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ANALYTICAL
Analytical Biochemistry 320 (2003) 273–280
www.elsevier.com/locate/yabio
BIOCHEMISTRY
Evaluation of the phosphorescentpalladium(II)–coproporphyrin labels in separation-free
hybridization assaysq
Martina Burke,a Paul J. O�Sullivan,a Aleksi E. Soini,b Helen Berney,c andDmitri B. Papkovskya,*
a Biochemistry Department/ABCRF, University College Cork, Lee Maltings, Cork,
Irelandb Arctic Diagnostics Oy, PO Box 51, FIN-20521 Turku, Finland
c National Microelectronics Research Centre, Lee Maltings, Prospect Row, Cork, Ireland
Received 3 April 2003
Abstract
Palladium(II)–coproporphyrin label and a set of corresponding monofunctional labeling reagents with different linker arms were
evaluated for labeling of oligonucleotides and subsequent use in hybridization assays. The properties of resulting oligonucleotide
probes including phosphorescence spectra, quantum yields, lifetimes, and labeling yields were examined as functions of the label
and oligonucleotide structures. Upon hybridization with complementary sequences bearing dabcyl, QSY-7, and rhodamine green
dyes, the probes displayed strong quenching due to close proximity effects. Intensity and lifetime changes of the phosphorescence,
distance, and temperature dependences were investigated in detail. The potential of the new label and probes for sensitive and
separation-free hybridization assays was discussed.
� 2003 Elsevier Science (USA). All rights reserved.
Keywords: DNA probes; Pd porphyrins; Time-resolved fluorescence; Hybridization assays; Phosphorescence; Proximity quenching
In recent years, fluorescently labeled oligonucleotides
and nucleic acid probes have found widespread use for
the detection of nucleic acid sequences in applications
such as fluorescence in situ hybridization, polymerasechain reaction (PCR), and gene arrays [1–7].
Separation-free hybridization assays, so-called real-
time PCR are of particular interest. The LightCycler
system [8] employs the intercalating dye (SybrGreen)
that alters its emission upon binding to DNA. The
formats based on fluorescence resonance energy trans-
fer, such as Taqman [9,10], molecular beacons [11], and
qFinancial support of this work by the Irish Research Foundation
‘‘Enterprise Ireland’’ (Grant IF/2001/008) and by the Higher Educa-
tional Authority is gratefully acknowledged.* Corresponding author. Fax: +353-21-274034.
E-mail address: [email protected] (D.B. Papkovsky).
0003-2697/$ - see front matter � 2003 Elsevier Science (USA). All rights res
doi:10.1016/S0003-2697(03)00383-X
scorpion primers [3,4], require dual-labeled oligonu-
cleotide probes or two probes hybridizing adjacent to
one another on the target sequence [12]. The intrinsic
quenching properties of nucleic acids, in particularguanine residues, have been exploited [13,14].
A major disincentive to real-time PCR is limited
throughput and high cost. Multiplexing [15,16] and al-
ternative amplification techniques have been investigated
to address this [17,18]. The potential of conventional
fluorescent labels in multiplexed hybridization assays is
limited and the detection of more than two labels in one
assay is difficult; their sensitivity is not always highenough, especially for nonexponential amplification
schemes [16]. Long-decay labels and time-resolved lu-
minescent detection can address these problems. Probes
based on fluorescent lanthanide chelates [14] and phos-
phorescent porphyrins [19] can complement conven-
tional fluorescent probes in multiplexed assays.
erved.
274 M. Burke et al. / Analytical Biochemistry 320 (2003) 273–280
In particular, the isothiocyanatophenyl derivative ofPt(II)–coproporphyrin (PtCP-NCS)1 has proven to be a
versatile reagent for phosphorescent labeling of bio-
molecules. Its protein conjugates have been applied to
sensitive immunoassays [20], time-resolved phosphores-
cence microscopy [21], and oxygen sensing [22].
