C O M M U N I C A T I O N S
Figure 3. Detection of the G insertion in âENaC gene sequences by the
fluorescence change of the Py2Lys-containing ODN probes, ODNLS Py2Lys).
(
ODNLS(WT) and ODNLS(G) (2.5 µM) were hybridized with 2.5 µM
ODNLS
(
Py2Lys) (50 mM sodium phosphate, 0.1 M sodium chloride, pH
7.0, room temperature, total volume 10 µL) in a 96 well microplate.
Fluorescence was observed using a fluorescence imager VersaDoc Imaging
System (BioRad) equipped with a 365 nm transilluminator. The image was
taken through a 380 nm long pass emission filter.
poor fluorescence of the ODNLS(
Py2Lys)/ODNLS(WT) duplex. The
hybridization of the Py2Lys-containing ODN with a target DNA
facilitates the determination of the presence/absence of insertion
polymorphisms located at a specific site on the target DNA by
simply mixing.
In conclusion, we have devised a facile method for the detection
of insertion polymorphisms by exploiting novel pyrene-labeled
ODN probes. We have designed a new pyrene-linking unit, Py2Lys,
which can distinguish the presence/absence of the base opposite
Py2Lys. The present method for insertion polymorphism detection
using Py2Lys-containing ODN probes constitutes a very facile and
accurate homogeneous assay.
Figure 2. (a) Fluorescence spectra of 2.5 µM ODN(Py2Lys) hybridized
with 2.5 µM ODN0 or ODN1(A) (50 mM sodium phosphate, 0.1 M sodium
chloride, pH 7.0, room temperature). Excitation was at 350 nm. Inset is the
fluorescence image of the solutions of ODN(Py2Lys)/ODN0 (left) and ODN-
(
Py2Lys)/ODN1(A) (right) duplexes illuminated with a 365 nm transillu-
minator. (b) Fluorescence intensities at 495 nm from ODN(Py2Lys)
hybridized with sequences possessing various insertion bases. The fluores-
cence was measured in the same way as described in a. (c) Variation of
absorption maxima of ODN(Py2Lys)/ODN0 and ODN(Py2Lys)/ODN1(A)
with solution temperature (10 µM strand concentration, 50 mM sodium
phosphate, 0.1 M sodium chloride, pH 7.0). (d) Intensities of fluorescence
from ODN(Py2Lys), ODN(Py2Orn), and ODN(Py2Dab) hybridized with
ODN0 or ODN1(A). The fluorescence was measured in the same way as
described in a.
Acknowledgment. We thank Dr. A. Yamane (Wakunaga
Pharmaceutical Co., Ltd.) for valuable discussions on the present
study.
Supporting Information Available: Detailed experimental data
on Py2Lys, Py2Orn, Py2Dab, and the related ODNs (PDF). This material
References
We next compared the fluorescence intensities of the pyrene-
labeled ODNs consisting of different amino acids, ODN(Py2Lys),
ODN(Py2Orn), and ODN(Py2Dab) (Figure 2d). ODN(Py2Orn) and
ODN(Py2Dab) hybridized with ODN1(A) are emissive, and the
fluorescence was stronger than those of the duplexes hybridized
with ODN0. However, these fluorescence intensities are much lower
than that of ODN(Py2Lys)/ODN1(A). Excimer fluorescence emis-
sion was strongly affected by the length of the amino acid side
chain that connects two pyrene chromophores. These experiments
concluded that ODN(Py2Lys) is most suitable for the detection of
an extra base in the target DNA among three ODN probes
containing different amino acids.
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The clear change in the fluorescence that depends on the
presence/absence of the inserted base opposite Py2Lys is very useful
for the detection of insertion polymorphisms. We tested the
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channel â-subunit (âENaC) associated with Liddle’s syndrome,
which is an autosomal dominant form of hypertension with variable
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and a premature stop codon (Table 1).6 We prepared the Py2Lys-
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in Supporting Information.
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containing probe ODNLS(
Py2Lys) and hybridized with âENaC gene
sequences, a wild-type ODNLS(WT), and a G-inserted mutant
ODNLS(G). The sample solutions were illuminated at 365 nm, and
the fluorescence images were taken through a 380 nm filter (Figure
3). The fluorescence emission from the duplex ODNLS
ODNLS(G) was very strong and clearly distinguishable from the
(
Py2Lys)/
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