Angewandte
Chemie
NH2
detected when dANO TP + dC TP were added to the
reaction mixture (Figure 4b). For tempT and dNTP mixes
involvinglabeled dUTP, the results were analogous. Taken
together, these data illustrate the applicability of the dNTP
conjugates in reliable SNP minisequencing through electro-
chemical detection of a single nitro- or amino-labeled
nucleobase specifically incorporated in the probe ON.
Other bioanalytical applications can involve, for example,
sandwich hybridization assays with nitro or amino end-
labeled reporter probes (RPs). Incorporation of multiple
modified bases in the RP single-stranded tail can provide
amplification of the electrochemical signal, and probes
bearingdiverse tags can be utilized for “multicolor” electro-
chemical DNA sensing.[2,3] Research focused on other ana-
lytical applications of nitro- or amino-modified nucleic acids
is now in progress.
2
of the conjugate aromatic system, the apparent redox
potentials of the amino or nitro labels responded to incorpo-
ration into ONs. Compared to the respective dNTP con-
jugates, reduction signals from ON-incorporated nitro labels
were shifted to more negative potentials and the oxidation
signals of the amino labels to more positive potentials
(Table 2). Although we do not have an explanation for this
dichotomy at this stage, the resulting changes in redox
potential are reproducible and analytically useful.
In principle, the irreversible electrochemistry of the nitro
and amino groups hampers utilization of ONs bearing these
tags in some specific detection systems based on reusable
redox-labeled recognition layers (for example, electrochem-
ical molecular beacons[12]). Nevertheless, practical bioanalyt-
ical applications of modified nucleic acids do not necessarily
require regeneration of the labeled nucleic acid molecules.
Other aspects, such as the possibility of parallel detection of
more labels, are often more important. Differences in
electrochemical processes that give rise to specific signals
produced by either nitro or amino DNA tags offer perfect
discrimination between the two labels (one label is irrever-
sibly reduced while the other is irreversibly oxidized, thus
their signals cannot overlap and cannot be mistaken). Hence,
both types of markers incorporated in the same DNA (ON)
molecule can be readily detected (see the Supporting
Information for details).
In conclusion, we have developed a facile single-step
synthesis of aminophenyl- and nitrophenyl-containingdNTPs
through cross-coupling reactions. The modified dNTPs were
efficiently incorporated by DNA polymerases to form NH2-
or NO2-modified ONs. Both types of modifications serve as
excellent electrochemical labels detectable by either oxida-
tion (NH2) or reduction (NO2), which allows perfect discrim-
ination between the two tags incorporated in the same DNA
molecule. In addition, the redox potentials of both labels
differ dependingon the nucleobase and respond to incorpo-
ration into ONs, which could be analytically useful.
Typical assays based on sequence-specific incorporation
of labeled dNTPs involve DNA “minisequencing” focused on
detection of point mutations or single-nucleotide polymorph-
isms (SNPs).[13] Figure 4 shows examples of PEX-based
Received: November 3, 2007
Published online: February 7, 2008
Keywords: DNA · electrochemistry · nucleobases · nucleosides ·
.
oligonucleotides
ˇ
[2] a) E. Palecek, F. Jelen, in Electrochemistry of Nucleic Acids and
Proteins.Towards Electrochemical Sensors for Genomics and
ˇ
Proteomics (Eds.: E. Palecek, F. Scheller, J. Wang), Elsevier,
Amsterdam, 2005, pp. 74 – 174; b) J. Wang, in Electrochemistry
of Nucleic Acids and Proteins.Towards Electrochemical Sensors
ˇ
for Genomics and Proteomics (Eds.: E. Palecek, F. Scheller, J.
Wang), Elsevier, Amsterdam, 2005, pp. 175 – 194; c) J. J. Good-
Figure 4. PEX probing of a SNP using nitro- and amino-labeled
nucleotides. The PEX reactions were conducted with templates tempA
[3] a) C. J. Yu, Y. J. Wan, H. Yowanto, J. Li, C. L. Tao, M. D. James,
Geisebrecht, J. J. Gooding, G. C. King, J.Am.Chem.Soc. 2004,
126, 4120 – 4121; c) S. S. W. Yeung, T. M. H. Lee, I.-M. Hsing, J.
ortemp C (sequences complementary to the synthesized stretches
NH2
shown) and a mixture of either a) dANO TP+dC TP or
2
b) dANH TP+dC TP (complemented with unlabeled dGTP). Sections
NO2
2
of voltammograms spanning the incorporated label signals are shown.
Otherdetails as in Figure 2.
ˇ
ˇ
Kostecka, M. Trefulka, L. Havran, E. Palecek, Anal.Chem.
A
probingof T/G SNPs in model ON templates temp and
[4] a) M. Pumera, M. T. Castaneda, M. I. Pividori, R. Eritja, A.
[5] a) S. O. Kelley, E. M. Boon, J. K. Barton, N. M. Jackson, M. G.
tempC (Table 1). The dNTP mix used for the PEX contained
labeled dATP or dCTP, one always bearinga nitro tagand the
NO2
other an amino tag. For the tempA and dANH TP + dC TP
2
mix, a well-defined NH2 peak but not NO2 peak was observed,
in agreement with specific incorporation of ANH (Figure 4a).
2
For tempC and the same dNTP mixture, the NO2 peak was
specifically detected, thus indicatingthe presence of G
(instead of T) in the template. Inverse responses were
Angew. Chem. Int. Ed. 2008, 47, 2059 –2062
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