In a previous study [19] we investigated the potential
of PtCP label and its oligonucleotide conjugates in
hybridization assays. Conditions for labeling of amino-modified oligonucleotides with PtCP-NCS were op-
timized to secure decent yields, and photophysical
properties of such oligonucleotide probes were investi-
gated. The capacity to detect labeled nucleic acids in
solution and agarose gels with picomolar sensitivity and
to monitor hybridization by proximity quenching was
demonstrated.
Pd(II)–coproporphyrin (PdCP) is another phospho-rescent porphyrin label and potential candidate for
sensitive and multiplexed binding assays [21,23], in-
cluding separation-free hybridization assays. Compared
to PtCP, the phosphorescent spectra of PdCP are red-
shifted for about 10–20 nm, and its emission lifetime is
about 10 times longer [24]. Therefore, PdCP and PtCP
labels can complement each other and be detected
selectively and with high sensitivity using time andwavelength discrimination.
At the same time, effects of MeCP label and carrier
biomolecule structures on the spectroscopic properties
of resulting conjugates have not been studied in suffi-
cient detail. For example, when PtCP and PdCP labels
were conjugated to proteins, significant quenching of the
phosphorescence and loss of activity were reported
[20,23,25]. It became possible to minimize these effectsand increase labeling yields by the rational design of
labeling reagent, by varying the length and hydrophi-
licity of the linker arm on PdCP [25]. In contrast,
spectroscopic properties of the PtCP-NCS label incor-
porated in DNA (labeled oligonucleotides and PCR
products) were found to be practically unaffected [19].
Properties of the PdCP label, which is more prone to
quenching due to its longer lifetime, in oligonucleotideconjugates have not been investigated as yet.
In this study, a set of derivatives of PdCP-NCS for
labeling of oligonucleotides and subsequent use in sep-
aration-free hybridization assays was evaluated. The
main objectives were to investigate the effects of the label
linker arm on the phosphorescent properties of resulting
conjugates, improve labeling yields, and develop prac-
tical hybridization assays based on the phosphorescentPdCP label. Using hybridization experiments in solu-
tion with two complementary oligonucleotide probes,
1 Abbreviations used: PtCP, platinum(II)–coproporphyrin; PtCP-
NCS, isothiocyanatophenyl derivative of PtCP; PdCP, palladium(II)–
coproporphyrin; RhG, rhodamine green; TEAA, triethylammonium
acetate; DMSO, dimethyl sulfoxide.
proximity quenching of PdCP with dabcyl, QSY-7, andrhodamine green (RhG) dyes was examined, including
intensity and lifetime changes and distance dependences.
Experimental
Materials
Triethylammonium acetate buffer (TEAA, 1M), pH
6.5, was from Alkem (Cork, Ireland). HPLC-grade
acetonitrile, dimethyl sulpfoxide (DMSO), tris(hydroxy-
methyl)aminomethane (Tris), HPLC glass vial inserts
(0.2ml), and 0.45-mm polyvinylidene fluoride dispos-
able filters were from Sigma–Aldrich (Dublin, Ireland);
0.22-lm filters were from Millipore (Watford, UK).
NAP-5 columns (DNA-grade Sephadex G-25) werefrom Amersham Pharmacia Biotech AB (Buckingham-
shire, UK). Acrylic cuvettes (3ml) optically clear on
four sides were from Sarstedt (Wexford, Ireland). All
salts and solvents were of analytical grade; solutions
were made using Millipore-grade water.
Monofunctionalized derivatives of palladium(II)–
coproporphyrin-I containing reactive 4-isothiocyana-
tophenyl moiety attached to the porphyrin macrocyclewith different spacer arms, synthesized according to So-
ini et al. [25] and having purity of >95% (HPLC), were
kindly provided by Arctic Diagnostics Oy (Turku, Fin-
land). Chemical structures of particular dyes abbreviated
asPdCP-NCS,PdCP-gly,PdCP-ala,PdCP-glu,PdCP-ca,
PdCP-glygly, and PdCP-caca, are given in Fig. 1.
