Two-dye and one- or two-quencher DNA probes for real-time PCR assay: Synthesis and comparison with a TaqMan™ probe

Abstract

A typical TaqMan™ real-time PCR probe contains a 5′-fluorescent dye and a 3′-quencher. In the course of the amplification, the probe is degraded starting from the 5′-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye–dye and dye–quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM–JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and “click chemistry” post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.
Figure
Fluorogenic TaqMan probes with various modifications for real-time PCR

Full text

1 23
Analytical and Bioanalytical
Chemistry
ISSN 1618-2642
Volume 404
Number 1
Anal Bioanal Chem (2012) 404:59-68
DOI 10.1007/s00216-012-6114-4
Two-dye and one- or two-quencher DNA
probes for real-time PCR assay: synthesis
and comparison with a TaqMan™ probe
Dmitry Y.Ryazantsev, Dmitry
A.Tsybulsky, Igor A.Prokhorenko,
Maksim V.Kvach, Yury V.Martynenko,
Pavel M.Philipchenko, et al.

1 23
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ORIGINAL PAPER
Two-dye and one- or two-quencher DNA probes for real-time
PCR assay: synthesis and comparison with a TaqMan™
probe
Dmitry Y. Ryazantsev &Dmitry A. Tsybulsky &Igor A. Prokhorenko &
Maksim V. Kvach &Yury V. Martynenko &Pavel M. Philipchenko &
Vadim V. Shmanai &Vladimir A. Korshun &Sergey K. Zavriev
Received: 11 December 2011 / Revised: 9 May 2012 /Accepted: 14 May 2012 /Published online: 19 June 2012
#Springer-Verlag 2012
Abstract A typical TaqMan™real-time PCR probe con-
tains a 5′-fluorescent dye and a 3′-quencher. In the course of
the amplification, the probe is degraded starting from the 5′-
end, thus releasing fluorescent dye. Some fluorophores (in-
cluding fluorescein) are known to be prone to self-quenching
when located near each other. This work is aimed at studying
dye–dye and dye–quencher interactions in multiply modified
DNA probes. Twenty-one fluorogenic probes containing one
and two fluoresceins (FAM), or a FAM–JOE pair, and one or
two BHQ1 quenchers were synthesized using non-nucleoside
reagents and “click chemistry”post-modification on solid
phase and in solution. The probes were tested in real-time
PCR using an ~300-bp-long natural DNA fragment as a
template. The structural prerequisites for lowering the probe
background fluorescence and increasing the end-plateau fluo-
rescence intensity were evaluated and discussed.
Keywords Real-time PCR efficiency .TaqMan probes .
Fluorescence quenching .Fluorescein .JOE
Introduction
Quantitative real-time PCR (qrtPCR) with fluorogenic
probes/primers is a powerful tool in molecular diagnostics.
There are several formats of qrtPCR using various types of
fluorescent DNA conjugates [1–8]. The perfect correlations
of the signal increase corresponding to the PCR product
accumulation were developed [1,9–12]. The main feature
of the success of qrtPCR is a design of both non-fluorescent
primers and fluorogenic DNA probes.
A widely used type of probe is the TaqMan™, an oligo-
nucleotide containing one fluorescent dye and one fluores-
cence quencher; the probe is complementary to a part of the
amplified sequence capable of the 5′→3′degradation by the
exonuclease activity of DNA polymerase, thus amplifying
the fluorescence intensity [13–16]. Each TaqMan probe can
be characterized by an original (background) and the
resulting (plateau) fluorescence intensity level. Properly
designed TaqMan probes should have low background and
high plateau fluorescence.
The approach leading to a background fluorescence de-
crease of the TaqMan probe is the moving of a dye or a
quencher from a terminal to an internal position in an oligo-
nucleotide. A distance decrease between a dye and a quencher
gives rise to a more efficient Förster resonance quenching
[17], thus diminishing the original probe fluorescence. The
method was used for several dye–quencher pairs: FAM–
TAMRA [ 18], BHQ1–FAM [ 19], and FAM–ZEN+IBFQ [20].
To increase the plateau fluorescence, AllGlo probes, con-
taining two identical dyes, were developed [21,22] and used
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-012-6114-4) contains supplementary material,
which is available to authorized users.
