coordination of Cu ions by both terpyridine moieties in
1
is evident by UV spectrophotometric titration, a character-
istic EPR signal (gpar = 2.23, Apar = 156 G), and destabilization
of a hybrid of 1 with short complementary DNA (see the
Supporting Information). Excess 1,10-phenanthroline (phen),
II
a high-affinity Cu chelator (complex formation constant
[
7]
II
II
logK = 9.2 for [Cu (phen)] versus logK = 9.1 for [Cu -
(
[
8]
tpy)] ), does not extract the metal ion from the copper(II)
complex of 1. However, the intensity of the UV band at
320 nm of the copper(II)-coordinated tpy decreases sponta-
neously when complementary 20 mer-DNA 3 is added to the
Scheme 1. Detection of DNA 3 (primary signal)by allosteric signal
transduction and catalytic amplification with formation of the fluores-
cent dye DCF. See text for abbreviations.
1-Cu/phen mixture (see the Supporting Information), and
[
*] N. Graf, Dr. M. Göritz, Prof. Dr. R. Krämer
Anorganisch-Chemisches Institut
Universität Heidelberg
Im Neuenheimer Feld 270, 69120 Heidelberg (Germany)
Fax: (+49)6221-54-8599
after one minute no further changes in absorbance are
observed. Apparently, formation of the rigid 1:3 DNA double
helix disrupts intramolecular bis(chelation) and destabilizes
the 1–Cu interaction.
The catalytic module of the nucleic acid detection assay
comprises the bidentate chelator and precatalyst 1,10-phen-
anthroline (phen), its cofactor copper(II), the nonfluorescent
substrate 2’,7’-dichlorodihydrofluorescein (DCFH), dioxygen
E-mail: roland.kraemer@urz.uni-heidelberg.de
[
**] N.G. and M.G. thank Baden-Württemberg for financial support
(
Landesgraduiertenstipendien). These authors contributed equally
to this work.
from air as an oxidant, and cysteamine (H NCH CH SH) as a
reducing cosubstrate. A plausible pathway for the formation
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2
2
2
Angew. Chem. Int. Ed. 2006, 45, 4013 –4015
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4013

Communications
of the fluorescent product 2’,7’-dichlorofluorescein (DCF)
includes the [Cu(phen)]-promoted generation of H O from
activity in the absence of complementary DNA (Figure 1a).
This observation indicates facile extraction of Cu ions from
II
2
2
cysteamine and O , followed by [Cu(phen)]-catalyzed H O
2
the complex with mono(tpy)-DNA 2 and confirms the
significant stabilization of 1-Cu by intramolecular bischela-
tion. The method is sensitive to single base mismatches: the
rate of DCF formation in the presence of DNA 4 (C/T
mismatch) is only slightly above the background level. At a
low concentration of 1-Cu, complementary DNA 3 is
detectable at 5 nm (250 fmol; Figure 1b).
After 12 h reaction time, the fluorescence of the 1 + 3
mixture (Figure 1a) can even be detected by the naked eye
(Figure 2). Turnover numbers of 20 and 5 have been
determined relative to copper(II) and analyte DNA 3,
respectively.
2
2
[
9]
oxidation of DCFH to DCF. Although it is likely that this
reaction also involves the formation of OH radicals or other
[
10]
aggressive oxygen species, no significant cleavage of oligo-
DNA is detectable by HPLC under assay conditions (see the
Supporting Information). We suggest that DCFH functions as
an antioxidant and protects DNA from degradation. DCFH
has been used as a fluorogenic probe to monitor the
[
11]
concentration of reactive oxygen species in living cells.
Formation of the product DCF was monitored by follow-
ing the increase in the relative fluorescence intensity at
5
23 nm with time (dI /dt). Figure 1a illustrates the sequence
rel
selectivity, and Figure 1b the sensitivity of the assay. Reagent
concentrations were optimized for a high signal-to-back-
ground ratio.
