Detection of mRNA of the cyclin D1 breast cancer marker by a novel duplex-DNA probe

Article abstract of DOI:10.1021/jm301838y

Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2′- deoxy-uridine, 6, as a fluorescent uridine analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red-shifted as compared to uridine.

Full text of DOI:10.1021/jm301838y

Article
pubs.acs.org/jmc
Detection of mRNA of the Cyclin D1 Breast Cancer Marker by a Novel
Duplex-DNA Probe
Meirav Segal,† Eylon Yavin,‡ Pinhas Kafri,§ Yaron Shav-Tal,§ and Bilha Fischer*,†
†Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
‡School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Ein Karem, Jerusalem 91120, Israel
§Faculty of Life Sciences and Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan 52900, Israel
S
* Supporting Information
ABSTRACT: Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, 6, as a fluorescent uridine
analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red-shifted as
compared to uridine. Here, we utilized 6 for preparation of labeled oligodeoxynucleotide (ODN) probes based on MS2 and
cyclin D1 (a known breast cancer mRNA marker) sequences. Cyclin D1-derived labeled-ssODN showed a 9.5-fold decrease of
quantum yield upon duplex formation. On the basis of this finding, we developed the ds-NIF (nucleoside with intrinsic
fluorescence)-probe methodology for detection of cyclin D1 mRNA, by which the fluorescent probe is released upon recognition
of target mRNA by the relatively dark NIF-duplex-probe. Indeed, we successfully detected, a ss-deoxynucleic acid (DNA) variant
of cyclin D1 mRNA using a dark NIF-labeled duplex-probe, and monitoring the recognition process by fluorescence
spectroscopy and gel electrophoresis. Furthermore, we successfully detected cyclin D1 mRNA in RNA extracted from cancerous
human cells, using ds-NIF methodology.
nucleosides with intrinsic fluorescence include 5-methyl
propargyl ether-2′-deoxyuridine,23 1; 5-benzothiophene-uri-
dine,3 2 (λmax 318 nm, λem 458 nm, Φ 0.035 in H2O), 2b
(λmax 316 nm, λem 431 nm, Φ 0.03 in H2O, a tricyclic cytosine
family;27 3 (λmax 395 nm, λem 505 nm, Φ 0.13 in H2O), and
4,28−30 (λmax 370 nm, λem 450 nm, Φ 0.3 in H2O); and a
tetracyclic cytosine analogue,31,32 5 (λmax 374 nm, λem 513 nm,
Φ 0.006 in 50 mM PBS containing 0.1 M sodium chloride at
pH 7.4) (Figure 1).
These fluorescent nucleosides were incorporated into
oligonucleotides which, in turn, were used as probes for
detection of genetic material. The photophysical properties of
nucleosides 1−5 remained the same or slightly changed upon
incorporation into an oligonucleotide and after oligonucleotide
hybridization.28−30
A great advantage of the above fluorescent pyrimidine
analogues is that their size and structure enable them to be
incorporated into nucleic acid with minimal disturbance of the
native structure as compared to fluorescent dyes covalently
linked to the backbone of the oligonucleotide.33
Recently, we reported on the promising properties of a novel
uracil nucleoside with intrinsic fluorescence (NIF), 5-((4-
methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, 6 (λabs 320 nm,
λem 478 nm, Φ 0.12 in H2O)34 (Figure 1). This fluorescent
uracil analogue bears a minimal chemical modification at a
position not involved in base-pairing, resulting in relatively long
absorption and emission wavelengths and sufficiently high
quantum yield. Analogue 6, exhibits quantum yield that is 3000-
fold larger than that of the natural uracil chromophore and
maximum emission which is 170 nm red-shifted as compared to
INTRODUCTION
■
The need to diagnose specific deoxynucleic acid (DNA) and
ribonucleic acid (RNA) sequences has created a demand for
recognition probes that respond with high signal-to-noise ratios
and low fluorescence background.1−3 For this purpose, various
fluorescent nucleoside analogues4−7 and oligonucleotide probes
have been developed.8,9 Most of these probes include extrinsic
fluorescent dyes10−12 for nucleic acid staining and labeling,
intercalating dyes, and minor groove binding dyes. These dyes
are used in various techniques for detecting genetic material in
DNA arrays,13 FISH,14 gels,15 virus particles,16 and cells by
fluorescence microscopy or electrophoresed gels.17
Methodologies using extrinsic fluorescent dyes are in many
cases limited due to experimental procedures required prior to
detection, poor solubility of dyes in water/phosphate buffer,
toxicity of certain dyes, or background fluorescence of
unreacted dyes. In addition, the large hydrophobic dye attached
to a nucleotide alters the efficiency of enzymatic incorporation
into DNA/RNA, resulting in different levels of labeling and
prohibits quantification of nucleic acids. Even a two-step
protocol involving first the incorporation of a slightly modified
nucleotide into a nucleic acid, e.g., aminoallyl modified bases,18
followed by covalent binding of fluorescent dyes, to allow the
synthesis of multiply labeled fluorescent oligomer hybridization
probes,19 requires laborious multiple serial labeling and
purifications to increase labeling and visualization efficiency.20
The limitations and complications of methodologies applying
extrinsic dyes for nucleic acids detection emphasize the need
for different fluorescent probes.
Over the past decade a number of nucleoside analogues with
intrinsic fluorescence, replacing natural DNA bases in
oligonucleotide probes, have been reported as an alternative
to dye-substituted nucleosides.21−26 For instance, pyrimidine
Received: December 13, 2012
© XXXX American Chemical Society
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Figure 1. Previously reported fluorescent pyrimidine nucleoside analogues.
uridine. In addition, because probe 6 adopts the anti
conformation and S sugar puckering, favored by B-DNA, it
makes a promising nucleoside analogue to be incorporated in
an oligodeoxynucleotide probe for detection of genetic material
by a hybridization assay.