Synthetic oligonucleotides, including 50- and 30-amino, 30-dabcyl, 30-rhodamine green, and 30-QSY-7modifications, were from MWG-Biotech (Ebersberg,
Germany) and from Genset (Evry, France), normally
matrix-assisted laser desorption ionization tested for
purity and molecular weight.
Labeling of oligonucleotides
Labeling was carried out using an optimized proce-dure described earlier for PtCP-NCS label [19]. The
PdCP-derivative was weighed and dissolved in DMSO
to give a concentration of 18mg/ml. A 3.5-ll aliquot of
this solution was added to a glass vial, followed by the
addition of amino-modified oligonucleotide (0.5mg/ml
in 0.1M Na–tetraborate, pH 9.5), to give a final volume
of 50 ll and approximately 14-fold molar excess of
dye (see Table 1). The reaction mixture was incubatedovernight with continuous shaking in a humid envi-
ronment at 37 �C.
The reaction mixture was loaded onto a NAP-5 col-
umn equilibrated with 0.1M Tris buffer, pH 7.4, con-
taining 1M NaCl. Unconjugated PdCP was retained on
the column, while fractions of PdCP–oligonucleotide
conjugate were collected (spectrophotometric control).
Fig. 1. Structures of PdCP derivatives (top) and pairs of oligonucleotides used in proximity experiments with PdCP label (bottom).
Table 1
Spectral and labeling properties and retention times of PdCP-NCS dyes and their 50LS oligonucleotide conjugates
Dye Labeling yield, %
(50LS)
Retention time,
dye (min)
Retention time,
conjugate (min)
Lifetime
50LS-PdCP (ms)
Quantum yield
dyeaQuantum yield
conjugatea
PdCP-NCS 90 18.47 14.64 1.15 0.18 0.26
PdCP-gly 68 18.02 13.70 1.16 0.18 —
PdCP-glu 34 16.51 12.91 1.15 0.17 0.13
PdCP-b-ala 56 17.89 13.43 1.14 0.18 0.19
PdCP-ca 60 18.49 14.09 1.24 0.18 0.19
PdCP-glygly 57 18.28 13.47 1.14 0.14 0.15
PdCP-caca 46 19.00 14.20 1.21 0.16 0.10
aAbsolute quantum yield; PdCP(III) in cetylmethylammonium bromide [23] was used as a reference.
M. Burke et al. / Analytical Biochemistry 320 (2003) 273–280 275
These fractions were desalted on a NAP-5 column equil-
ibrated with 0.1M TEAA, pH 6.5, and dried by vacuum
centrifugation. The pellet was redissolved in a minimal
volumeof 0.1MTEAA, pH6.5, and separatedby reverse-
phase HPLC. An 1100 Series system (Agilent) consisting
of a quaternary pump, diode-array photometric detector,
autosampler, and Supelcosil LC-18 column (25 cm�4.6mm, 5 lm; Supelco) was used. A 0–50% gradient(20min at 2ml/min) of acetonitrile in 0.1M TEAA, pH
6.5, was used for separation. The fractions containing
PdCP–oligonucleotide conjugate (spectral identification
on the diode-array detector) were collected, spectrally
analyzed, aliquoted, and stored at )70 �C.
Spectral measurements
Absorption spectra, range 240–600 nm,weremeasured
using an HP8453 diode-array UV–VIS spectrophoto-
meter and Chemstation software (Hewlett–Packard).
Emission spectra and lifetimes were measured on a
LS50B luminescence spectrometer using FLwinlab
software and FLDM software, respectively (Perkin–Elmer). Time-resolved phosphorescence measurements
on a microscale (50–200 ll) were carried out on a 532-
nm-laser-based ArcDia TR-plate fluorometer (Arctic
Diagnostics, Finland) described in detail elsewhere [26].