D. Y. Ryazantsev :I. A. Prokhorenko :V. A. Korshun (*):
S. K. Zavriev (*)
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Miklukho-Maklaya 16/10,
117997 Moscow, Russia
e-mail: korshun@ibch.ru
e-mail: szavriev@ibch.ru
D. A. Tsybulsky :M. V. Kvach :Y. V. Martynenko :V. V. Shmanai
Institute of Physical Organic Chemistry,
Surganova 13,
220072 Minsk, Belarus
P. M. Philipchenko
Primetech LLC,
Surganova 13,
220072 Minsk, Belarus
Anal Bioanal Chem (2012) 404:59–68
DOI 10.1007/s00216-012-6114-4
Author's personal copy

in rtPCR assays [22–26]. If two identical fluorophores pos-
sessing a small Stoke’s shift are located within the Förster
distance, a quenching caused by resonance energy migration
can occur [17]. Apparently, the self-quenching mechanism
contributes substantially to the reduction of the AllGlo
background fluorescence. In the course of the PCR, probe
degradation occurs and two dye residues separate from each
other to produce plateau fluorescence, doubled in compari-
son with a standard dye–quencher TaqMan probe. Thus,
AllGlo probes should have higher background and plateau
fluorescence vs. TaqMan probes.
We employed modern conjugation chemistry methods to
construct qrtPCR probes containing two fluorescent dyes
and quencher(s) to combine the advantageous features of
both classic TaqMan and AllGlo probes with low back-
ground and high plateau fluorescence level.
Results and discussion
In the last decade, azide–alkyne cycloaddition reaction,
“click chemistry,”became a powerful method for synthesiz-
ing modified oligonucleotides [27–30]. To introduce
modification in non-terminal positions, we applied 5-
(octadiynyl)-dU phosphoramidite 1[31,32]instandard
oligonucleotide synthesis. The “click”reaction is performed
on a solid phase or in solution. Scheme 1illustrates both the
ways for the introduction of internal fluorescein into a DNA
probe.
Modification on solid phase
Oligonucleotide synthesis is stopped after the synthetic
cycle introducing the 5-(octadiynyl)-dU unit. Oligomer 2
reacts with an azidoalkyl derivative of O-protected fluo-
rescein 3in dimethyl sulfoxide (DMSO) in the presence
of a Cu(I) catalyst to give product 4. The latter contains
fluorescein (FAM) as a non-fluorescent acyl-protected resi-
due suitable for oligo synthesis resumption. After automated
synthesis, the oligonucleotide is cleaved and deprotected as
usual, affording internally modified conjugate 5(ammonia
easily cleaves cyclohexylcarbonyl groups from FAM and
fluorescence restores).
Modification in solution
Phosphoramidite 1is used to introduce an internal alkyne.
The deprotected and purified oligomer 6reacts in an
aqueous solution with polar FAM azide 7/Cu(I) catalyst to
yield the desired conjugate 5(Scheme 1). Similarly, the
fluorescence quencher BHQ1 can be introduced into oligo-
nucleotides as internal modification 9(Scheme 2). Azido
derivative 8(Primetech) is suitable for both “click”on solid
support and in solution.
Scheme 1 Internal modification of oligonucleotides with FAM using “click chemistry”: on solid phase and in solution. Fluorescent FAM residues
are shown in green
60 D.Y. Ryazantsev et al.
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For terminal modifications, phosphate, BHQ1, and FAM
solid supports (Glen Research), 6-FAM phosphoramidite
[33] and 6-JOE phosphoramidite [34], were used. Alterna-
tively, the alkyne support 10 (see Electronic supplementary
material (ESM)) and “click”in solution were used for the
introduction of the FAM dye into the 3′-terminal position of
a probe (Scheme 3). To introduce two BHQ1 into the 3′-end
of a probe, a combination of BHQ1 support (Glen Research)
and BHQ1 phosphoramidite [35] was used. Moreover, the
alkyne support 13 (see ESM) was prepared and used for
“click”modification with the BHQ1 azide 8(Scheme 3).