Here, we report labeling 15-mer oligodeoxynucleotides by
one or several 6 monomers and the photophysical properties
and stability of the related duplexes. We compare the
fluorescence of labeled single strands to that of monomer 6
and the corresponding double strands. In addition, we analyze
the dependence of photophysical properties of labeled
oligodeoxynucleotides on the number of labels and the
neighboring nucleobases. Finally, we demonstrate the use of
monomer 6 for the preparation of a novel ds-NIF probe and for
the detection of cyclin D1 breast cancer mRNA marker in total
RNA extract from human cancerous cells.
for the detection of mRNA, we initially incorporated NIF
analogue 5-((4-methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine,
6, replacing 2′-deoxy-thymidine, into ssDNA, which is
complementary to the target mRNA. The mRNA we chose
to focus on was cyclin D1. The cyclin D1 protein is a major
player in the control of the cell cycle.35 The cyclin D1 gene has
been convincingly implicated in oncogenesis,36 and it is highly
expressed in many cancer types and has been specifically
implicated in breast cancer.37 In addition, we also tested a
unique repetitive RNA sequence called MS238 that is inserted
into genes for the labeling of specific mRNAs.39 Because of the
repetitive nature of the sequence, it is possible to obtain a
strong signal via the binding of several identical probes to the
sequence repeats.
We first prepared the protected and activated nucleoside
phosphoramidite, 10, starting from 5-I-2′-deoxy-uridine, 7.
Thus, 5-iodo-2′-deoxyuridine, 2, was protected with 4,4′-
dimethoxytrityl group at C5′-OH, to obtain 8 in 85% yield,
which was then employed as a substrate in a Suzuki coupling
reaction with trans-2-(4-methoxy-phenyl)-vinyl boronic acid to
give 9 in 86% yield. Finally, 5′-DMTO-3′-O phosphoramidite,
RESULTS
■
Incorporation of 5-((4-Methoxy-phenyl)-trans-vinyl)-
2′-deoxy-uridine, 6, into Oligodeoxynucleotides
(ODNs). To develop fluorescent oligodeoxynucleotide probes
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a
Scheme 1. Synthesis of Phosphoramidite 10
a
Reaction conditions: (a) 4,4′-dimethoxytrityl chloride (1.2 equiv), pyridine, 80% yield; (b) trans-2-((4-methoxy)-phenyl) boronic acid (1.25 equiv),
Na2CO3 (3 equiv), TPPTS (0.25 equiv), Pd(OAc)2 (0.05 equiv), CH3CN:H2O (2:1), 86% yield; (c) N,N′-diisopropylcyanoethyl-phosphoramidite
chloride, TEA, CH2Cl2, 75% yield.
Table 1. Photophysical Properties of Labeled MS2-Derived ODNs (1, 3, 5, 7, 9, 11) and Their Corresponding Duplexes (2, 4, 6,
a
8, 10, 12) in PBS Buffer
DNA sequence
(MS2-ODN)
λabs max
(nm)
λem max
(nm)
Tm
(°C)
entry
Φ
unmodified duplex (control)
3′-ATCCTAGATTACTTG-5′
5′-TAGGATCTAATGAAC-3′
3′-AUCCTAGATTACTTG-5′
3′-AUCCTAGATTACTTG-5′
5′-TAGGATCTAATGAAC-3′
3′-AUCCTAGATTACTTG-5′
3′-AUCCTAGATTACTTG-5′
5′-TAGGATCTAATGAAC-3′
3′-ATCCTAGAUTACTTG-5′
3′-ATCCTAGAUTACTTG-5′
5′-TAGGATCTAATGAAC-3′
3′-ATCCTAGATUACTTG-5′
3′-ATCCTAGATUACTTG-5′
5′-TAGGATCTAATGAAC-3′
3′-ATCCTAGATTACUTG-5′
3′-ATCCTAGATTACUTG-5′
5′-TAGGATCTAATGAAC-3′
3′-AUCCUAGAUUACTTG-5′
3′-AUCCUAGAUUACTTG-5′
5′-TAGGATCTAATGAAC-3′
260
43
49
45
64
59
46
60
1
2
331
330
461
460
0.020
0.025
3
4
331
333
463
456
0.018
0.023
5
6
334
334
459
455
0.013
0.023
7
8
334
334
463
459
0.009
0.029
9
332
329
462
489
0.150
0.013
10
11
12
328
325
462
459
0.008
0.014
a
Tm values are given for all duplexes. Bold U denotes fluorescent deoxyuridine label.
10, was prepared upon treatment of 9 with phosphoramidite
chloride to obtain compound 10 in 75% yield (Scheme 1).