Measurements were carried out under the following
276 M. Burke et al. / Analytical Biochemistry 320 (2003) 273–280
conditions: laser flash duration, 20 ls; gate time, 700 ls;delay time, 200 ls; number of flashes, 1000 (integration
time, 0.45 s). For measurement of luminescence decay
curve on this instrument, channel width was set at 2 lsand the number of points at 300. Lifetimes were calcu-
lated using single-exponential decay fit. Quantum yields
were calculated by a method described by O�Riordan
et al. [26].
Hybridization experiments
Oligonucleotide sequences were selected so that the
reporter of PdCP label could be evaluated when the
quencher was 0, 3, 6, 8, 10, 12, 15, and 18 bases apart in a
duplex. PdCP was attached to the 50 end of the sequences
TCT ATT TAA CCC CAG ACG (50LS-18mer), ATT
TAA CCC CAG ACG (50LS-15mer), TAA CCC CAGACG (50LS-12mer), and A CCC CAG ACG (50LS-10mer), and to the 30 end of TCT ATT TAA C (30LS-10mer), TCT ATT TAA CCC (30LS-12mer), TCT ATT
TAA CCC CAG (30LS-15mer), and TCT ATT TAA
CCC CAG ACG (30LS-18mer). RhG, QSY-7, and dab-
cyl were attached to the 30 end of the complementary
sequence CGT CTG GGG TTA AAT AGA (30-ALS).Hybridization and quenching experiments were car-
ried out on the LS50B spectrometer in 3-ml cuvettes.
The phosphorescence of a 10 nM solution of PdCP-la-
beled oligonucleotide in hybridization buffer (1M NaCl,
10mM Tris, 10mM MgCl2, 0.1M Na2SO3, pH 7.6) was
monitored prior to and after the incremental addition of
quencher-labeled or amino-modified complementary
oligonucleotide to a final concentration of 20 nM. Ex-
citation was at 396 nm and emission at 668 nm with anadditional 515 nm cutoff filter; slit widths were 5 and
15 nm, respectively; delay and gate times were 200 and
1000 ls. Lifetime of the PdCP–oligonucleotide conju-
gates was measured before and after the addition of
complementary oligonucleotide, using the short phos-
phorescence decay option of FLDM software (Perkin–
Elmer). Hybridization experiments were also performed
in black 96-well plates, on the ArcDia TR-plate fluo-rometer, which can simultaneously measure the intensity
and lifetime of PdCP.
Fig. 2. Chromatogram of PdCP-b-ala-18mer reaction mixture monitored at 2
NCS at 17.5min, its conjugate at 13.4min, and PdCP-18mer breakdown pr
Results and discussion
Labeling of oligonucleotides with PdCP-NCS derivatives
Standard PdCP-NCS [27] and its six analogs [25],
each possessing the porphyrin ring structure, an iso-
thiocyanatophenyl group, and a unique linker unit
connecting the two moieties (Fig. 1), were applied to
labeling of amino-modified oligonucleotides. All theselabeling reagents are pure and stable compounds, which
can spontaneously react with primary amino groups
under mild conditions [20]. The linkers of different
length and hydrophilicity were aimed to improve water
solubility and reactivity of corresponding labeling re-
agents, minimize interactions of the conjugated label
with the oligonucleotide backbone, and maximize the
efficiency of such probes in hybridization assays basedon proximity quenching.
Using the conditions previously described for
PtCP-NCS [19], successful labeling of five different
amino-modified oligonucleotides, 50LS, 50LS-12mer,
50LS-10mer, 30LS-10mer, and 30LS-12mer, was achieved
for all the PdCP derivatives. Labeling yields of up to 90%
were achieved, though the reagents with longer and more
hydrophilic linkers produced lower labeling yields, thanstandard PdCP-NCS and PtCP-NCS [19]. Comparison
of labeling yields for 50LS oligonucleotide is shown in
Table 1. Possibly the longer, more charged arms interfere
with labeling, by repelling the DNA. There are particu-
larly low labeling yields for PdCP-glu and PdCP-caca.