We used a 27-nucleotide-long probe sequence within a
291-bp PCR-amplified fragment of the gene of translation
elongation factor 1αFusarium avenaceum as a model DNA
template [19](see“Experimental”). Combining the 3′-
modifying solid supports, the 5′-modifying phosphoramidites,
5-(octadiynyl)-dU phosphoramidite, and “click chemistry”
techniques, a standard TaqMan probe P1 and 21 ofits versions
(P2–P20, P23, and P24), together with two AllGlo-type
probes (P21 and P22), were synthesized (Table 1).
Scheme 4shows examples of the strategies used for the
synthesis of highly modified fluorogenic DNA probes. To
produce probes containing two identical internal modifica-
tions, the oligonucleotide was assembled and deblocked and
then the FAM azide was attached using Cu(I) catalyzed
cycloaddition in solution (Scheme 4a). The approach was
used for the probes containing two T
FAM
modifications (P6
and P8–P11). To produce probes containing two different
internal modifications (P15–P20), the oligonucleotide syn-
thesis was stopped after the synthetic cycle introducing the
first 5-(octadiynyl)-dU unit; the “click”modification involv-
ing the terminal alkyne was performed on the solid phase
CuSO4 / ascorbate /
TBTA / H2O / DMSO
12
10
11
- solid support
N
DmtO 7
2. conc. NH3, 60oC
1. Oligo synthesis
O
O
O
O
N
H
N
O
OH
O
P
OO
O
N
O
OH
O
P
OO
O
N
NN
N
H
O
O
OH
O
O2C
OO
OO
O
O
DmtO
O
13 O
O
N
H
OO
OO
14
O
P
OO
ON
NN
NN
OCH3
NO2
N
H
N
O
N
N
NN
N
N
N
OCH3
NO2
O
HN
O
N
N
N
O
OH
Scheme 3 Introduction of the 3′-terminal FAM or BHQ1 pair using alkynyl supports and “click chemistry.”Fluorescent FAM residue is shown in
green; BHQ1 dyes are shown in bold
Scheme 2 Introduction of internal BHQ1 on 5-(octadiynyl)-dU in oligonucleotides using “click chemistry.”BHQ1 dye is shown in bold
Two-dye and one- or two-quencher DNA probes for qrtPCR 61
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and the synthesis was then continued upon completion. The
oligonucleotide was isolated and the second “click”modi-
fication was carried out in solution (Scheme 4b). “Click”in
solution with FAM azide 7resulted in excellent yields of
modification. In contrast, “click”on solid support considerably
reduces the isolated amount of the probe, e.g., typical yields of
internally BHQ1-modified probes P15–P20 were approx. 5 %
as compared with P1–P14 probes. 3′-Modifications in probes
P22 and P23 were introduced using “click”in the solution
procedure. All the probes were purified by PAGE and rpHPLC
and their structures were confirmed by matrix-assisted laser
desorption–ionization time-of-flight (MALDI-TOF) mass
spectrometry (Table 1and “Experimental”).
Probes P1–P24 have the same nucleotide sequence and
were designed to study various dye–dye and dye–quencher
interactions, as well as the modification influence on probe
degradation by DNA polymerase. All the probes were tested
and compared in qrtPCR under identical conditions (see
“Experimental”and ESM Figs. S1–S8). Figure 1shows
the background fluorescence and the fluorescence increase
in the end point of the PCR for each probe.
Surprisingly, both AllGlo-type probes, P21 and P22, pre-
pared using a different chemistry showed a high starting
fluorescence and its gradual decrease, not a sigmoid increase,
in the course of PCR (Table 1and ESM Figs. S7and S10).
FAM dyes were used in AllGlo probes [22]. Moreover, FAM
dyes are prone to considerable self-quenching on DNA [36].
Therefore, our results suggest that the 27-nucleotide-long
sequence used in this study cannot be terminally modified
with FAM to yield a valid AllGlo-type probe.
Remarkably, the background fluorescence of all probes is
not higher than that of the standard TaqMan probe P1 (Table 1
and Fig. 1). This is obvious for probes P2 and P3, also having
one FAM residue attached slightly closer to the 3′-quencher. In
contrast, probes P4–P11 and P15–P20 contain twoFAM res-
idues; their combined fluorescence appears less than the fluo-
rescence of one FAM in P1. The fact indicates that in addition
to FAM–BHQ1 quenching in an oligonucleotide, there is a
quenching interaction between FAMs. The nature of the inter-
action is most likely an electronic excited energy migration
increasing the probability of non-radiative relaxation. Two
FAM residues in probe P4 are located close to each other;
thus, contact (exitonic or Dexter) quenching is also possible.