Phosphoramidite, 10, was used as a monomer for the
preparation of 15-mer oligodeoxynucleotides (ODNs) which
contain a single label or multilabels replacing T at different
positions of the oligonucleotide sequence. Two series of labeled
15-mer deoxyoligonucleotides were prepared: (1) ODNs
derived from MS240,41 bacteriophage RNA sequence (Table
1) and (2) ODNs derived from cyclin D142 mRNA sequence
(Table 2). We chose a 15-mer long oligonucleotide because 15-
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Table 2. Photophysical Properties of Labeled Cyclin D1-Derived ODNs (13, 15, 17, 19, 21, 23, 25, 27, 29) and Their
a
Corresponding Duplexes (14, 16, 18, 20, 22, 24, 26, 28, 30) in PBS Buffer
DNA sequence
(cyclin D1-ODN)
λabs max
(nm)
λem max
(nm)
Tm
(°C)
entry
Φ
unmodified duplex (control)
3′-CTTGTTCGAGTTCAC-5′
5′-GAACAAGCTCAAGTG-3′
3-CUTGTTCGAGTTCAC-5′
3′-CUTCTTCGAGTTCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CTUGTTCGAGTTCAC-5′
3′-CTUGTTCGAGTTCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CTTGUTCGAGTTCAC-5′
3′-CTTGUTCGAGTTCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CTTGUTCGAGTTCAC-5′
3′-CTTGTUCGAGTTCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CTTGTTCGAGUTCAC-5′
3′-CTTGTTCGAGUTCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CTTGTTCGAGTUCAC-5′
3′-CTTGTTCGAGTUCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CUTGTTCGAGTUCAC-5′
3′-CUTGTTCGAGTUCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CUTGTUCGAGTUCAC-5′
3′-CUTGTUCGAGTUCAC-5′
5′-GAACAAGCTCAAGTG-3′
3′-CUUGUUCGAGUUCAC-5′
3′-CUUGUUCGAGUUCAC-5′
5′-GAACAAGCTCAAGTG-3′
260
49
50
36
41
55
45
50
51
50
47
13
14
327
324
459
455
0.034
0.090
15
16
327
327
460
455
0.040
0.080
17
18
327
327
457
453
0.050
0.140
19
20
327
326
466
452
0.110
0.300
21
22
327
327
458
447
0.100
0.250
23
24
329
327
460
448
0.060
0.260
25
26
328
328
463
453
0.130
0.020
27
28
328
328
460
457
0.150
0.016
29
30
325
325
459
458
0.005
0.004
a
Tm values are given for all duplexes.
mer is the minimal length which would ensure specific binding
to target MS2 or cyclin D1 sequences.
would trigger a significant quantum yield change upon duplex
formation.
Photophysical Characterization of MS2-Derived La-
beled ODNs 1−12. In the MS2-derived ODN series (entries
1−12, Table 1), six labeled single-strand oligonucleotides were
synthesized. For every oligomer, the corresponding duplex was
prepared. All studied ODNs, either single- or double-strand,
were excited at 325−334 nm and emitted at 455−463 nm,
except for the double strand labeled at position 13, which
emitted at 489 nm. The position 4-labeled ODN (entry 11),
exhibited a very low Φ value of 0.008, however this value
increased 2-fold upon duplex formation (entry 12). The
quantum yield also increased 3-fold in monolabeled ODN at
position 10 (entry 7) upon duplex formation (entry 8). The Φ
value of three monolabeled ODNs at position 2, 5, or 9 (entries
1, 3, and 5) remained unchanged upon duplex formation (entry
2, 4, and 6). The ODN monolabeled at 13 position displayed
an exceptionally large 11.5-fold reduction of quantum yield
upon duplex formation, changing from 0.15 (entry 9) to 0.013
(entry 10).
ODNs containing the modified uridine monomer(s) were
prepared on solid-phase according to conventional DNA
synthesis protocol. The coupling yields were always higher
than 95%. Deprotection of the oligomers was performed with
aqueous 33% NH4OH at 37 °C over 24 h. Purification of the
oligonucleotides was achieved on Poly-Pak II column. The
oligonucleotides were detritylated with 2% TFA, and further
purification was performed by reversed-phase HPLC. The
purified oligomers were characterized by MALDI-TOF mass
spectrometry.
Because emission of fluorescent nucleosides within oligonu-
cleotides is sensitive to neighboring nucleobases,27,31 we
incorporated nucleoside 6 at various positions of the studied
15-mer oligonucleotides while keeping all the other bases the
same. In addition, differently multilabeled oligonucleotides
were tested.
ODN labeling included: (1) MS2 derived ODNs labeled at
positions 2 or 5 or 9 or 10 or 13, and tetra-labeled at 2, 5, 9, and
10 (Table 1), and (2) cyclin D1 ODNs derived labeled at
positions 2 or 3 or 5 or 6 or 11 or 12, dilabeled at positions 2
and 6, or 2 and 12, trilabeled at positions 2, 6, and 12, and hexa-
labeled at positions 2, 3, 5, 6, 11, and 12 (Table 2).
These series of oligomers were used to explore whether our
monomer of choice, 6, when incorporated into a ss-ODN,
Photophysical Characterization of Cyclin D1-Derived
Labeled ODNs 13−30. In the series of cyclin-D1 derived
ODNs (entries 13−30), nine labeled single-stranded oligonu-
cleotides were synthesized. For every oligomer, the correspond-
ing duplex was prepared (Table 2).
All studied ODNs, either single- or double-strand, were
excited at 325−329 nm and emitted at 447−460 nm (Figure 2).
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5-((4-methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, 6, labeled
ODNs for detection of ssDNA, we selected an ODN exhibiting
a large change of fluorescence upon hybridization/dehybridiza-
tion. Specifically, we selected a cyclin D1-derived ODN labeled
at positions 2, 6, and 12 (entry 27), the fluorescence of which is
reduced 9.5-fold upon duplex formation (Figure 2). Fur-
thermore, we devised a NIF-duplex probe composed of a 15-
mer ODN (entry 28):35-mer duplex (Figure 4). This ds probe
Figure 2. Emission spectra of ODN 27 (ssDNA) vs 28 (dsDNA).
Six monolabeled fluorescent ssODNs (entries 13−23)
exhibited Φ values of 0.034−0.11, however, these values
increased 2−4-fold upon duplex formation (entries 14−24).
