Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe

Article abstract of DOI:10.1016/j.bmc.2014.03.033

A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2′-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2′-deoxy-uridine nucleoside, dU^TO, (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2′-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU^TO at various positions. dU^TO-2′-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU^T can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU ^TO oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

Full text of DOI:10.1016/j.bmc.2014.03.033

Bioorganic & Medicinal Chemistry 22 (2014) 2613–2621
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Detection of cyclin D1 mRNA by hybridization sensitive
NIC–oligonucleotide probe
Marina Kovaliov a, Meirav Segal b, Pinhas Kafri c, Eylon Yavin b, Yaron Shav-Tal c, Bilha Fischer a,
⇑
a Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
b School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Ein Karem, Jerusalem 91120, Israel
c Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange
(TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have
developed a phosphoramidite monomer, 10, and prepared a 20-OMe-RNA probe, labeled with 5-(trans-N-
hexen-1-yl-)-TO-20-deoxy-uridine nucleoside, dUTO, (Nucleoside bearing an Inter-Calating moiety, NIC),
for selective mRNA detection. We investigated a series of 15-mer 20-OMe-RNA probes, targeting the
cyclin D1 mRNA, containing one or several dUTO at various positions. dUTO-20-OMe-RNA exhibited up
to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus
that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of
Received 6 February 2014
Revised 18 March 2014
Accepted 20 March 2014
Available online 29 March 2014
Keywords:
mRNA diagnosis
RNA probe
Fluorescent probe
Cyclin D1
a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/
ll). Further-
more, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluores-
cence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/ll).
Breast cancer
Additionally, dUT can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding
the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dUTO oligonucle-
otides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the
diagnosis of breast cancer.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Probes modified with one type of fluorophore, were designed
for homogeneous assays, in which hybridization with the target
Homogeneous fluorescence-based assays for detection of nu-
cleic acids,1 are widely used in various studies in molecular biology
and biochemistry, for example single-nucleotide polymorphism,2
real-time PCR3,4 and detection of cellular RNA.5 Detection of nu-
cleic acids is highly important for diagnostics of genetic and infec-
tious diseases, discovery of gene-targeted drugs, and other
biomedical studies. Fluorescent nucleobases provide interesting
opportunities for sensing the target DNA or RNA in extract mix-
tures as well as in living cells.6,7 To become an effective tool for
hybridization analysis, the synthetic oligonucleotide (ONs) must
produce measurable hybridization-induced differences in fluores-
cence intensity. Labeled-single-stranded oligonucleotides having
such properties would provide the basis for development of
hybridization assays, suitable for the determination of comple-
mentary DNA and RNA targets.
nucleic acids leads to increase or decrease of fluorescence.8,9,5
Among these fluorophores organic dyes such as cyanines, fluor-
ecseins and rhodamines were used for developing of these hybrid-
ization probes.10,11
Another approach for modulation of fluorescence upon hybrid-
ization involves an intercalative transduction agent in nucleic acid
hybridization assays, such as thiazole orange (TO). TO is a chromo-
phore composed of a benzothiazole derivative and a quinolinium
ring linked via a monomethine bridge,12 which has low fluores-
cence in the free form.13 TO has been reported to provide dramat-
ically enhanced fluorescence upon dsDNA binding, because of the
interruption of the twisting motion around the central methane
bridge (U changes from 0.0002 alone in aqueous solution to 0.1–
0.4 with DNA).13 Recently hybridization sensitive probes, labeled
with TO, have been developed, including PNA FIT (forced intercala-
tion) probes,14 and ECHO probes that are, doubly TO labeled
hybridization-sensitive DNA probes, controlled by exitonic interac-
tions of the dyes.15 Although TO labeled PNA probes showed
increased fluorescence upon hybridization with complementary
⇑
Corresponding author. Tel.: +972 35318303; fax: +972 36354907.
E-mail address: Bilha.Fischer@biu.ac.il (B. Fischer).
http://dx.doi.org/10.1016/j.bmc.2014.03.033
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2614
M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
DNA or RNA they suffer from several disadvantages, such as their
lack of duplex stability and their costly synthesis.16
Previously, we reported on duplex-DNA probes labeled with 5-
(p-methoxy-cinnamyl)-uridine for the detection of cyclin D1
mRNA.8 This NIF (Nucleoside with Intrinsic Fluorescence) probe
exhibited high quantum yield (
U 0.12) and maximum emission
(478 nm) which is 170 nm red shifted as compared to uridine.
The fluorescence of the NIF-uridine analogue was quenched when
incorporated into an oligonucleotide and was quenched even more
(9.5-fold) upon duplex formation. Hence, cyclin D1 derived ds-NIF
probe was incubated with RNA total extracts of cancerous cells
containing high levels of cyclin D1 mRNA. Consequently, fluores-
cence increased due to de-hybridization of the ds-NIF probe, fol-
lowed by hybridization to target mRNA (cyclin D1), and release
of the ss-NIF probe. This process led to a 3-fold increase in fluores-
cence within 90 min at room temperature.
Here we attempted to increase signal-background ratio upon
Figure 2. Schematic presentation of the principle of mRNA homogeneous detection
using ssNIC-dUTO probe.
hybridization by
a different approach- applying fluorescent
Nucleosides bearing an Inter-Calating moiety (NIC) probes. Hybrid-
ization of NIC probe to a cyclin D1-derived mRNA was compared to
our previously developed ds-NIF probes.
Specifically, we describe the synthesis and photopysical proper-
ties of TO labeled 20-OMe-RNA probes in which dUTO monomer, 1,
replaces uridine (Fig. 1). We compared the fluorescence of singly or
doubly labeled single strands to that of the corresponding duplexes
with complementary DNA and RNA. In addition, we analyzed the
fluorescent properties of mismatched duplexes in a dUTO opposite
position, to deduce base-paring specificity and to study the hybrid-
ization properties of monomer 1. Finally, we demonstrated the use
of dUTO-labeled oligonucleotide probes for the detection of cyclin
D1, a breast cancer mRNA marker, in total RNA extract from human
cancerous cells.
whereas the quantum yield of unbound dye is very weak.13,17 In
addition TO has relatively long absorption and emission wave-
lengths kabs 500 nm and kem = 530 nm, respectively. Therefore, we
selected the TO dye as an intercalator of choice and conjugated it
to uridine via a six carbons linker, at uridine C5 position to pre-
serve the natural uridine H-bonding pattern. A six-carbon long
hexenyl linker was selected to allow intercalation of TO, located
at the end of the linker, between the stacked bases in the duplex
of oligonucleotide probe-target.