Indeed, its background fluorescence is inferior to one of
probes P5–P9, where two FAMs are separated by several
nucleotides. Probes P10 and P11 have FAM pairs close to
BHQ1; thus, their background fluorescence is greatly reduced.
Excitonic quenching in linear terminally labeled DNA
probes was reported once as an addition to the Förster
energy transfer (FRET) type of quenching [37]. Moreover,
Dexter dynamic quenching by an occasional collision of
dyes is also possible [38,39]. AllGlo-type probes P21 and
P22 show high fluorescence, thus confirming that contact
Table 1 Structures and characteristics of fluorogenic probes
Number Probe structure, 5′→3′
a
MALDI MS,
calc/found
b
Relative background
fluorescence, I
0Pi
/I
0P1
c
Relative fluorescence
increase, I
fPi
/I
0Pi
c
Relative fluorescence addition
(I
fPi
−I
0Pi
)/(I
fP1
−I
0P1
)
c
T
m
/ΔT
m
(°C)
d
P1 FAM -TCATTCGAAACGCATTCATTACCCCGC-BHQ1 9,230/9,224 1.00 1.56 1.00 69.7/0.0
P2 (T
FAM
)CATTCGAAACGCATTCATTACCCCGC-BHQ1 9,243/9,236 0.88 1.58 0.90 69.9/+0.2
P3 TCA(T
FAM
)TCGAAACGCATTCATTACCCCGC-BHQ1 9,243/9,245 0.82 1.56 0.83 69.2/−0.5
P4 FAM -(T
FAM
)CATTCGAAACGCATTCATTACCCCGC-BHQ1 9,779/9,771 0.42 1.38 0.29 69.8/+0.1
P5 FAM -TCA(T
FAM
)TCGAAACGCATTCATTACCCCGC-BHQ1 9,801
e
/9,800 0.81 1.63 0.91 68.9/−0.8
P6 (T
FAM
)CA(T
FAM
)TCGAAACGCATTCATTACCCCGC-BHQ1 9,814
e
/9,815 0.92 1.53 0.87 68.9/−0.8
P7 FAM -TCAT(T
FAM
)CGAAACGCATTCATTACCCCGC-BHQ1 9,801
e
/9,798 0.77 1.91 1.25 68.9/−0.8
P8 (T
FAM
)CAT(T
FAM
)CGAAACGCATTCATTACCCCGC-BHQ1 9,814
e
/9,814 0.72 1.70 0.89 68.7/−1.0
P9 TCA(T
FAM
)TCGAAACGCA(T
FAM
)TCATTACCCCGC-BHQ1 9,814
e
/9,816 0.72 1.43 0.55 66.6/−3.1
P10 TCATTCGAAACGCAT(T
FAM
)CA(T
FAM
)TACCCCGC-BHQ1 9,814
e
/9,810 0.10 1.30 0.05 66.4/−3.3
P11 TCATTCGAAACGCAT(T
FAM
)CAT(T
FAM
)ACCCCGC-BHQ1 9,814
e
/9,812 0.09 1.21 0.03 66.4/−3.3
P12 JOE-(T
FAM
)CATTCGAAACGCATTCATTACCCCGC-BHQ1 9,908/9,903 0.02 0.30 −0.02 69.7/0.0
P13 JOE-TCA(T
FAM
)TCGAAACGCATTCATTACCCCGC-BHQ1 9,908/9,902 0.18 1.80 0.26 69.4/−0.3
P14 JOE-TCAT(T
FAM
)CGAAACGCATTCATTACCCCGC-BHQ1 9,908/9,907 0.24 2.56 0.65 69.0/−0.7
P15 FAM -TCAT(T
FAM
)CGAAACGCATTCA(T
BHQ1
)TACCCCGCp 10,009/10,000 0.51 1.63 0.57 65.8/−3.9
62 D.Y. Ryazantsev et al.
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Table 1 (continued)
Number Probe structure, 5′→3′
a
MALDI MS,
calc/found
b
Relative background
fluorescence, I
0Pi
/I
0P1
c
Relative fluorescence
increase, I
fPi
/I
0Pi
c
Relative fluorescence addition
(I
fPi
−I
0Pi
)/(I
fP1
−I
0P1
)
c
T
m
/ΔT
m
(°C)
d
P16 FAM -TCAT(T
FAM
)CGAAACGCA(T
BHQ1
)TCATTACCCCGCp10,009/10,004 0.32 2.01 0.58 65.9/−3.8
P17 FAM -TCAT(T
BHQ1
)CGAAACGCATTCA(T
FAM
)TACCCCGCp10,009/10,010 0.35 1.64 0.40 66.3/−3.4
P18 FAM -TCAT(T
BHQ1
)CGAAACGCA(T
FAM
)TCATTACCCCGCp10,009/10,001 0.25 1.98 0.44 66.3/−3.4
P19 FAM -TCAT(T
BHQ1
)CGAAACGCA(T
FAM
)TCATTACCCCGC-BHQ1 10,484/10,480 0.14 1.96 0.24 66.1/−3.6
P20 FAM -TCAT(T
FAM
)CGAAACGCA(T
BHQ1
)TCATTACCCCGC-BHQ1 10,484/10,475 0.48 1.27 0.23 65.2/−4.5
P21 FAM -TCATTCGAAACGCATTCATTACCCCGC-FAM(1) 9,243/9,249 3.35 0.61 −2.31 69.9/+0.2
P22 FAM -TCATTCGAAACGCATTCATTACCCCGC-FAM(2) 9,404/9,408 3.60 0.90 −0.65 70.0/+0.3
P23 FAM -TCATTCGAAACGCATTCATTACCCCGC-(BHQ1)
