Evaluation of the phosphorescent palladium(II)–coproporphyrin labels in separation-free...

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.

mail to: [email protected]

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


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.


(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.


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.


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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