Yet, the hexa-labeled ODN retained its fluorescent quantum
yield value upon duplex formation (entries 19 and 29). The Φ
value of dilabeled and trilabeled ODN (entry 25, Φ 0.13), and
entry 27, Φ 0.15) decreased by a factor of 6.5 and 9.5,
respectively.
Circular Dichroism Measurements of Duplexes of
Cyclin D1-Derived ODNs 16−30. We used CD spectroscopy
to analyze the secondary structure of duplexes containing 5-((4-
methoxy-phenyl)-trans-vinyl)-2′-deoxy-uridine, Figure 3). The
Figure 4. ds-NIF probe methodology for detection of ssDNA or
mRNA. (A) Preparation of ds-NIF probe from fluorescent ss-DNA
and a longer complementary ssDNA. (B) Application of ds-NIF probe
for the detection of target ssDNA/mRNA.
(Figure 4A) is relatively dark. When this ds-NIF probe is added
to target ssDNA or mRNA, it undergoes dehybridization and
concomitant by rehybridization with the target, with release of
the fluorescent NIF-ssODN (Figure 4B). In this way, the ds-
NIF probe indicates the presence of the target ssDNA or
mRNA.
Specifically, we hybridized ODN 27 (Φ 0.15) with a 35-mer
ODN, the middle part of which is complementary to ODN 27,
to obtain double-stranded DNA, 28 (Φ 0.06). To this duplex
we added at room temperature another 35-mer ODN (5
equiv), representing target DNA, which is complementary to
the first mentioned 35-mer. At this point, we observed a time-
dependent increase in fluorescence intensity, indicating the
release of the fluorescent 15-mer ODN and a concomitant
formation of a 35:35-mer duplex (Figure 5). In this way, we
achieved a significant signal:background ratio (13:1), indicating
clearly the presence of target oligonucleotide.
In addition, we validated the ds-NIF probe methodology by
monitoring the dehybridization−rehybridization process and
concomitant release of the fluorescent probe 27 by gel
electrophoresis (Figure 6). The gel picture depicts 7 lanes:
In lane 1: 15-mer fluorescent ODN 27. Lanes 2 and 3: 35-
mer ODNs complementary to each other. Lane 4: 15-mer
ODN 27:35-mer duplex (blue arrow). Lane 5: 35-mer:35-mer
ds. Lane 6: 15-mer:15-mer duplex. Lane 7: 15-mer:35-mer
duplex upon addition of complementary 35-mer ODN. Clearly,
Figure 3. CD spectra of cyclin-D1 derived ODN duplexes, 16−30.
CD spectra exhibit positive bands at 271 and 220 nm as well as
a negative band at 245 nm. The general appearance of the CD
signal between 300 and 200 nm resembles that of a common
right-handed helical B-DNA conformation, which is normally
characterized by a positive band centered at 275 nm, a negative
band at 240 nm, and a band which could be positive or negative
at 220 nm.43
Thermostability of Duplexes. The feasibility of the use of
the above ODNs for detection of genetic material depends on
the thermostability of the related duplexes.
The Tm values of almost all hybrids of our new fluorescent
ODNs with the complementary DNA strands are 45−61 °C,
indicating that they are thermally stable (Tables 1, 2). In all
MS2-derived sequences, Tm values were increased by 6−21 °C
as compared to the corresponding parent duplex (control). In
cyclin D1, six sequences showed an increase in Tm of 1−6 °C
(entries 14, 20, 24, 26, and 28) whereas four sequences showed
a 2−10 °C decrease (entries 16, 18, 22, and 30).
Proving the Applicability of ds-NIF Probe for
Detection of Target ssDNA. To prove the applicability of
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Figure 7. Levels of cyclin D1 mRNA expression were three times
higher in U2OS cells overexpressing GFP-cyclin D1 than in wild-type
U2OS cells (WT), as measured by quantitative RT-PCR. Glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were
used as a control.
were the RNA extract alone and RNA extract with MS2 ODN
probe, 10. Upon addition of the probe to RNA extract
containing high levels of cyclin D1, we observed a time-
dependent increase in fluorescence intensity up to 3-fold vs the
ds-NIF probe fluorescence after 90 min (signal:background
ratio 3:1). This fluorescent enhancement indicates the release
of the fluorescent 15-mer ODN, 27, and the formation of a
DNA:cyclin D1 RNA duplex (Figure 8).
Figure 5. Time-dependent fluorescence of 50 μM ds-NIF probe in the
presence of target ssDNA in PBS buffer.
Figure 6. Gel electrophoresis monitoring of ds-NIF probe method-
ology for the detection of target ssDNA visualized by ethidium
bromide.
the band of 15-mer:35-mer duplex weakened (red arrow) and
the band of 35-mer:35-mer ds became stronger (green arrow),
indicating dehybridization of 15-mer:35-mer duplex and
rehybridization to obtain 35-mer:35-mer ds. Indeed, these
data support the occurrence of the mechanism of the ds-NIF
probe methodology as observed by fluorescence monitoring
(Figure 5).
In conclusion, upon addition of complementary 35-mer
single-strand to double-stranded DNA, 28, 15:35-mer, we
observed an increase in fluorescence intensity, indicating the
release of the fluorescent 15-mer single-strand and formation of
a 35:35-mer duplex. The reason for this rehybridization is
probably formation of a more stable duplex (35:35-mer) than
duplex 28 (15:35-mer).