2
10,411/10,402 0.40 1.19 0.13 69.3/−0.4
P24 FAM -TCATTCGAAACGCATTCATTACCCCGC-BHQ1-BHQ1 9,783/9,775 0.20 2.32 0.48 69.5/−0.2
a
p—3′-phosphate; other modifications
Two-dye and one- or two-quencher DNA probes for qrtPCR 63
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quenching is negligible in the case of this probe sequence.
Consequently, FRET is probably the main quenching mech-
anism in the presented probes, with minor participation of
excitonic and Dexter contact mechanisms. However, the
exact share of each mode is not relevant because every type
of quenching is not functioning after probe degradation.
In the course of rtPCR, the probe is degraded from the 5′-
end. Probes P1–P3 have one FAM residue to be detached.
The fluorescence increase for probes P2 and P3 is not higher
than for P1; therefore, they are less susceptible for degrada-
tion by DNA polymerase. Melting studies show that probes
P1–P3 have a very similar duplex stability (Table 1). The
conclusion can be drawn that nucleoside T
FAM
modification
is more inhibitive for the exonuclease activity of the DNA
polymerase than the non-nucleotide 5′-FAM modification.
Probe P4 shows a weak fluorescence increase (Table 1and
Fig. 1). An explanation is that FAM residues are not separated
when the probe is degraded because the DNA polymerase is
not able to hydrolyze the phosphate diester between 5′-FAM
and T
FAM
. By combining two modifications, inhibition of
polymerase exonuclease activity becomes possible. Alterna-
tively, the “bitten”piece of oligonucleotide may contain
several nucleosides, retaining 5′-FAM and T
FAM
together.
This is consistent with a probe degradation study [13].
Probes P10 and P11 indicate no fluorescence increase in
the PCR. This confirms the nature of 5′-degradation of a
probe: after the detachment of several nucleosides, the
remaining part of the probe, still containing two FAMs and
one BHQ1, is no longer able to form a duplex at the
temperature of the elongation (67 °C).
On the contrary, probes P5–P9, carrying two FAMs sepa-
rated by several nucleosides, are capable of enzymatic degra-
dation. As expected, probe P9 releases only one FAM;
therefore, its fluorescence increase is minimal among double-
FAM - l a be led probe s P 5 –P9 (Table 1and Fig. 1). The best
results are shown by probe P7. It differs from probe P5 by
one nucleotide position. Probe P7 has similar background
fluorescence and a better fluorescence increase (137 % as
compared to P5). This effect, resulting from one nucleotide
distance increase, might be attributed to the ability of the
enzyme to cut off a short oligonucleotide from a probe rather
than individual nucleotides [13]. Indeed, dichloro-2′,7′-dime-
thoxy-6-carboxyfluorescein (JOE) containing probes P12–P14
has similar duplex stability; however, the increase of the pla-
teau fluorescence correlates with the distance between FAM
and JOE: P14> P13>>P12. Thus, a degradation product con-
taining simultaneously FAM and JOE is probably typical not
only for probe P12 but also for P13 (it is noteworthy that P14
shows 255% of the fluorescence increase as compared to P13).