Although oxidation of DCFH by 1-Cu in the presence of
excess phen is very slow, the rate increases dramatically in the
presence of complementary DNA 3. The activity is about two
II
thirds that of a “DNA-free” phen/Cu mixture, thus indicat-
[
12]
ing formation of the active [Cu(phen)] catalyst. In contrast
to 1-Cu, a 2/Cu/phen mixture displays significant catalytic
Figure 2. Top: Reaction solutions containing DNA 1 (right)and 1+3
(left)under UV light after 12 h (for the conditions see Figure 1a).
Bottom: Increase in the relative fluorescence intensity with time:
a)DNA 1, b)DNA 1+3.
Our prototypical DNA sensor does not approach the low
[
5a–e]
detection limits of some reported systems
since the
sensitivity is limited to the nanomolar range by uncatalyzed
background oxidation of the fluorogenic substrate DCFH. In
À1
addition, the low catalytic turnover rate (2 h ) limits the
efficiency of the signal amplification. The catalytic module
must be optimized to achieve better sensitivity and more
effective amplification. The signal-to-background ratio could
be slightly improved from 3:1 to 5:1 at lowest target DNA
concentrations when the precatalyst phen was replaced by 5-
amino-1,10-phenanthroline. We are currently exploring alter-
native chemical and biochemical catalytic systems, such as the
activation of apoenzymes by their metal-ion cofactors.
Figure 1. Increase in the relative fluorescence intensity from the initial
level (0–10 min)with time (d I /dt, l =503 nm, l =523 nm): pH 7
rel
ex
em
(
10 mm 3-(N-morpholino)propanesulfonic acid (MOPS) buffer) 258C,
DCFH (10 mm), cysteamine (100 mm), NaCl (0.1m)as well as a)5 mm
phen, 0.5 mm CuSO , and 2 mm of the indicated DNA oligonucleoti-
4
de(s)or b)0.2 mm phen, 0.02 mm CuSO , 0.05 mm 1, and various
4
amounts of 3.
4
014 www.angewandte.org
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Angewandte
Chemie
In conclusion we have described a homogeneous-phase
chemical method for DNA detection which combines an
allosteric signal transduction with a catalytic amplification
step. The system resembles the initial steps of biological signal
cascades such as hormone action. For example, the primary
chemical signal adrenaline is (in a simplified scheme)
allosterically transduced by a membrane receptor, thus
resulting in the activation and release of receptor-associated
G protein. The latter then combines with adenylate cyclase
and activates this enzyme for catalytic generation of cAMP
from ATP. Such biological cascades can be a source of
inspiration for the design of artificial, modular detection
systems which amplify chemical signals strongly and rapidly.
Both reaction types show a complicated dependence of rate on
the copper concentration. We found that phen/copper(II) is a
much more efficient catalyst than free copper(II) ions for the
oxidation of DCFH.
[
10] D. S. Sigman, D. R. Graham, V. DꢀAurora, A. M. Stern, J. Biol.
Chem. 1979, 254, 12269 – 12272; B. C. Bales, T. Kodama, Y. N.
Weledji, M. PitiØ, B. Meunier, M. M. Greenberg, Nucleic Acids
Res. 2005, 33, 5371 – 5379.
[11] See for example: S. L. Hempel, G. R. Buettner, Y. Q. OꢀMalley,
D. A. Wessels, D. M. Flaherty, Free Radical Biol. Med. 1999, 27,
146 – 159.
[
12] For simplicity, we denote the active species [Cu(phen)], although
both [Cu(phen)] and [Cu(phen) ] are expected to form and
2
catalyze the oxidation of DCFH (see the Supporting Informa-
tion). The catalytic activities of the in situ prepared complexes
[
Cu(tpy) ] and [Cu(tpy)] are negligible. A 1:1:1 mixture of tpy,
2
II
Received: December 5, 2005
Revised: February 24, 2006
Published online: May 9, 2006
phen, and Cu in the absence of any DNA has only 10% of the
activity of a 1:1 phen/Cu mixture under the reaction conditions
indicated for Figure 1a.