Proving the Applicability of ds-NIF Probe for
Detection of Cyclin D1 in RNA Cell Extracts. Next, we
proved the ds-NIF probe methodology by monitoring the
fluorescence intensity of probe 28 in RNA extracted from the
human U2OS osteosarcoma cell line (Figure 7). The ds-NIF
probe was added to total cell RNA extracts containing high
levels of cyclin D1 from a cell line stably overexpressing a cyclin
D1 gene, 1842 ng/μL.
In addition, probe 28 was added to extracts of cells
expressing basal levels of cyclin D1 found in U2OS cells.
Fluorescent ssODN 27 was used as a positive control, while ds-
NIF probe, 28, was the negative control. Additional controls
Figure 8. Time-dependent fluorescence of 50 μM ds-NIF/ssNIF
probes, 28/27 in PBS buffer, in the presence of RNA cell extract
containing high levels of cyclin D1.
Yet, addition of ODN 28 to the RNA extract of cells
expressing basal levels of cyclin D1 did not result in any
increase of fluorescence intensity (Supporting Information p
S1).
Furthermore, addition of the MS2-based probe, ODN 10, to
the RNA extract of cells expressing high levels of cyclin D1, did
not result in any elevation of intensity (Supporting Information
p S2).
In this way we demonstrated qualitatively the efficiency of
the ds-NIF probe method for the detection of breast cancer
marker, cyclin D1, in RNA extracted from cancerous cells.
DISCUSSION
■
Selection of ODNs for Validation of the ds-NIF Probe
Methodology. To validate the proposed ds-NIF probe
method, we first examined a NIF probe containing monomer
6 on an existing gene system expressing mRNA containing
MS2 24 sequence repeats44 which may result in amplification of
the mRNA signal. Hence, the binding of up to 24 probe
molecules to each mRNA molecule is expected. MS2 is a
mRNA, but we studied its DNA variant because of its higher
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stability. Next, we applied our methodology to the detection of
cyclin D1, a mRNA marker of breast cancer.45 We selected a
15-mer ODN from the cyclin D1 mRNA sequence, which is
specific to this mRNA only and does not appear in any other
endogenous mRNA. This 15-mer ODN was selected by Basic
Local Alignment Search Tool (BLAST) and has six bases of
adenine which are complementary to the fluorescent
thymidine-like monomer 6.
The Fluorescence of Monomer 6 is Quenched upon
its Incorporation into Monolabeled MS2 and Cyclin D1
ssODNs. Monolabeled MS2 ODNs (entries 1, 3, 5, and 7)
showed a decreased quantum yield as compared to that of
monomer 6 up to 13-fold. In those monolabeled ODNs, one of
monomer 6 neighboring nucleobases was adenine. Likewise,
monolabeled cyclin D1 ODNs (entries 13, 15, 17, and 23)
exhibited fluorescence quenching as compared to Φ value of
monomer 6 itself. In those monolabeled ODNs, one of
monomer 6 neighboring nucleobases was thymine. Yet, when
the neighboring nucleobases were C or G (entries 9, 19, and
21), the quantum yield remained high (Φ 0.1−0.15) as for
monomer 6 itself.
derived ODN duplexes series, we observed a clear dependence
of Tm on the bases adjacent to the NIF monomer, 6.
In the MS2 series, the presence of an A and a C on either
side of 6 results in a modest increase in Tm (ΔTm = +2 °C for
AU*C and ΔTm = +6 °C for CU*A, entries 11 and 13 in Table
2, respectively). However, changing C to T results in a dramatic
increase in Tm: ΔTm = +21 °C for AU*T and ΔTm = +16 °C
for TU*A (entries 15 and 17 in Table 2, respectively). In fact,
this is quite a remarkable increase in Tm for a single
substitution.
Interestingly, we have also observed a correlation between
ΔTm and Φ values. Namely, Φ is slightly increased for the
AU*C and CU*A ODNs upon duplex formation, whereas Φ is
doubled and even tripled for the AU*T and TU*A ODNs
(ΔTm = +16 °C to +21 °C). It is possible that increased
thermal stability reduces the accessibility of water molecules to
the NIF probe, 6, resulting in less quenching and, as a
consequence, higher Φ values.
In the cyclin D1-derived ODNs series, we also noticed the
effect of neighboring bases on Tm, although the effect is less
pronounced.
Thus, having a C and a T as neighboring bases results in a
modest increase in Tm: CU*T (entry 13, Table 2) ΔTm = +1
°C and TU*C (entries 19 and 23, Table 2) result in ΔTm = +6
°C and +1 °C, respectively. Replacing C with G leads to a
significant decrease in thermal stability where TU*G (entry 15,
Table 2) and GU*T (entries 17 and 21, Table 2) exhibit a ΔTm
−10 °C ΔTm’s values of −8 °C and −4 °C, respectively.
Application of the ds-NIF Probe Methodology for the
Detection of Cyclin D1 mRNA. The limitations of current
standard practice for the diagnosis of DNA or RNA emphasize
the urgent need for conceptually different fluorescent
probes.47,48
Therefore, we demonstrated here the ability of ds-NIF probe
methodology to detect cyclin D1 mRNA in cancerous cell
extract. This methodology is centered on nucleoside with
intrinsic fluorescence, monomer 6, which is incorporated into
oligodeoxynucleotides that subsequently hybridize to form ds-
NIF probe probes. This methodology may be applied in those
cases where the ss-NIF-DNA probes exhibit significantly
different photophysical properties than those of ds-DNA
(RNA):NIF-DNA. The unique photophysical properties and
Tm values of DNA:NIF-DNA vs mRNA:DNA duplexes may be
harnessed to detect target mRNA (Figure 4).