Previously reported studies on PNA and DNA probes in which
the carboxyalkyl linker was attached to the TO fluorophore, had
shown the desired increase in the fluorescence upon hybridization
(up to 30-fold).18,16,19–21 However, in these studies a TO moiety
was attached to the oligonucleotide post synthetically or as a base
surrogate. Our goal here was to develop 20-deoxy uridine conju-
gate, dUTO, 1, which can be incorporated anywhere in the sequence
by automatic synthesis, and will result in the fluorescence
enhancement upon intercalation of the moiety, following duplex
formation with the complementary strand. We used single-
stranded dUTO-labeled 20-OMe-RNA probes for the detection of cy-
clin D1 mRNA, a marker for breast cancer.
The cyclin D1 gene has been convincingly implicated in onco-
genesis.22–26 Cyclin D1 is a major player in the control of the cell
cycle27,28 and its expression is regulated at several levels, including
mRNA transcription.29 Among the dozen known human cyclin
genes, only cyclin D1 has been implicated in oncogenesis25 and
its overexpression was shown to cause mammary cancer in trans-
genic mice,30 and has been specifically implicated in breast can-
cer.31 Furthermore, cyclin D1 overexpression has been observed
in other types of cancer in which no amplifications or transloca-
tions occur.
2. Results
2.1. Design of dUTO-labeled-oligonucleotide probes
Previously, we reported on promising dsDNA probes labeled
with 5-(p-methoxy-cinnamyl)-uridine NIF-probe for the specific
detection of cyclin D1 mRNA.8 ds-NIF probes specifically identified
the target mRNA, however the change of emission intensity rela-
tively to background fluorescent intensity was insufficiently high.
Therefore, here we implemented an alternative approach which
may trigger a more significant change in the fluorescent intensity
upon hybridization of the probe to target mRNA. For this purpose
we designed a nucleoside bearing a chromophore moiety, serving
as an intercalator (NIC probe). The fluorescence of the latter was
expected to be activated upon hybridization to the target mRNA
(Fig. 2). The same target mRNA, cyclin D1, was chosen here, for
the sake of comparison of the efficiency of the current NIC method
to that of the previous NIF method.
One of the important properties of TO, is that the quantum yield
has been reported to increase upon intercalative binding to DNA,
We expected that our dUTO-labeled probes would show
enhancement of emission upon hybridization with target mRNA
in total RNA extract, to allow the detection of cyclin D1 in cancer-
ous cells that overexpress this biomarker. To test the applicability
of dUTO-labeled oligonucleotide probes for the detection of cyclin
D1 in cancerous cell extracts, we selected a 15-mer sequence that
targets nucleotides 644–658 of cyclin D1 mRNA. We chose a 15-
mer long oligonucleotide since this is the minimal length that
would ensure specific binding to target mRNA. The specificity of
the sequence was assessed by BLAST (basic local alignment search
tool). In addition to singly labeled oligonucleotides (ONs), several
di-labeled ONs were prepared to examine the effect of more than
one label on fluorescence of the single-strand and of the resulting
duplex. We used 20-OMe-RNA as a backbone of the dUTO-labeled
N
O
N
S
HN
O
N
HO
O
OH
1
Figure 1. TO-modified 20-deoxy-uridine nucleotide, dUTO
.

M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
2615
oligonucleotides because 20-OMe-RNA is known to exhibit a higher
affinity for RNA targets than the corresponding DNA. In addition,
20-OMe-RNA is chemically more stable than either DNA or RNA
and is resistant to degradation by RNA- or DNA-specific
nucleases.32,33
similar photophysical properties as TO (kabs 504 nm, kem 530 nm,
0.0002 in PBS buffer). For this purpose we synthesized dT7dUTO-
dT7 oligonucleotide probe, as a model capable of binding to the
polyA tail of mRNA. Specifically, we wanted to monitor any change
in fluorescent emission of ss-dUTO-oligonucleotide, upon selective
hybridization to the complementary target RNA.
U
2.2. Synthesis of dUTO phosporamidite, 9
Emission spectra of dT7dUTOdT7, showed weak fluorescence
emission, upon excitation at 510 nm, at room temperature in PBS
buffer. However, upon addition of complementary RNA, A15, a
19-fold increase in fluorescence was observed (Fig. 3). Next, we
tested whether dT7dUTOdT7 probe can detect mRNA in heteroge-
neous RNA solution. The fluorescent emission was measured in
the presence of mRNAs, containing complementary polyA tail, in
total RNA cell extracts at a range of concentrations (30, 15 and
The synthesis of dUTO nucleoside, 1, was based on a conven-
tional dye synthetic protocol.34–36 The thiazole orange moiety with
an alkenyl linker was achieved by reaction of lepidine, 2, under re-
flux in acetonitrile, for 4 days with trans-6-chloro-1-hexene-1-
ylboronic acid pinacol ester 3, to give compound 4 as a yellow solid
in 85% yield (Scheme 1). Boronic acid pinacol ester, 4, was coupled
to DMT-protected 5-I-dU, 5, under Suzuki conditions37 using
TPPTS, Na2CO3 and Pd(OAc)2 in CH3CN/H2O (2:1), to obtain deriva-
tive 6 as a green solid in 88% yield. 2-Methylthio-N-meth-
ylbenzothiazolium iodide, 7, was condensed with compound 6 in
the presence of triethylamine in absolute ethanol to give the thia-
zole dye 8 in 83% yield as a red solid (Scheme 2).38
Compound 1, was obtained in 80% yield upon treatment of 8
with 3% trichloroacetic acid in dichloromethane. Finally, dUTO
phosphoramidite monomer, 9, required for oligonucleotide synthe-
sis, was obtained in 78% yield, upon treatment of 8 with phoph-
oramidite chloride and diisopropylethylamine. 31P NMR spectrum
showed two typical phosphoramidite signals at 148 ppm, for two
diasterioisomers. The above monomer 9 allowed the facile incor-
poration of dUTO into oligonucleotides at any internal or terminal
position via a conventional oligonucleotide synthesis.
10 ng/lL). The mRNA extract was obtained from human U2OS ost-
reosarcoma cell line. As a control, we used complementary cDNA,
obtained from the mRNA total extract of the same cells, and ran-
dom RNA strand 50-GACGAUGGAGGGGCCGGACUCGUCAUACUC
CUGCU-30. The mRNA total cell extract was added after 3 min of
excitation at 510 nm, to monitor if there are changes in fluorescent
emission of the probe during the excitation. We measured the
change in fluorescence emission at 535 nm, after addition of total
RNA extract or cDNA, at room temperature, upon excitation at
510 nm (Fig. 4).