Keywords: allostery · copper · DNA · fluorescence ·
.
signal amplification
[
1] L. Zhu, E. V. Anslyn, Angew. Chem. 2006, 118, 1208 – 1215;
Angew. Chem. Int. Ed. 2006, 45, 1190 – 1196.
[
2] For a comprehensive overview on synthetic allosteric receptors
and catalysts, see: L. Kovbasyuk, R. Krämer, Chem. Rev. 2004,
1
04, 3161 – 3187.
3] N. C. Gianneschi, S. T. Nguyen, C. A. Mirkin, J. Am. Chem. Soc.
005, 127, 1644 – 1645.
4] Q. Wu, E. V. Anslyn, J. Am. Chem. Soc. 2004, 126, 14682 –
[
[
2
14683; see also L. Zhu, V. M. Lynch, E. V. Anslyn, Tetrahedron
2004, 60, 7267 – 7275.
[
5] Heterogeneous enzyme-based methods: a) F. Patolsky, Y. Weiz-
mann, I. Willner, J. Am. Chem. Soc. 2002, 124, 770 – 772; b) F.
Patolsky, E. Katz, I. Willner, Angew. Chem. 2002, 114, 3548 –
3
552; Angew. Chem. Int. Ed. 2002, 41, 3398 – 3402; c) T. Niazov,
V. Pavlov, Y. Xiao, R. Gill, I. Willner, Nano Lett. 2004, 4, 1683 –
687; d) F. Patolysky, R. Gill, Y. Weizmann, T. Mokari, U. Banin,
1
I. Willner, J. Am. Chem. Soc. 2003, 125, 13918 – 13919;
homogeneous enzyme-based methods: e) A. Saghatelian,
K. M. Guckian, D. A. Thayer, M. R. Ghadiri, J. Am. Chem.
Soc. 2003, 125, 344 – 345; f) V. Pavlov, B. Shlyahovsky, I. Willner,
J. Am. Chem. Soc. 2005, 127, 6522 – 6523; g) P. Simon, C.
Dueymes, M. Fontecave, J.-L. DØcout, Angew. Chem. 2005, 117,
2824 – 2827; Angew. Chem. Int. Ed. 2005, 44, 2764 – 2767;
Ribozyme-based: h) M. N. Stojanovic, P. de Prada, D. W.
Landry, ChemBioChem 2001, 2, 411 – 415; i) S. Sando, T.
Sasaki, K. Kanatani, Y. Aoyama, J. Am. Chem. Soc. 2003, 125,
15720 – 15721; j) J. S. Hartig, I. Gruene, S. H. Najafi-Shoushtari,
M. Famulok, J. Am. Chem. Soc. 2004, 126, 722 – 723; k) S. Sando,
A. Narita, K. Abe, Y. Aoyama, J. Am. Chem. Soc. 2005, 127,
5300 – 5301; catalysis templated by nucleic acids: l) Z. Ma, J. S.
Taylor, Proc. Natl. Acad. Sci. USA 2000, 97, 11159 – 11163; m) J.
Brunner, A. Mokhir, R. Krämer, J. Am. Chem. Soc. 2003, 125,
1
2410 – 12411.
6] M. Göritz, R. Krämer, J. Am. Chem. Soc. 2005, 127, 18016 –
8017.
[
1
[
[
7] P. Mitchell, H. Sigel, J. Am. Chem. Soc. 1978, 100, 1564 – 1570.
8] A. Odani, H. Masuda, K. Inukai, O. Yamauchi, J. Am. Chem.
Soc. 1992, 114, 6294 – 6300.
[
9] Copper-promoted formation of H O from O and cysteamine:
2
2
2
T. Kamidate, I. Kuniya, T. Segawa, H. Watanabe, Chem. Lett.
992, 887 – 890. Copper-catalyzed oxidation of DCFH to DCF in
1
the presence of O2 and reducing agents: D. E. Ryan, J.
Holzbecher, Int. J. Environ. Anal. Chem. 1971, 1, 159 – 168.
Angew. Chem. Int. Ed. 2006, 45, 4013 –4015
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
4015