The ds-NIF probe method demonstrated here involves the
preparation of a duplex of NIF-DNA and a longer
complementary DNA, the duplex is termed NIF probe, which
is the actual probe. This ds-NIF probe is relatively dark, i.e., has
a low Φ value (Figure 4A). Next, this probe is allowed to
interact with the target mRNA, resulting in the formation of
DNA:mRNA duplex and the release of the fluorescent ss-NIF-
DNA, thus indicating the presence of the target mRNA (Figure
4B).
We found that the fluorescence of the NIF analogue 6 was
quenched in most cases upon incorporation into a single-strand
ODN, up to 24-fold. Yet, fluorescence quantum yield of the
related duplexes increased, decreased, or remained unchanged
as compared to the corresponding labeled-ss-ODNs. On the
basis of the 9.5-fold reduction of fluorescence upon duplex
formation with cyclin D1 derived ODN 27 (Table 2), we
designed a NIF probe consisting of a duplex of ODN 27 with a
longer complementary strand for the detection of cyclin D1
mRNA in the presence of a ss-DNA variant of cyclin D1 mRNA
Fluorescence is Quenched Dramatically when Two
Adjacent Labels are Introduced to Cyclin D1 and MS2
ODNs. The fluorescence of ss-cyclin D1 hexa-labeled ODN
(entry 29) and MS2 tetra-labeled ODN (entry 11) was
quenched dramatically compared to that of monomer 6. In
these two oligomers, there were two adjacent monomers 6. The
close proximity between two monomers 6 may possibly cause
collisions in the excited state.46 Following this event, energy
may possibly be emitted as heat and not as a photon emission,
which may result in quenching.
Cyclin D1 Multilabeled ODNs Showed Decreased Φ
Value upon Duplex Formation. In cases of di- and trilabeled
ss ODNs (entries 25 and 27, respectively), Φ value was
decreased upon duplex formation (entries 26 and 28). Possibly,
intramolecular interactions between the labels in the duplex
form might be the cause for this phenomenon, although
quenching by water molecules might also contribute to the
overall fluorescence quenching. It is also notable that even in
the ss-DNA, addition of multiple labels tends to decrease Φ, as
exemplified by ODN 29 that bears six labels and shows a
negligible quantum yield of 0.005.
CD Spectra of Labeled DNA Sequences Reveal
Features Characteristic of B-DNA. Circular dichroism
measurements of the series of labeled ds-ODNs sequences
(entries 2−40), where neighboring bases of monomer 6 vary,
all resemble B-DNA form. Thus, monomer 6 does not seem to
distort the duplex conformation (Figure 3).
Incorporation of Monomer 6 in ODNs Usually
Increases the Stability of the Resulting Duplexes.
Development of a fluorescent probe, the application of which
is based on hybridization, requires not only suitable photo-
physical properties in ss- and ds-DNA but also duplex stability.
In all MS2-derived labeled duplexes, Tm increased on the
average by 10.8 °C as compared to the unmodified duplex. In
cyclin D1-derived labeled duplexes, Tm increased on the
average by 2.5 °C as compared to that of the unmodified
duplex. The increased stability of these duplexes may indicate
favorable stacking interactions of monomer 6 as compared to
those of T itself with its neighboring nucleobases.
Thermal Stability (Tm) is Highly Sensitive to Neighbor-
ing Bases of Monomer 6. In both MS2 and cyclin-D1
G
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Journal of Medicinal Chemistry
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analytical reverse-phase column (Gemini 5μ C-18 110A, 150 mm ×
4.6 mm, 5 μm, Phenomenex, Torrance, CA, USA) in 0.1 M TEAA
buffer: CH3CN 97:3 to 75:25 in 30 min (1 mL/min). Purity was
determined by analytical HPLC and confirmed >95% pure. Thermal
denaturation curves measurements were performed on a Cary 300
spectrophotometer (Varian Inc.). The extinction coefficients for the
modified oligonucleotides were approximated by the linear combina-
tion of the extinction coefficients of the natural nucleotides and the
extinction coefficient of the modified nucleoside. To account the base
stacking interactions, this linear combination was multiplied by 0.9 to
give the final extinction coefficients for the oligomers. The individual
extinction coefficients at 260 nm used were εT = 8400 M−1 cm−1, εC =
7050 M−1 cm−1, εG = 12 010 M−1 cm−1, εA = 15 200 M−1 cm−1, and
εmodified U = 13 500 M−1 cm−1.
Synthesis. 1-(5-((Bis(4-methoxyphenyl)(phenyl)methoxy)-
methyl)-4 hydroxytetrahydrofuran-2-yl)-5-iodopyrimidine-2,4-
(1H,3H)-dione, 8. Compound 7 (2 g, 5.65 mmol) was dissolved in
dry pyridine (29 mL). The pyridine was evaporated to half volume.
DMT-Cl (2.3 g, 6.78 mmol) was added, and the orange solution was
stirred for 19 h under nitrogen atmosphere at RT. After 19 h, a new
spot was observed on TLC (98:2 CH2Cl2:MeOH). Cold water (80
mL) was added to the orange solution, and the mixture was extracted
twice with CH2Cl2 (2 × 100 mL). The two organic phases were
combined and dried over Na2SO4. The solvent was evaporated, and
the product was purified on a silica gel column (98:2 CH2Cl2:MeOH)
to yield compound 8 as a white solid in 84% (3.14 g). 1H NMR (300
MHz, CD3CN): δ 8.00 (s, 1H) 7.37−6.86 (m, 13H), 6.13(t, J = 3.5
Hz, 1H), 4.42−4.40 (m, 1H), 3.96−3.94 (m, 1H), 3.76 (s, 6H), 3.4−
3.25 (m, 2H), 1.94 (m, 2H). 13C NMR (300 MHz, CDCl3) δ 127.88,
128.15, 129.25, 135.52, 135.64, 144.50, 144.60, 150.52, 158.65, 160.7,
127.09, 113.46, 113.19, 87.00, 86.77, 85.89, 77.68, 77.26, 76.83, 72.42,
69.02, 65.89, 63.74, 55.34, 41.43, 15.311 ppm. λmax (CH3CN) = 278
nm ESI+ MS m/z: (C30H29IN2O7) 679 (MNa+). Anal. Calcd for
C30H29IN2O7: C, 30.18; H, 4.22; I, 35.44; N, 7.82; O, 22.34. Found: C
29.88, H 4.02, I, 35.24; N 8.05, O 22.81.