The probe became highly fluorescent almost immediately (30 s)
after the addition of the total RNA extract, indicating hybridization
with the polyA tails of mRNAs. Moreover, fluorescence is linearly
dependent on the concentration of mRNA extracts. Upon addition
of cDNA fluorescence intensity increased slightly, due to incom-
plete reverse transcriptase reaction and the presence of mRNA in
cDNA. Likewise, we observed no increase of fluorescence intensity
upon addition of a scrambled 30 nt RNA. We concluded, that the
increase of the fluorescence intensity of dUTO-labeled duplex oc-
curred, due to the specific hybridization of dT7dUTOdT probe to
the target polyA.
2.3. Synthesis of dUTO-labeled oligonucleotide probes
Phosphoramidite 9 in dry dichloromethane was used in auto-
mated oligonucleotide synthesis, and the coupling time for the
dUTO monomer was prolonged to 10 min. Cleavage of dUTO-labeled
oligonucleotide from the solid support was achieved by employing
fast deprotection, with 1:1 (v/v) 33% NH4OH and 33% methylamine
in ethanol at 65 °C, 10 min. All modified oligonucleotides were
purified by HPLC and their identity was confirmed by MALDI-TOF
mass spectroscopy.
Encouraged by these results we synthesized and studied the
fluorescent properties of several single stranded, dUTO-labeled, cy-
clin D1 derived oligo nucleotides ON1-4 (Table 1). Corresponding
duplexes were prepared for all oligomers. The fluorescence of
probes ON1-4 was measured before and after hybridization with
target DNA and RNA. Table 2 summarizes absorption and emission
maxima, quantum yields, and Tm values of ON1-4 duplexes with a
complementary strand. Measurements were performed that at
First, to study the fluorescent properties of dUTO-labeled oligo-
nucleotide upon hybridization with target RNA, we synthesized as
a model dT7dUTOdT7 capable of binding to the polyA tail of mRNA.
Next, we synthesized four oligonucleotides, ON1-4, derived from
cyclin D1 sequence and labeled with dUTO at different positions:
4 or 10, di-labeled at positions 4 and 11; or 10 and 11 (Table 1).
0.5 lM in PBS buffer (pH 7.4), at room temperature. The absorption
bands of dUTO-ONs were monitored at 483–512 nm. The emission
spectra showed a single broad band at about 540 nm. ON1-4 du-
plexes of with complementary RNA showed enhancement of fluo-
rescence up to 6-fold versus that of ssON1-4. On the contrary, only
a slight enhancement of TO emission was observed upon hybrid-
ization ON1-4 with DNA as target.
2.4. Fluorescence properties of dUTO-labeled oligonucleotide
probes
Our main interest pertains to the detection of mRNA in compex
environments, such as total RNA cell extract. Therefore, first we
wanted to investigate the fluorescent properties of dUTO in a nucle-
oside, an oligonucleotide, and upon binding to a specific target in
homogeneous solution. dUTO-modified nucleoside, 1, showed
The most significant DU was observed for ON2-RNA duplex
where quantum yield of dUTO increased by 7-fold. However, almost
no change of quantum yield was observed for ON2-DNA duplex
(Fig. 5). The increase of fluorescence of dUTO upon hybridization
with complementary RNA, unlike the corresponding DNA, sug-
gested that TO might intercalate with 20-OMe-RNA-RNA hybrid
in a different manner than that of 20-OMe-RNA-DNA duplex. Gen-
erally, TO dye binds to the duplex through intercalation between
base pairs of the duplex and/or major- or minor- groove bind-
ing.39,40 To establish the mode of TO binding in our probes we mea-
sured CD spectra of ON2 and the corresponding duplexes with DNA
and RNA (Fig. 6). The single strand (ON2), and the duplexes showed
negative and positive signals at 450–550 nm, which coincide with
TO’s absorption band. These signals might be due to different bind-
ing modes of the dye to the oligonuclotide.41,42 Negative Cotton
effect has been assigned to the intercalating mode of binding,
O
B
O
B
3
Cl
N
O
O
Cl
N
ACN, RT, 4 days
2
4
Scheme 1. Synthesis of reagent for the preparation of dUTO
.

2616
M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
O
N
O
N
I
N
NH
NH
O
O
a
DMTO
S
DMTO
O
O
OH
OH
6
5
N
O
S
N
N
S
HN
7
b
N
O
DMTO
O
c
d
OH
8
N
O
N
N
S
HN
O
N
O
S
N
HN
HO
O
N
O
DMTO
OH
O
O
1
O
P
CN
N
9
Scheme 2. Synthesis of dUTO phosphoramidite, 9a. aReagents and conditions: (a) TPPTS, Pd(OAc)2, Na2CO3 (3 equiv), CH3CN/H2O (2:1); (b) 7, TEA, EtOH, reflux, 3 h; (c) 3% TCA
in DCM, rt, 0.5 h; (d) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2, rt, 3 h.
Table 1
dUTO-labeled cyclin D1-derived ONs
60
dUTO-20-OMe-RNA
ONs
Sequence 50 ? 30
RNA extract 30 ng/µL
50
40
30
20
10
RNA extract 15 ng/µL
RNA extract 10 ng/µL
cDNA
Unmodified
ON1
ON2
ON3
ON4
CACUUGAGCUUGUUC
CACUUGAGCdUTOUGUUC
CACdUTOUGAGCUGUUC
CACdUTOUGAGCUdUTOGUUC
CACUUGAGCdUTOdUTOGUUC
GAACAAGCTCAAGTG
random RNA
Complementary
0
5
10
Time (min)
15
20
Figure 4. Measurements of time-dependent fluorescence of dT7dUTOdT7 probe
(0.5 M) after adition of mRNA cell extract (30, 15 and 10 ng/ L), cDNA (30 ng/ L)
and scrambled random RNA (50-GACGAUGGAGGGGCCGGACUCGUCAUACUCCUGCU-
30) (0.25
M), in PBS buffer (pH 7.4, 25 °C), kex 510 nm, kem 535 nm. Total RNA extract
l
l
l
40
35
30
25
20
15
10
5
l
dT7-dUTO-dT7
dT7-dUTO-dT7 + 15dA
was added at 3 min.
and it was assumed that the positive effect was caused by TO bind-
ing to the DNA/RNA groove.43,44 The clear negative Cotton effect
observed for ON2-RNA duplex indicates a predominant TO-binding
through intercalation. In contrast, ON2-DNA hybrid showed a posi-
tive Cotton effect, indicating a less efficient intercalation and dye
binding to the major or minor groove.45,46
The ON1 exhibited only a modest enhancement in quantum
yield upon hybridization with DNA and RNA, 1.6 and 2.7-fold,
respectively. Likewise, only slight enhancements of quantum
yields were observed for duplexes of dUTO doubly-labeled oligonu-
cleotides ON3 and ON4, with either DNA or RNA.