According to the results with probes P12–P14, JOE is a
very efficient quencher for FAM in contact and energy
transfer mode. JOE fluorescence addition changes P14>
P13>>P12, similar to FAM fluorescence (ESM Fig. S11).
b
Average (not monoisotopic) mass; calculated [M+H]
+
masses are given. The most intense peaks in the mass spectra (data not shown) come from typical fragmentation of BHQ1-containing
oligonucleotides in the MALDI mass spectrometer:
For the P5–P11, prone to Na
+
addition, major fragmentation peaks correspond to [M + Na]
+
, with the above splitting-off fragment replaced by OH (data not shown)
c
I
0P1
0starting (background) fluorescence intensity of probe P1; I
0Pi
0starting (background) fluorescence intensity of probes P1–P20; I
fP1
0final (plateau) fluorescence intensity of probe P1; I
fPi
0final (plateau)
fluorescence intensity of probes P1–P20. The values were calculated using data obtained in a PCR experiment with high amounts of a target (25,000 copies), duration of 45 cycles, mean of two repeats, sigmoid
fit of fluorescence curve (see ESM Fig. S9)
d
Melting temperature of the duplex with complementary DNA oligonucleotide 5′-GCG GGG TAA TGA ATG CGT TTC GAA TGA-3′determined using SYBR Green I fluorescence (see
“Experimental”); ΔT
m
is given in comparison with the value for the standard TaqMan probe P1
e
Calculated [M+Na]
+
masses
64 D.Y. Ryazantsev et al.
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The structure of the FAM pair in probes P15–P16 is similar
to P7; the quencher, however, is inserted closer to fluorophores.
Unfortunately, T
BHQ1
modification considerably reduces the
probe melting temperature (approx. 4 °C), thus making the
duplex less stable at the temperature of the elongation step.
Probes P7, P14, P16, P18, P19, and P24 show reliably
higher values of the relative fluorescence increase than the
standard TaqMan P1. Probe P14 has a better value (2.56 vs.
1.56 for P1), thus indicating that JOE is an efficient addi-
tional FAM quencher (cf. similar to probe P3; Table 1).
The arrangement of dyes is important for background
fluorescence. To evaluate this factor, probes P15–P18,
possessing two FAMs and one BHQ1 within a nucleo-
tide sequence, were designed (Table 1). Their fluores-
cent properties are similar (Table 1and Fig. 1and ESM
Fig. S5). Probes P19 and P20 have two FAMs and two
BHQ1. A remarkable difference is observed in their
background fluorescence (more than three times), con-
firming that FAM–BHQ1–FAM–BHQ1 in probe P19 is
an appropriate arrangement for effective quenching.
p p pp
p
p
oligo
synthesis
oligo
synthesis
oligo
synthesis
deblocking
deblocking
'click'
modification
'click'
modification
'click'
modification
a)
b)
p - phosphate
-BHQ1
-FAM
-terminalalkyne
- solid support
- oligonucleotide
P8
P16
Scheme 4 Preparation of doubly labeled probes using “click chemistry.”aSynthesis of probe P8: “click”in solution. bSynthesis of probe P16:
stepwise “click”on solid phase and in solution
-6000
-5000
-4000
-3000
-2000
-1000
0
1000
2000
3000
4000
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P23 P24
Fig. 1 Relative background fluorescence (yellow) and fluorescence
increase after 45 cycles (green). For convenience of comprehension,
the background fluorescence values are shown as negative (Y-axis
labels are arbitrary units). Fluorescence increase is the difference
between the final and the starting plateau levels for sigmoidally fitted
fluorescence intensity/PCR cycle curve (in the case of probes with a
non-sigmoidal dependence, P10–P12, the difference between the final
and the starting fluorescence is given). Data were collected on the FAM
channel of a rtPCR cycler (for mean values from two repeats, see
“Experimental”). Error values of the data are within ±10 % (highest
limits of possible errors coming from UV/Vis measurements of DNA
probes, dilution procedures, and fluorescence reading in the rtPCR
cycler based on multiple repetitive experiments with several probes)
Two-dye and one- or two-quencher DNA probes for qrtPCR 65
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Unfortunately, this probe poorly degrades under the
conditions used for PCR elongation.