(probe 28). Indeed, when tested, this duplex dehybridized and
released the significantly more fluorescent ODN 27 within 80
min at room temperature, thus indicating the presence of target
oligonucleotide.
The ds-NIF-DNA Probe Methodology Is Useful for
Detection of Cyclin D1 mRNA in Cancerous Cell total
RNA Extract. The above ds-NIF-DNA probe, 28, underwent
dehybridization in cell RNA extracts containing high levels of
cyclin D1 mRNA and released the significantly more
fluorescent ODN 27 within 90 min at room temperature,
thus indicating the presence of target cyclin D1. Furthermore,
the fluorescence intensity of this probe remained unchanged in
the presence of low levels of cyclin D1 in the extract. In
addition, the fluorescence intensity did not increase upon
addition of a MS2 ds-NIF probe into the RNA extract
containing high levels of cyclin D1. The results confirm the
usefulness of ds-NIF probe methodology for the diagnosis of
breast cancer subtype by using extracts of biopsies.
CONCLUSIONS
■
In summary, we propose a novel probe for the efficient
detection of cyclin D1 mRNA in a total RNA cell extract. This
new methodology may have several benefits over current
procedures. For instance, synthetic procedures currently
performed on the genetic material in the biological sample
prior to detection may be circumvented. Instead, ready-made
fluorescent nucleosides may be used for automatic synthesis of
the probe. In addition, no toxic or water insoluble fluorescent
dyes are used. Furthermore, background fluorescence of
unreacted probe is not significant, which may be especially
useful when working with cells. The suitability of the new
methodology for additional applications will be reported in due
course.
(E)-1-(5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4 hy-
droxytetrahydrofuran-2-yl)-5-(4-methoxystyryl)pyrimidine-2,4-
(1H,3H)-dione, 9. Water−acetonitrile (6: 3 mL) was added through a
septum to nitrogen-purged round-bottom flask containing 8 (1 g,
1.523 mmol), trans-2-(4-methoxy-phenyl)-vinylboronic acid (338.86
mg, 1.9 mmol), Pd(OAc)2 (17.24 mg, 0.076 mmol), TPPTS (216.42
mg, 0.38 mmol), and Na2CO3 (480 mg, 4.56 mmol). The mixture was
stirred under reflux for 4 h. A new spot was observed on TLC (9:1
CHCl3: MeOH) while starting material disappeared. Acetonitrile was
removed, water was added and the crude residue was freeze-dried. The
product was purified on a silica gel column (90:10 CHCl3:MeOH),
EXPERIMENTAL SECTION
General. Compounds were characterized by nuclear magnetic
■
resonance using Bruker AC-200, DPX-300, or DMX-600 spectrom-
1
eters. H NMR spectra were measured at 200, 300, or 600 MHz.
Phosphoramidite monomer was characterized also by 31P NMR in
CD3CN, using 85% aq H3PO4 as an external reference on Bruker AC-
200. Chemical shifts are expressed in ppm. Nucleosides were analyzed
under ESI (electron spray ionization) conditions on a Q-TOF
microinstrument (Waters, UK). MALDI-TOF mass spectra of
oligonucleotides were measured with a mass spectrometer in a
negative ion mode with HPA matrix. 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). All
moisture sensitive reactions were carried out in flame-dried reaction
flasks with rubber septa, and the reagents were introduced with a
syringe. All reactants in moisture sensitive reactions were dried
overnight in a vacuum oven. Absorption spectra were measured on a
UV-2401PC UV−vis recording spectrophotometer (Shimadzu, Kyoto,
Japan). Emission spectra were measured using Aminco-Bowman series
2 (AB2) luminescence spectrometer (Thermo Electron Corporation,
Markham, Ontario, Canada). Absorption and fluorescence spectra
were recorded in PBS buffer containing 100 mM NaCl and 10 mM
phosphate. Primary oligonucleotides synthesis was carried out on an
ABI DNA/RNA synthesizer (Forster City, USA) on a 1 μmol scale by
standard automated solid-phase method using natural β-cyanoethyl-
phophoramidite bases. Cleavage from the solid support and
deprotection were carried out in 33% NH4OH at 37 °C for 24 h.
Purification of oligonucleotides was achieved on a Poly-Pak II column
(Glen Research, Sterling, VA). Final purification of oligonucleotides
was achieved on an HPLC (Merck−Hitachi) system, using an
1
yielding compound 9 in 88.5% (893 mg) as a white solid. H NMR
(300 MHz, CD3CN): δ 7.80 (s, 1H) 7.48−6.72 (m, 11H), 7.21−6.98
(m, 8H), 6.31−6.26 (m, 2H), 4.52 (m, 1H), 4.00 (m, 1H), 3.99 (s,
3H), 3.75 (s, 6H), 3.49−3.15 (m, 2H) ppm. 13C NMR (300 MHz,
CDCl3) δ 40.14, 40.29, 40.57, 40.84, 55.41, 55.52, 55.58, 64.23, 70.87,
84.88, 86.23, 111.88, 113.7, 114.44, 119.31, 127.27, 127.62, 128.21,
128.36, 128.47, 130.11, 130.24, 130.41, 136.01, 137.55, 145.18, 149.89,
158.59, 159.171, 162.63 ppm. λmax (CH3CN) = 327 nm. ESI+ MS m/
z: (C39H38N2O8) 685 (MNa+). Anal. Calcd for C39H38N2O8: C, 59.33;
H, 6.64; N, 7.69; O, 26.34. Found: C, 59.61; H, 6.39; N, 7.54; O,
26.46.