0
500
550
600
Wavelength (nm)
650
700
Figure 3. Emission spectra of dT7dUTOdT7 probe alone and with complementary
A15, (0.5 M) measured in PBS buffer (pH 7.4, 25 °C), kex 510 nm.
l

M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
2617
stability of the duplexes (Table 2). Tm values of all dUTO-ONs du-
plexes with complementary DNA and RNA strands were 47–49,
and 55–57 °C, respectively. In most cyclin D1-derived labeled du-
plexes Tm increased by 1–6 °C as compared to the unmodified du-
plex for DNA and RNA duplexes (Table 2). There is the clear
difference between duplexes in which the complementary strand
Table 2
Photophysical properties of ON1-4 and the corresponding duplexes with comple-
mentary DNA and RNAa
20-OMeRNAs (ONs)
kmax (nm)
U
kem (nm) Tm (°C)
ON1
ss
511, 492
dsRNA-DNA 512, 488
dsRNA-RNA 510, 488
513, 490
dsRNA-DNA 512, 488
dsRNA-RNA 510, 488
510, 487
dsRNA-DNA 510, 488
dsRNA-RNA 509, 481
510, 486
dsRNA-DNA 510, 488
dsRNA-RNA 508, 483
0.18
0.30
0.41
0.04
0.037 532
0.285 537
0.030 539
0.046 540
0.051 541
0.018 539
0.015 541
0.026 541
540
540
537
533
49
55
was DNA (DTm = +1–3 °C) or RNA (DTm = 6–9 °C). These results
ON2
ON3
ON4
ss
indicate that the introduction of dUTO to 20-OMe-RNA strands sta-
bilizes the corresponding DNA or RNA duplexes. We concluded
that the thiazole orange moiety binds to the duplexes through
47
56
ss
48
57
intercalation and stabilizes the duplex structure by additional
stacking interactions.
p-
ss
47
55
46
51
2.6. Effect of dUTO labeling on matched versus mismatched
duplexes
Unmodified duplex dsRNA-DNA
dsRNA-RNA
a
To test whether ON1-4 can be used to sensitively detect se-
quence differences such as polymorphism, we investigated the
changes of the fluorescence intensity of mismatched duplexes of
ON2. When the base opposite to dUTO in ON2 was changed to a
mismatched base (C/U/G), the quantum yields of the mismatched
hybrids decreased by 14–27% versus those of the fully matched hy-
brid (Table 3). The small change in fluorescence suggests that a de-
crease in the RNA binding ability of dyes caused by a mismatched
base pair is not significant (Fig. 7). Probably, the mismatched base
pair partially destabilize the duplex structure, but does not affect
the intercalation of TO dye to duplex binding site. It is noteworthy
that the emission of the probes in the presence of mismatched RNA
exhibits small change, indicating that the discrimination ability of
dUTO is limited.
All duplexes and single strand were measured at 0.5 lM, PBS buffer (pH 7.4,
25 °C), kex = 490 nm. Tm values are given for all duplexes.
140
ss-ON2
120
100
80
60
40
20
0
ds-ON2-(+DNA)
ds-ON2-(+RNA)
2.7. Detection of cyclin D1 in RNA cell extracts by using dUTO
labeled ON2
-
500
550
600
Wavelength (nm)
650
700
We applied ON2 that exhibited the largest change of fluores-
cence intensity upon hybridization, to detect cyclin D1 mRNA in
total RNA extracted from human U2OS ostreosarcoma cell line.
Specifically, the fluorescence of ON2 probe was measured, before
and after addition of RNA cell extract from a cell line stably over-
expressing cyclin D1 gene versus wild-type (WT) U2OS cells (Fig
7). Levels of cyclin D1 mRNA expression, were 3 times higher in
U2OS cells overexpresing GFP-cyclin D1 than in WT U2OS cells.8
The fluorescence of ON2 was measured in PBS buffer for 4 min
and exhibited no change. After 4 min RNA extract containing high
levels of cyclin D1 was added. As a controls we measured the fluo-
rescence intensity of RNA extract alone (see Fig. S8, SI), and of ex-
tracts of cells that expressing basal level of cyclin D1. The emission
intensity of dUTO increased almost immediately (1 min) upon RNA
extracts addition at room temperature (Fig. 8), indicating a rapid
hybridization reaction between single stranded ON2 probe and
the target RNA. This experiment was performed in duplicates and
on two different extract batches on three separate days using the
same concentrations of ss-NIC probe and RNA extracts. The
Figure 5. Emission spectra of ss-ON2, and ds-ON2/DNA(RNA) (0.5
PBS buffer (pH 7.4, 25 °C), kex = 490 nm.
l
M) measured in
20
ssON2
dsON2-DNA
dsON2-RNA
15
10
5
0
-5
-10
-15
200
300
400
500
600
Wavelength (nm)
Table 3
Hybridization of ON2 with matched and mismatched ONs with and their photo-
Figure 6. CD spectra of ss-ON2, and ds-ON2/DNA(RNA) (2.5
PBS buffer (pH 7.4, 25 °C).
lM) were measured in
physical propertiesa
Sequence 50 ? 30
Tm (°C)
U
ON2
CACdUTOUGAGCUGUUC
GAA CAA GCU CAA GUG
GAA CAA GCU CAC GUG
GAA CAA GCU CAU GUG
GAA CAA GCU CAG GUG
0.040
0.285
0.245
0.233
0.208
2.5. Effect of dUTO-labeling on duplex stability
dsRNA-A
dsRNA-C
dsRNA-U
dsRNA-G
56
54
53
53
Development of hybridization sensitive fluorescent probe re-
quires not only different potophysical properties in labeled-single
strand versus labeled-duplex, but also duplex stability. Thus we
assessed the effect of modified dUTO-oligonucleotides on the
a
All duplexes and single strand were measured at 0.5
25 °C), kex = 490 nm. Tm values are given for all duplexes.
l
M, PBS buffer (pH 7.4,

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M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
The novel NIC probes were based on dUTO-labeled 20-OMe-RNA
120
100
80
60
40
20
0
probes hosting of the 2-deoxy-uridine bearing a TO moiety that
was attached via six-carbon hexenyl linker at 5-position. The NIC
probes were designed to be used for the detection of cyclin D1
mRNA, a breast cancer marker. We selected a 15-mer sequence
from the cyclin D1 mRNA sequence, which is specific to this mRNA
only and does not appear in any other endogenous mRNA. We
incorporated dUTO at various positions within the selected se-
quence and produced mono or doubly dUTO-labeled oligonucleo-
tides. Finally, we compared the ability of these dUTO-labeled 20-
OMe labeled probes to that of ds-NIF probes,8 to detect cyclin D1
mRNA.