Their linker arms have a high number of charged groups.
In the case of PdCP-glu, it is the only derivative to
possess a charged side chain and yields the lowest la-beling results. Perhaps removing the charged groups
could reduce the inhibition and increase labeling yields.
A two-step purification process described earlier for
the oligonucleotide conjugates with PtCP-NCS em-
ployed reverse-phase HPLC of the reaction mixture
followed by chromatography of the conjugate fractions
on a NAP-5 column, to remove traces of unconjugated
porphyrin [19]. However, the PdCP-NCS derivativesproduced more complex HPLC patterns, with a number
of peaks corresponding to the porphyrin breakdown
60 nm. The peaks were identified as oligonucleotide at 9.9min, PdCP-
oduct in the remaining peaks.
M. Burke et al. / Analytical Biochemistry 320 (2003) 273–280 277
(hydrolysis) products, appearing close to the conjugatepeak (Fig. 2) and contaminating the latter. To obtain
pure conjugates, separation on a NAP-5 column, which
eliminates free (unconjugated) porphyrin, was per-
formed prior to reverse-phase HPLC.
Fig. 4. Phosphorescence decay curves for PtCP- and PdCP-labeled
oligonucleotides (LS18, 10 nM) measured at 381/648 and 396/668 nm
(excitation/emission), respectively.
Spectral properties
The excitation and emission spectra of Pt- and PdCPare given in Fig. 3; PdCP is slightly red-shifted com-
pared to PtCP. Similar to PtCP-NCS, conjugation with
oligonucleotides was found to have little effect on the
phosphorescent properties of the PdCP label. Emission
lifetimes of the free dyes of approximately 1.15ms were
practically unaffected by the oligonucleotide. The stan-
dard PdCP-NCS derivative has a higher quantum yield
than the other PdCP derivatives, in particular glygly andcaca, both in the free and in the conjugated form (see
Table 1).
Phosphorescence decay curves for equimolar oligo-
nucleotide conjugates of PtCP and PdCP measured
under optimal conditions on the LS50B spectrometer
are given in Fig. 4. It is clear that PtCP gives a more
intense, though shorter-lived signal. Using the ArcDia, a
time-resolved fluorescent plate reader with pulsed 532-nm laser excitation, the detection limits for PtCP and
PdCP were <1 and 60 pM, respectively. While intensity
readings for PtCP were higher, the longer delay time
used for PdCP provided lower background counts
yielding a similar signal-to-noise ratio. The sensitivity of
both porphyrin labels is significantly better than that for
conventional DNA probes based on short-decay fluors
and prompt fluorescence detection. Differences in spec-tral and decay characteristics can be used for the selec-
tive detection of PtCP and PdCP labels in solution.
Simultaneous detection of PtCP- and PdCP-labeled
oligonucleotide probes by time-resolved fluorescence is
currently under investigation.
Fig. 3. Emission and excitation spectra of PtCP and PdCP in cetyl-
trimethylammonium bromide. Excitation maxima occur at 380 and
396nm, a second excitation peak occurs at 535 and 454nm, and
emission maxima occur at 648 and 668 nm for PtCP and PdCP,
respectively.
The spectral properties of PdCP (spectra, lifetime,
and quantum yield) are not affected by the lengthened
linker arms or by conjugation to amino-modified oligo-
nucleotides (see Table 1).
Proximity quenching of PdCP-labeled oligonucleotides
upon hybridization
A simple model involving two complementary oli-
gonucleotides, one labeled at its 50 end with PdCP and
the other labeled at its 30 end with dabcyl, QSY-7, or
RhG dyes, was used to evaluate the quenching effects atclose proximity.