In addition, probes P23 and P24 containing 3′-BHQ1 pairs
were synthesized (Table 1). Interestingly, the compact quencher
pair in probe P24 has a vivid advantage over two freely long-
linked BHQ1 molecules in probe P23: the background fluores-
cence for P24 is halved and the fluorescence increase is tripled
as compared to P23. However, these probes show a remarkable
fluorescence decrease during the early PCR cycles (ESM
Fig. S8) and normal fluorescence increase during cycles 20–
45. The nature of this spectral behavior remains unclear.
Thus, the structure–fluorescence property relationships and
correlations may appear useful for the design of linear fluoro-
genic qrtPCR probes (analogues of TaqMan and AllGlo).
Moreover, the data presented provide a reason for the assump-
tion that multi-dye/multi-quencher qrtPCR DNA probes would
be more efficacious in a nonlinear format (e.g., secondary-
structured Molecular Beacons, junction probes [40], transcrip-
tion factor beacons [41], etc.) by improving their reproducibil-
ity at low target concentrations. The study of such probes is in
progress, and the results will be reported elsewhere.
Experimental
Chemicals
FAM phosphoramidite [33], JOE phosphoramidite [34],
BHQ1 phosphoramidite [35], and 5-alkyne dT phosphora-
midite [31,32] were prepared as reported. BHQ1 and FAM
solid supports were from Glen; FAM azide, FAM
CHC
azide,
and BHQ1 azide were from Primetech LLC (Minsk, Belarus).
Other reagents and solvents were from commercial suppliers
and used as received.
Fluorogenic DNA probes
TaqMan probes were prepared using oligonucleotide syn-
thesis on a BiosSet ASM-800 instrument on a 200-nmol
scale using standard manufacturer’s protocols and “click”
procedures on solid support and in solution.
“Click”on solid support: general procedure
The solid support carrying the synthetic oligonucleotide
stopped after the synthetic cycle introducing the first 5-
(octadiynyl)-dU modification was suspended in the solution of
Cu(CH
3
CN)
4
PF
6
(1 mg), TBTA (2 mg), and DIEA (10 μL) in
DMSO (0.5 mL). Dye azides (1 mg) were added in the mix-
tures: BHQ1 azide for the modification of P15, P16, and P20;
FAM
CHC
azide for the modification of P17, P18, and P19. The
reaction mixtures were left overnight in the dark, centrifuged,
washed with DMSO (2×1 mL) and acetone (2×1 mL), and
dried. The modified solid supports were placed in an oligonu-
cleotide synthesizer for further oligonucleotide synthesis.
“Click”in solution: general procedure
PAGE and RP-HPLC purified and desalted oligonucleotides (1
OD260 units) containing one or two alkyne residues were evap-
orated to dryness, dissolved in milliQ water (30 μL),and1M
TEAA buffer (10 μL, pH 7.0), DMSO (40 μL), and 10 mM dye
azide solution in DMSO (5 μL) were added. The “click”catalyst
consisted of 10 mM CuSO
4
in water (5 μL), 10 mM TBTA in
DMSO (5.5 μL), and 10 mM sodium ascorbate in water (5 μL),
which was prepared separately and added to each reaction
mixture. Reaction mixtures were degassed by bubbling argon,
shook vigorously overnight in the dark, then 1 M LiClO
4
(50 μL) was added, followed by acetone (1 mL). Precipitated
oligonucleotides were collected by centrifugation, the super-
natant was discarded, and oligonucleotides were dissolved in
milliQ water and purified by reversed-phase HPLC.