+RNA
+RNA mismatch (G)
+RNA mismatch (C)
+RNA mismatch (U)
Fluorescence intensities (which correlate with quantum yield)
of the ONs and the corresponding duplexes were dependent on
the location of the label in the ON. In particular, ON2 in which
the dUTO was close to the end of the oligonucleotide exhibited
500
550
600
Wavelength (nm)
650
700
Figure 7. Emission spectra of ON2 probe hybridized with RNA (match, mismatch-G,
mismatch-C, mismatch-U), measured at 0.5 lM, kex = 490 nm, in PBS buffer (pH 7.4,
low quantum yield in the single-strand (
U 0.04) and high quantum
yield in duplex with RNA (
U 0.285). On the contrary, ON1, in which
25 °C).
dUTO was located in the middle of the oligonucleotide, exhibited
similar high quantum yield in the single-strand and RNA duplex
(
U
0.18–0.41). No additive effect was observed for multi-labeled
ONs. Moreover, di-labeled ON3 and ON4 exhibited very low quan-
tum yields in duplexes with DNA and RNA (e.g., ON3, 0.046,
0.051 and ON3, 0.015, 0.026, respectively). This phenomenon
U2OS cells overexpressing cyclin D1
WT U2OS cells
1.39
1.19
0.99
0.79
0.59
0.39
0.19
-0.01
U
U
may be explained by excitonic interactions between neighboring
TO moieties.18
We studied the dependence of the thermo-stability of the la-
beled-20-OMe-DNA and -RNA duplexes on the position of dUTO la-
bel and number of labels. We found that incorporation of monomer
1 in ONs increased the stability of the resulting duplexes. In most
labeled duplexes, Tm increased by 3–6 °C as compared to the
unmodified duplex. The increased stability of these duplexes and
their enhanced fluorescence intensities versus the corresponding
ssONs, may indicate that the six-carbon hexenyl linker is long en-
ough to lead to favourable stacking interactions through intercala-
tion of the TO moiety.
Generally, the binding of TO to double stranded oligonucleotide
involves several binding modes, the most important being interca-
lation and groove binding. The duplexes of ON2 with RNA were
more emissive and stable as compared to duplexes with DNA.
The results of Tm and CD measurements suggested that the TO
dye binds to RNA duplexes mainly through intercalation and thus
thermally stabilizes the duplex structure.
These probes were designed to be used for the detection of spe-
cific mRNA. Therefore, after proving their ability to detect selectively
polyA tails in total RNA cell extract by dT7dUTOdT7, we demon-
strated the ability of the optimal dUTO-labeled probe, ON2 to detect
selectively cyclin D1 mRNA in an extract from cancerous U2OS cells.
An immediate increase in fluorescence up to 2.5-fold was observed
following the addition of the probe at room temperature to total
RNA extract of cyclin D1 overexpressing U2OS cells versus WT
0
5
10
15
20
25
30
35
time (min)
Figure 8. Representative measurements of time-dependent fluorescence of ON2
after addition of mRNA extracted from cyclin D1 overexpressed and WT U2OS cells.
Conditions: 0.5 lM probe in 80 lL PBS buffer (pH 7.4, 25 °C), and 250 ng/lL (after
addition) total RNA extract, kex = 510 nm, kem = 535 nm. RNA extract was added at
4 min.
fluorescent enhancement upon hybridization, ranged from 2.3 to
2.6-fold for RNA extracted from cyclin D1-over expressing cells
as compared to that of WT cells. This is noteworthy considering
that the overexpression of cyclin D1 is only 3 times higher in the
overexpressing cells versus WT U2OS cells.8 These results demon-
strate qualitatively the efficiency of our dUTO probe for the selec-
tive detection of a breast cancer marker, cyclin D1, in total RNA
extracted from cancerous cells.
3. Discussion
U2OS RNA extract (both in concentration of 250 ng/ll) indicating
the specific recognition of the cyclin D1 mRNA by the probe. It is
noteworthy, that the level of cyclin D1 in overexpressing U2O2 cells
is only ca. 3-fold higher than the basal level in WT cells.
Our main goal here was to improve the sensitivity of our previ-
ously published fluorescent NIF probes.8 For this purpose we tar-
geted the preparation NIC probes by incorporation of dUTO
monomer, 1, into an oligonucleotide. We suggested that single
stranded probe, labeled with TO intercalator, would increase fluo-
rescence signal upon hybridization to the target, due to its affinity
to dsDNA.47 In duplex-NIF probes, the fluorophore is quenched
upon duplex formation, and becomes fluorescent when released
from the duplex by strand displacement process with the target.
This process resulted in ca. 3-fold increase of fluorescence inten-
sity. Here we targeted a better signal to noise ratio by using nucle-
oside-bound TO, known to increase emission by tens to thousands
times upon ds-intercalation.
The design of dUTO-labeled ONs proposed here is different from
recently reported singly-labeled probes.14,15 Unlike the latter
probes, here monomer 1 is used in conventional phosphoramidite
solid phase oligonucleotide synthesis, without need for any addi-
tional post-synthetic modification of oligonucleotides. Further-
more, it can be substituted anywhere in the probe’s sequence
and in multiple positions. Substitution of the linker-TO moiety at
C5- position of 20-deoxy uridine, allows the preservation of the
natural H-bonding pattern of U/T and does not hinder the duplex
formation and stability. Furthermore the incorporation of dUTO
increased the stability of the corresponding RNA duplexes.

M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
2619
In contrast to NIF-probe, dUTO-labeled NIC-probe, ON2, demon-
strated remarkable ability to detect a small concentration of spe-
under reduced pressure to give a yellow–green solid (500 g, 92%).
1H NMR (600 MHz, CDCl3) d 10.32 (d, 1H, J = 6 Hz), 8.35–8.34 (m,
1H), 8.26–8.25 (m, 1H), 8.18–8.15 (m, 1H), 7.98–7.96 (m, 2H),
6.55–6.52 (m, 1H), 5.41 (d, 1H, J = 18 Hz), 5.27 (t, 2H, J = 6 Hz),
3.00 (s, 3H), 2.26–2.24 (m, 2H), 2.10–2.07 (m, 2H), 1.69–1.66 (m,
2H), 1.24 (s, 12 H) ppm. 13C NMR (100 MHz, CDCl3) d 157.8,
152.4, 149.3, 137.0, 135.2, 129.8, 129.4, 126.8, 123.4, 118.6, 83.1,
57.5, 34.9, 29.4, 24.9, 24.8, 24.7, 20.4 ppm. HRMS ES+ m/z: calcd
for C22H31BNO2 [M]+ 352.245, found 352.244.
cific mRNA, cyclin D1 mRNA, in total RNA extract (250 ng/
lL vs
1842 ng/ L, respectively). In addition, the maximum emission
l
wavelength of NIC probe, is longer than that of NIF-probe,
540 nm versus 480 nm, thus allowing detection beyond the auto-
fluorescence range of cells.