Dabcyl and QSY-7 have a broad effective range of
absorption and are used as quenchers for various flu-
orophores. Probes incorporating these molecules have
lower background fluorescence than those that incor-
porate fluorescent quenchers [28].
For all these dyes, strong quenching of PdCP emis-
sion was observed upon hybridization. Maximalquenching (up to 94% of the initial signal) was observed
for QSY-7 (Fig. 5), which is greater than that for a
Fig. 5. The residual phosphorescence intensity (396/668 nm) of PdCP-
labeled oligonucleotide (50LS-18mer, 10 nM) upon hybridization with
QSY-7/RhG/dabcyl/unlabeled complement (30ALS, twofold molar
excess), in hybridization buffer with sulfite, 22 �C. Residual intensity
was calculated with respect to the intensity of PdCP prior to the
addition of complement.
278 M. Burke et al. / Analytical Biochemistry 320 (2003) 273–280
europium chelate and Cy5 pair under similar conditions[29]. For Dabcyl and RhG maximal quenching was 87
and 77%, respectively (Fig. 5). Quenching of PdCP-la-
beled oligonucleotide by its unlabeled complement oli-
gonucleotide (7%) was less than that for the
conventional fluorophores such as fluorescein which
loses 26% of its signal on hybridization [12].
Oligonucleotide probes labeled with the different
PdCP-NCS derivatives showed patterns of proximityquenching similar to those of the three quencher dyes
used in this study. However, the quenching effect on
PdCP-gly was lower for dabcyl and QSY-7 and higher
for RhG where PdCP and the quencher are 0 bases apart
Table 2
Percentage residual intensity of 10 nM PdCP-labeled oligomers (kex ¼ 396 n
amino-modified complement (30ALS) in hybridization buffer (1M NaCl, 10
PdCP derivative No. of bases between
PdCP and quencher
molecule
Dabcyl Q
PdCP-NCS 0 13
3 —
6 —
8 55
10 90
12 103
15 100
18 99
PdCP-gly 0 19
6 23
12 96
PdCP-glu 0 13
6 14
8 52
10 89
12 96
PdCP-b-ala 0 9
6 27
8 51
10 78
12 93
PdCP-ca 0 11
6 27
8 53
10 77
12 91
PdCP-glygly 0 10
6 27
8 51
10 81
12 90
PdCP-caca 0 11
6 31
8 56
10 82
12 90
The residual intensity of PdCP-labeled oliogonucleotides when QSY-7/Rh
hybridization scheme) is given.
(Table 2). The most obvious explanation for this wouldbe structural influences of the linker unit, which may
influence the position and orientation of PdCP. Com-
pared to PdCP-NCS with the shortest linker unit,
PdCP-gly linker unit contains an additional peptide
bond increasing its hydrophilicity and reducing flexi-
bility [30]. PdCP-b-ala differs from PdCP-gly by a single
methylene group. Perhaps these differences are enough
to give a more favorable orientation between PdCPand RhG and a less favorable orientation between
dabcyl and QSY-7. Studies of the distance dependence
of quenching of labeled oligonucleotides upon hybridi-
zation (see Fig. 1 for schematic diagram) revealed that
m, kem ¼ 668 nm) on titration with 2X QSY-7/RhG/dabcyl-labeled or
mM Tris, pH 7.6) and sulfite at 22 �C
SY-7 RhG NH2-modified
6 23 93
10 26 96
8 33 107
39 31 106
69 74 97
80 88 86
84 93 96
84 87 92
15 4 97
12 25 —
81 103 —
9 18 85
12 50 —
33 26 —
86 80 —
91 97 —
8 16 90
15 24 —
31 26 —
71 76 —
90 96 —
6 19 95
34 27 —
32 27 —
73 68 —
81 89 —
10 16 93
20 22 —
31 25 —
79 75 —
84 71 —
8 18 97
17 29 —
34 27 —
78 77 —
83 89 —
G/dabcyl are positioned at varying distances away from each other (see
Fig. 7. Residual intensity of PdCP-labeled 50LS-18mer and 50LS-12mer
after hybridization with 2M excess of RhG-labeled complement po-
sitioning PdCP at zero and six bases from RhG at increasing tem-
peratures. Conditions were the same as those in Fig. 5. Residual
intensity was calculated with respect to the intensity of PdCP prior to
the addition of complement.