HPLC purification was performed using an Agilent 1100
instrument (Waters Sunfire C18 5.0 μm, 4.6×250-mm col-
umn, elution of 1 mL/min, 0→40 % MeCN in 0.1
MNH
4
OAc for 40 min). Typical retention times of the
fluorogenic probes listed in Table 1range 30–35 min. Modi-
fied oligonucleotides were characterized by MALDI-TOF
mass spectrometry (Bruker Microflex instrument) in positive
ion mode using a mixture (1:1, v/v) of 2,4,6-dihydroxyaceto-
phenone (40 mg/mL in MeOH) and aqueous diammonium
hydrogen citrate (80 mg/mL) as a matrix premixed just before
loading the samples onto a plate.
Real-time PCR
As a model, the qrtPCR earlier optimized system containing
the plasmid pTZ-Fat carrying a fragment of F. avenaceum
elongation factor 1α(GeneBank accession no. JF278604)
downstream Fat65F primer 5′-GGT CGC TTA TCT GCA
CTC GGA-3′and upstream primer 5′-GTC ACT CGA GTG
GCG GGG TAA G-3′(Fig. 2) were used [19].
5'-GGTCGCTTATCTGCACTCGGAACCCGCCAAACCTGGCGGGGTATCACCACGACATCTT
GCTAACTCTTGACAGACCGGTCACTTGATCTACCAGTGCGGTGGTATCGACAAGCGAAC
CATCGAGAAGTTCGAGAAGGTTAGTCAATATCCCTTCGATTACGCGCGCTCCCATCGATT
CCCACGACTCGCTCCCTCATTCGAAACGCATTCATTACCCCGCTCAAGTCCGAAAATT
TTGCGGTGCGACCGTGATTTTTTTTGGTGGGGTATCTTACCCCGCCACTCGAGTGAC-3'
Fig. 2 Nucleotide sequence of the DNA fragment of the translation elongation factor 1αgene of F. avenaceum (nucleosides 67–357). Primer
positions are underlined; the sequence complementary to fluorogenic probes is highlighted
66 D.Y. Ryazantsev et al.
Author's personal copy

PCR assay was performed on a DT-96 instrument (DNA
Technology, Moscow, Russia) as follows: 94 °C, 90 s (1 cycle);
94°Cfor10s,64°Cfor15s,and67°Cfor10s(45cycles)—
FAM and JOE detection channel. The reaction mixture (35 μL)
contained 75 mM Tris–HCl, 20 mM ammonium sulfate,
0.01 % Tween-20, 1 mM of each dNTP, 1 μM primers,
0.2 μM of a fluorescent DNA probe, 2.5 U of Taq polymerase,
and DNA template (25,000 copies), pH 8.8 [19]. The experi-
ments were repeated twice and analyzed by the geometric
method (C
p
) using DNATechnology software. The background
fluorescence of every sample well was taken into account. As a
template, the plasmid pTZ-Fat containing the cloned translation
elongation factor 1αgene fragment (from F. avenaceum)was
used. In quantitative experiments, tenfold dilutions of the DNA
template (2,500, 250, and 25 copies) were employed.
EvaGreen melting was performed on the same instrument
(FAM detection channel) under similar conditions, except
8 pmol of the probe, 12.5 pmol of both primers, and 1×
EvaGreen (Biotium, USA) were added to each reaction
mixture (35 μL). Melting temperatures were calculated as
the first derivative maximum.
Conclusions
Briefly, we used two-step “click”reactions to synthesize
qrtPCR probes containing two dyes (FAM and FAM or FAM
and JOE) and one or two fluorescence quenchers, BHQ-1. The
probes show lower background fluorescence as compared with
the standard TaqMan probe P1. However, their end point
fluorescence increase is substantially lower, presumably due
to the reduced probe degradation in the course of qrtPCR. An
exception is probe P7—its fluorescence increase is higher. The
JOE dye appears as a good fluorescence quencher for FAM,
reducing the background fluorescence four to five times
(probes P13 and P14). The alternate arrangement of FAMs
and BHQ1 is favorable for efficient quenching (probe P19).
Acknowledgments The research was supported by the Molecular
and Cellular Biology Program of the Russian Academy of Sciences,
Russian Foundation for Basic Research (project no. 10-04-00998), and
the Ministry of Industry and Trade of the Russian Federation (contract
no. 11411.0810200.13.B24). The authors are grateful to Irina A.
Stepanova and Elena V. Nozhevnikova for their help in data analysis
and Tatyana E. Chernichko for the reading of the manuscript.
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