4. Conclusions
5.2. Synthesis of 5-(trans-4-methyl-N-(hexen-1-yl-)-
In conclusion, we have designed a series of fluorescent oligonu-
cleotide probes based on the incorporation of dUTO, uridine ana-
logue 1. The hybridization-sensitive, quencher free fluorescent
NIC-probe, ON2 proved to be applicable for the specific detection
of a minute amount of a breast cancer marker, cyclin D1mRNA in
quinolinium)-50-DMT-uridine (6)
h Water/acetonitrile (8/4 mL) was added through a septum to a
nitrogen–purged round bottom flask containing 5-I-50-DMT-Uri-
total RNA cell extract (250 ng/l
l) from cancerous U2OS cells. dUTO
dine,
6 (1 g, 1.25 mmol), compound 3 (736 mg, 1.90 mmol),
Pd(OAc)2 (17.2 mg, 0.076 mmol), TPPTS (216 mg, 0.38 mmol) and
Na2CO3 (106 mg, 4.56 mmol). The mixture was stirred under reflux
for 3 h and monitored by TLC (8:2 DCM/MeOH). The solvents were
evaporated. The deep green residue was separated on a silica gel
column (5% MeOH in DCM). Product 7 was obtained as a green so-
lid (870 mg, 88%). 1H NMR (600 MHz, CDCl3): d 9.92 (d, 1H,
J = 6 Hz), 9.04 (s, 1H), 8.32 (d, 1H, J = 8 Hz), 8.26 (d, 1H, J = 6 Hz),
8.16 (t, 1H, J = 7.5 Hz), 7.94 (t, 1H, J = 7.5 Hz), 7.78 (d, 1H,
J = 6 Hz), 7.74 (s, 1H), 7.40 (d, 2H, J = 7.5 Hz), 7.28–7.23 (m, 6 H),
7.17–7.16 (m, 1H), 6.79–6.78 (m, 4 H), 6.35–6.34 (m, 1H), 6.24–
6.19 (m, 1H), 5.69 (d, 1H, J = 15 Hz), 5.07–5.05 (m, 2H), 4.71–4.72
(m, 1H), 4.05 (m, 1H), 3.73 (s, 6H), 3.42–3.41 (m, 1H), 3.33–3.32
(m, 1H), 2.92 (s, 3H), 2.46–2.39 (m, 2H), 1.94–1.92 (m, 4H), 1.50–
1.42 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) d 162.3, 158.5,
158.0, 149.6, 148.6, 144.6, 136.9, 136.1, 135.7, 135.5, 131.5,
130.1, 129.9, 129.1, 128.0, 127.9, 127.8, 127.0, 126.8, 123.3,
121.6, 119.1, 113.2, 112.5, 86.6, 85.5, 84.7, 76.8, 63.3, 57.6, 55.3,
53.4, 40.7, 32.2, 29.2, 25.2, 24.4, 20.5 ppm. HRMS ES+ m/z: calcd
for C46H48N3O7 [M]+ 754.349, found 754.349.
monomer preserves the natural H-bonding pattern and can be used
as a monomer in solid-phase oligonucleotide synthesis, without
additional need for post-synthetic modification of ON probes.
These results support the potential usefulness of ON2 probe for
the diagnosis of breast cancer.
5. Experimental section
Reagents and solvents were purchased from commercial
sources and were used without further purification. All moisture
sensitive reactions were carried out in flame-dried reaction flasks
with rubber septa, and the reagents were introduced with a syr-
inge. All reactants in moisture sensitive reactions were dried over-
night in a vacuum oven. Progress of reactions was monitored by
TLC on precoated Merck silica gel plates (60F-254). Visualization
was accomplished by UV light. Medium pressure chromatography
was carried out using automated flash purification system (Biotage
SP1 separation system, Uppsala, Sweden). Compounds were char-
acterized by nuclear magnetic resonance using Bruker AC-200,
DPX-300 and DMX-600 spectrometers. 1H NMR spectra were mea-
sured at 200, 300 and 600 MHz. Phosphoramidite monomer was
characterized also by 31P NMR in CD3CN, using 85% aq H3PO4 as
an external reference on Bruker AC-200, at 80 MHz. Chemical shifts
are expressed in ppm, downfield from Me4Si (TMS), used as inter-
nal standard. Compounds were analyzed under ESI (electron spray
ionization) conditions on a Q-TOF micro-instrument (Waters, UK).
Unmodified oligonucleotides were purchased from Integrated DNA
Technologies (Coralville, Iowa, USA). Modified oligonucleotides
were synthesized by standard automated solid-phase method on
an AKTA OligoPilot (GE healthcare) an ABI DNA/RNA synthesizer
(Forster City, USA). MALDI-TOF mass spectra of oligonucleotides
were measured with mass spectrometer in a negative ion mode
with THAP matrix. Absorption spectra were measured on a UV-
2401PC UV–vis recording spectrophotometer (Shimadzu, Kyoto, Ja-
pan). Emission spectra were measured using Cary Eclipse Fluores-
cence Spectrophotometer. Absorption and fluorescence spectra
were recorded in PBS buffer containing NaC1 8.0 g, KC1 0.2 g, Na2-
HPO4 1.15 g, KH2PO4 0.2 g, in water 100 mL (pH 7.4).
5.3. Synthesis of 5-(trans-N-(hexen-1-yl-)-TO)-50-DMT-uridine
(8)
2-Methylthio-N-methylbenzo thiazolium iodide, 5, (81 mg,
0.25 mmol) was added to compound 7 (200 mg, 0.25 mmol) in
absolute ethanol (5 mL) and Et3N (0.05 mL). The mixture was
heated under reflux, under N2 atmosphere. After 3 h, TLC analysis
(CH2Cl2/CH3OH 8:2) indicated the absence of starting material.