M. Burke et al. / Analytical Biochemistry 320 (2003) 273–280 279
the most efficient quenching of the PdCP signal wasobserved where no nucleotide bases separated the ac-
ceptor molecule and the PdCP label. The nature of the
linker unit did not have a great impact on the quenching
observed, though some anomalies were observed, e.g.,
the limited quenching of PdCP-glu by RhG at 6 bases
apart, which is restored at 8 bases apart. Studies of the
effect of distance on quenching show that where there
are 8 bases separating the acceptor the PdCP quenchingcan occur, but if this distance is increased to 10 bases the
quenching is almost eliminated. Between 3 and 8 bases,
the DNA between the PdCP and the quencher is single
stranded and by its nature flexible, whereas at all other
distances the intervening DNA is double stranded. It is
possible that separating the PdCP and the quencher by
single-stranded DNA would give a higher degree of
quenching. The mechanism of quenching of PdCP is stillopen to question, but initial investigations show that
static quenching is probable, where increasing temper-
ature decreases quenching [31]. In all cases, quenching
disappears when PdCP and the quencher dye are more
than 8–10 bases apart from each other in a duplex
(Table 2).
Hybridization and proximity quenching of the PdCP
label were usually accompanied by some reduction in itsemission lifetime. Lifetime changes were higher when the
complement was labeled with dabcyl, QSY-7, or RhG
than with amino-modified complement (Fig. 6).
Temperature effects on the emission of PdCP-labeled
oligonucleotides and on quenching by RhG-labeled
complement were examined. When the labels were at
zero and six bases apart, a significant decrease in
quenching was observed with increasing temperature(Fig. 7). The difference in the temperature profiles be-
tween the 18mer (zero bases apart) and the 12mer (six
bases apart) was probably due to the lower melting
temperature for the 12mer. The calculated melting
Fig. 6. Phosphorescence lifetime changes for PdCP-labeled oligonu-
cleotides (50LS-18mer, 5 nM) upon hybridization with QSY-7/RhG/
dabsyl/unlabeled complement (labels are zero bases apart) measured
on ArcDia TR-plate fluorometer: excitation at 532 nm; emission at
650 nm. Other conditions are the same as those in Fig. 5.
temperature of the 18mer and 12mer were 48.8 and
43.4 �C, respectively [32].
Conclusions
Long-decay PdCP proved to be a viable label for
sensitive detection of nucleic acids. Successful labeling
of a series of oligonucleotides with PdCP-NCS was
achieved.
The phosphorescent properties of the PdCP labels
were not significantly affected by conjugation to amino-
modified oligonucleotides. Labeled oligonucleotidesdemonstrated strong (up to 94%) quenching in solution
upon hybridization with complementary sequences la-
beled with dabcyl, QSY-7, and RhG dyes. The quench-
ing occurred when the quencher was less than 8–10 bases
apart from PdCP, i.e., at close proximity, and was ac-
companied by a 10–20% decrease of emission lifetime.
The PdCP derivatives with longer and more hydro-
philic linker units were unable to improve labeling yieldsfor the oligonucleotides or emission and quenching
properties of the oligonucleotide probes. The standard
PdCP-NCS with the shortest-arm reagent provided the
best results.
PdCP-based oligonucleotide probes have potential in
separation-free hybridization assays; they can supple-
ment the PtCP-based oligonucleotide probes.
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