The solvent was removed under reduced pressure. A red solid
was obtained after purification on a silica gel chromotography
(DCM/MeOH, 8:2), (156 mg, 66%). 1H NMR (700 MHz, DMSO-d6):
d 11.41 (s, 1H), 8.79–8.78 (m, 1H), 8.57 (d, 1H, J = 8 Hz), 8.08–
8.06 (m, 1H), 8.05–8.03 (m, 1H), 7.98–7.96 (m, 1H), 7.79 (d, 1H,
J = 8 Hz), 7.75–7.73 (m, 1H), 7.63–7.60 (m, 1H), 7.58 (s, 1H) 7.43–
7.41 (m, 1H), 7.34–7.31 (m, 3H), 7.22–7.19 (m, 6H), 7.14–7.12
(m, 1H), 6.92(s, 1 H), 6.82–6.81 (m, 4 H), 6.21–6.17 (m, 2H), 5.61
(d, 1H, J = 16 Hz), 5.32 (d, 1H, J = 4 Hz), 4.54–4.51 (m, 2 H), 4.36–
4.32 (m, 1H), 4.02 (s, 3H), 3.89–3.88 (m, 1H), 3.68 (s, 6H), 3.16–
3.13 (m, 2H), 2.27–2.25 (m, 1H), 2.19–2.17 (m, 1H), 1.86–1.83
(m, 2H), 1.75–1.73 (m, 2H), 1.26–1.23 (m, 2H) ppm. 13C NMR
(175 MHz, DMSO-d6): d 162.1, 160.0, 158.1, 149.4, 144.2, 140.4,
136.9, 135.3, 130.2, 129.5, 127.7, 127.6, 126.7, 124.5, 124.2,
123.8, 122.8, 121.8, 113.1, 112.9, 111.0, 107.8, 88.1, 85.7, 85.6,
84.1, 70.3, 63.4, 54.9, 53.9, 39.6, 33.8, 32.4, 28.2, 25.5 ppm. HRMS
ES+ m/z: calcd for C54H53N4O7S [M]+ 901.363, found 901.364.
5.1. Synthesis of trans-4-methyl-N-(hexene-1-ylboronic acid
pinacol ester-)-quinolinium chloride (4)
Lepidine (200 mg, 1.4 mmol), trans-6-chloro-1-hexene-1-ylbo-
ronic acid pinacol ester (684 mg, 2.8 mmol) and NaI (cat.) were dis-
solved in freshly distilled acetonitrile (10 mL). The mixture was
heated under reflux for 4 days under nitrogen atmosphere. The sol-
vent was evaporated and the residue was dissolved in a minimal
amount of DCM and Et2O was till the product precipitated. The
product was filtered under vacuum, washed with Et2O and dried
5.4. Synthes of 5-(trans-N-(hexen-1-yl-)-TO)-uridine (1)
To a solution of 8 (50 mg, 0.05 mmol) in dichloromethane
(1 mL) was added 3% trichloroacetic acid in dichloromethane

2620
M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
10,000 Mꢀ1 cmꢀ1
,
e
C-3 -OMe = 9050 Mꢀ1 cmꢀ1
,
e
G-3 -OMe = 13,700 Mꢀ1
0
0
(2 mL) and the mixture was stirred for 30 min at room tempera-
ture. The resulting mixture was evaporated and purified by silica
gel chromatography (DCM/MeOH, 7:3), to yield 9 as a red solid
(37 mg, 80%). 1H NMR (700 MHz, DMSO-d6): d 11.34 (s, 1H),
8.81–8.79 (m, 1H), 8.64 (d, 1H, J = 7.7 Hz), 8.14–8.13 (m, 1H),
8.06–8.05 (m, 1H), 7.99–7.95 (m, 1H), 7.96 (s, 1H) 7.80–7.79 (m,
1H), 7.76–7.73 (m, 1H), 7.63–7.60 (m, 1H), 7.44–7.42 (m, 1H),
7.39–7.38 (m, 1H), 6.40–6.37 (m, 1H), 6.16–6.14 (m, 1H), 6.07 (d,
1H, J = 16 Hz), 5.22 (d, 1H, J = 4 Hz), 5.06 (t, 1H, J = 4 Hz), 4.63–
4.61 (m, 2 H), 4.25–4.23 (m, 1H), 4.02 (s, 3H), 3.79–3.77 (m, 1H),
3.62–3.55 (m, 2H), 2.16–2.08 (m, 4H), 1.90–1.86 (m, 2H), 1.50–
1.48 (m, 2H) ppm. 13C NMR (175 MHz, DMSO-d6): d 163.0, 162.0,
160.0, 159.9, 149.4, 148.4, 144.2, 140.4, 136.9, 136,2, 133.1,
131.5, 130.6, 129.7, 128.1, 126.7, 125.7, 124.4, 124.1, 123.7,
122.8, 121.9, 118.0, 112.9, 110.7, 107.8, 107.7, 88.1, 87.3, 84.1,
70.3, 61.0, 53.8, 45.6, 39.7, 33.7, 32.2, 28.2, 25.6 ppm. HRMS ES+
m/z: calcd for C33H35N4O5S [M]+ 599.232, found 599.229.
cmꢀ1
,
e
A-3 -OMe = 15,400 Mꢀ1 cmꢀ1 and
e
dU
= 11,000 Mꢀ1 cmꢀ1. The
TO
0
extinction coefficients for the modified oligonucleotides were
approximated by the linear combination of the extinction coefficients
of the natural nucleotides and the extinction coefficient of the mod-
ified nucleoside. To account the base stacking interactions, this linear
combination was multiplied by 0.9 to give the final extinction coef-
ficients for the oligomers. The extinction coefficients of the duplexes
(eD) are less than the sum of the extinction coefficients of their com-
plementary strands (eS1, eS2), due to hyprochromic effect that should
be taken into account.48 Therefore, the extinction coefficients were
calculated by the following equation:
eD ¼ ð1 ꢀ h260nmÞðeS1
þ
eS2
Þ
h260nm ¼ 0:287fAT þ 0:059fGC
where fAT and fGC are the fractions of the AT and GC base pairs,
respectively.
5.5. Synthesis of 5-(trans-N-(hexen-1-yl-)-TO)-30-(2-cyanoethyl-
N,N-diisopropylphosphoramidite)-50-DMT-uridine (9)
5.8. Hybridization of oligonucleotides
Solutions of labeled single-strands were mixed at room temper-
ature with an equimolar amount of the complementary single
strand oligonucleotides in PBS buffer (pH 7.4). Samples were
hybridized by heating to 90 °C for 5 min and subsequently allowed
to cool to room temperature over 2 h prior to measurements.
Compound 8 (200 mg, 0.16 mmol) was dissolved in CH2Cl2
(4 mL) in a flame dried two-neck flask under N2. Diisopropylethyl-
amine (0.48 mmol, 0.083 mL) and 2-Cyanoethyl N,N-dii-
sopropylchlorophosphoramidite (54 lL, 0.2 mmol) were added to
this solution. The clear yellow solution was stirred at rt for 19 h.
The solvent was evaporated and the crude was immediately sepa-
rated on silica gel using hexane/EtOAc (2:8) as an eluent containing
3% TEA. Compound 10 was obtained as a red solid (137 mg, 78%).
1H NMR (200 MHz, CDCl3): d 8.76–8.73 (m, 2H), 7.78–7.74 (m,
1H) 7.51–7.22 (m, 11H), 6.85–6.71 (m, 8H), 6.42–6.37 (m, 2H),
6.15–6.10 (m, 1H), 4.81–4.55(m, 1H), 5.58 (d, 1H, J = 16 Hz),
4.64–4.62 (m, 1H), 4.38–4.10 (m, 2H), 4.05 (s, 3H), 3.76 (s, 6H),
3.63–3.48 (m, 4H), 3.33–3.09 (m, 2H), 2.50–2.47 (m, 4H), 1.72–
1.50 (m, 4H) 1.17–1.01 (m, 12H) ppm. 31P NMR (80 MHz, CD3CN):
d 148.61, 148.33 ppm. HRMS ES+ m/z: calcd for C63H70N6O8PS [M]+
1101.471, found 1101.473.
5.9. Absorption and fluorescence measurements
Absorption spectra of the probes were measured in PBS buffer
containing 8.0 g NaC1, 0.2 g KC1, 1.15 g Na2HPO4, 0.2 g KH2PO4,
in water 100 mL (pH 7.4). The concentrations of the oligonucleo-
tides were in the range of 2–2.5 lM. Absorbance was kept less than
0.05 AU in order to avoid inner filter distortion. Samples were mea-
sured in a 10 mm quartz cell. The fluorescence measurement con-
ditions of oligonucleotides included 710 V sensitivity and a 5 nm
slit. Samples were measured in PBS buffer (pH 7.4). kex = 490 or
510 nm, kem = 500–700 nm range. The concentration of the sam-
ples was in the range of 0.4–0.5
lM. Samples were measured in a
5.6. Oligonucleotide synthesis, work-up and purification
10 mm quartz cell. The quantum yields (
U
) of the single-stranded
and double-stranded oligonucleotides were calculated from the
observed absorbance and the area of the fluorescence emission
band. The fluorescence quantum yields of all oligonucleotides were
determined relative to fluorescein in 0.1 M NaOH (kex 494 nm, kem
The ON probes were assembled by DNA/RNA synthesizer apply-
ing phosphoamidite method. CPGs (3 lmol, pore size 500 Å) sup-
port and commercially available phosphoramidite nucleosides
were used. The synthesized dUTO modified nucleoside phophoram-
idite 9 was used at 0.1 M in dry DCM. The ONs were cleaved from
the support with 1:1 (v/v) 33% NH4OH and 33% methylamine in
ethanol at 65 °C, for 10 min. The crude product was purified by
HPLC on a C-18 column, and eluted with a linear gradient of 5–
40% acetonitrile in 0.1 M TEAA (pH = 7), over 30 min at a flow rate
of 3 mL/min. The oligomers were converted to the sodium salt
using CM Sephadex C-25 equilibrated in NaCl and washed well
with water. The identity of the ONs was determined by MALDI-
TOF mass spectroscopy: dT7dUTOdT7 calcd for C173H203N32O103P14-
S ([M+Na])+ 4864.75, found 4865.15 ON1 calcd for C178H226N51-
521 nm,
U 0.54) The quantum yield was calculated according to
the following equation:
UF
¼
URI=IR ꢁ ODR=OD ꢁ g2
=gR
Here,
U and UR are the fluorescence quantum yield of the sam-
ple and the reference, respectively, I and IR are areas under the
fluorescence spectra of the sample and of the reference, respec-
tively, OD and ODR are the absorption values of the sample and
the reference at the excitation wavelength, and
g and gR are the
refractive index for the respective solvents used for the sample
and the reference.
O106P14
S
([M+H])+ 5238.92, found 5237.98; ON2 calcd for
C
178H226N51O106P14S ([M+H])+ 5238.92, found 5238.32; ON3 calcd
for C201H233N53O105P14S2 ([M+Na])+ 5592.05, found 5592.67; ON4
calcd for C201H233N53O105P14S2 ([M+Na])+ 5592.05, found 5594.11.
The yields of ONs were in the range of 95–98%.
5.10. CD measurements
CD spectra of fluorescent probes were measured in PBS buffer
(pH 7.4) at 2.5
lM concentration, by using 1 cm quartz cell.
5.7. Concentration and extinction coefficients calculations
5.11. Thermal denaturation measurements (Tm
)
Concentrations of oligonucleotides were calculated using the fol-
The Tm values of duplexes were measured in. The absorbance of
the samples was monitored at 260 nm and the temperature ranged
from 20 to 85 °C with heating rate of 1 °C/min.
lowing extinction coefficients (OD260/
tion coefficients used were
dT = 8400 Mꢀ1 cmꢀ1
cmꢀ1 dG = 12,010 Mꢀ1 cmꢀ1
lmol): The individual extinc-
e
,
,
e
dC = 7050 Mꢀ1
0
,
e
e
dA = 15,200 Mꢀ1 cmꢀ1
,
eU-3 -OMe
=

M. Kovaliov et al. / Bioorg. Med. Chem. 22 (2014) 2613–2621
2621
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Human U2OS osteosarcoma cells were maintained in low glu-
cose Dulbecco’s Modified Eagle’s Medium (DMEM, Biological
Industries, Israel) containing 10% fetal bovine serum (FBS, HyClone,
Logan, UT). The clone overexpressing cyclin D1 contained a stable
integration of a GFP-cyclin D1 plasmid under the control of the
CMV promoter. RNA was extracted from wild-type U2OS cells
and cyclin D1 overexpressing cells using TRI Reagent Sigma–Al-
drich protocol. cDNA (1 lg RNA) was synthesized using the
ReverseAid™ First Strand cDNA Synthesis Kit (Fermentas) with oli-
go-dT as a primer.
9. Astakhova, I. V.; Ustinov, A. V.; Korshun, V. A.; Wengel, J. Bioconjugate Chem.
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Fluorescence measurements of ON2 probe, in RNA extracts
were performed at room temperature in PBS buffer (pH 7.4). The
final concentration of the probe was 0.5
tion of the total RNA extracts was 250 ng/
l
l
M, while the concentra-
L. The fluorescence was
measured upon excitation at 510 nm. Samples were measured in a
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Supplementary data (fluorescence and absorption spectra of
dUTO and ON1–4, selection of the probe’s sequence, time depen-
dent fluorescence spectra upon addition of ON2 to total RNA cell
extracts and NMR-spectra of compounds 1–9) associated with this
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