(E)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-(4-
methoxystyryl) 2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
tetrahydrofuran-3-yl cyanomethyldiisopropylphosphoramidite, 10.
Nucleoside 9 (200 mg, 0.3 mmol) was dissolved in CH2Cl2 (3 mL) in
a flame-dried two-neck flask under N2. Diisopropylethylamine (0.54
mmol, 0.13 mL) and phosphoramidite chloride (83.65 μL, 1.25 equiv)
were added to this solution. The clear yellow solution was stirred at
RT for 19 h. The solvent was evaporated, and the crude residue was
immediately separated on a silica gel column using hexane:EtOAc
(2:8) as an eluent containing 3% TEA. Compound 10 was obtained in
1
78% yield (200 mg) as yellow oil. H NMR (200 MHz, CD3CN): δ
7.83 (s, 1H) 7.51−7.22 (m, 11H), 6.95−6.67 (m, 8H), 6.3−6.25 (m,
2H), 4.81−4.55(m, 1H), 4.07−4.04 (m, 1H), 3.66 (s, 3H), 3.61 (s,
H
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Journal of Medicinal Chemistry
Article
6H), 3.59−3.56 (m, 4H), 2.50−2.47 (m, 4H) ppm. 31P NMR
(CD3CN): δ 148.54 ppm. ESI+ MS m/z: (C48H55N4O9P) 862
(MNa+).
UV Measurements. Absorption spectra of all oligonucleotides
were determined in PBS buffer (pH 7.4). The concentrations of the
oligonucleotides were determined by UV absorption measurement at
260 nm and were found to be at 0.1−5.5 μM range. Absorbance was
kept less than 0.5 AU in order to avoid inner filter distortion. Samples
were measured in a 10 mm quartz cell.
Fluorescence Measurements. The measurement conditions of
oligonucleotides and RNA extracts included 610 V sensitivity and a 4
nm slit. Samples were measured in PBS buffer (pH 7.4). The
concentration of the samples was in the range of 0.1−5.5 μM. Samples
were measured in a 10 mm quartz cell.
Quantum Yield Measurements. The quantum yield of each
oligonucleotide was calculated from the observed absorbance and the
area of the fluorescence emission band. The fluorescence quantum
yields of all oligonucleotides were determined relative to the quantum
yield of quinine sulfate (0.58) in 0.1 M H2SO4 according to eq 1.
7.4). The concentration of the samples was 50 μM. Molar ratio of
ODN 28 (15:35):complementary ssDNA (35-mer) was 1:5. Samples
were measured in a 10 mm quartz cell. All measurements were
performed at room temperature.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C NMR of compounds 9 and 10, Tm values of dsODN
and fluorescence graphs. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 972-3-5318303. Fax: 972-3-6354907. E-mail: bilha.
fischer@biu.ac.il.
Notes
The authors declare no competing financial interest.
ΦF = ΦEI/IR × ODR /OD × η2/η
(1)
R
ACKNOWLEDGMENTS
■
We gratefully acknowledge Dr. Gadi Miller for his help in
performing a gel electrophoresis experiment.
Where R = reference, I = integration of the peak, and η = refractive
index of the solvent.
Hybridization of Oligonucleotides. Solutions of labeled single-
strands were mixed at room temperature with an equimolar amount of
the complementary single strand oligonucleotides in PBS buffer.
Samples were heated to 90 °C for 3−5 min and thereafter annealed by
slow cooling to 25 °C.
ABBREVIATIONS USED
■
ODN, oligo deoxynucleic acid; NIF, nucleoside with intrinsic
fluorescence; CD, circular dichroism; DMT, 4,4-dimethoxy-
trityl (4,4′- dimethoxyltriphenylmethyl); PBS, phosphate
buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydro-
genase
Melting Temperature Measurements. The Tm values of
duplexes were measured in PBS buffer (pH 7) containing 10 mMNaCl
and 100 mM phosphate. The absorbance of the samples was
monitored at 260 nm from 20 to 85 °C with heating rate of 1 °C/min.
CD Measurements. Circular dichroism (CD) spectra were
obtained using a 60 DS CD spectrometer (Aviv Associates, Lakewood,
NJ). All measurements were made at 25 °C. Samples were measured in
a 10 mm quartz cell. Spectra were obtained in PBS buffer (pH 7)
containing 10 mM NaCl and 100 mM phosphate. All spectral data
reported here were obtained at a 0.1−0.5 mM concentration of duplex.
Gel Electrophoresis. Agarose gel (1 g) was added to a solution of
100 mL of 0.5% TBE (Tris borate EDTA) in microwave (1 s). The
agarose gel was poured into the electrophoresis apparatus and set aside
for 20 min for solidification. At this time, the samples were diluted
with ethidium bromide and loaded into the gel. The samples were run
for 45 min and power setting of 150 V. The concentration of the
samples was 0.5 μM.
RNA Cell Extract. Human U2OS osteosarcoma cells were
maintained in low glucose 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-Aldrich protocol.
For RT-PCR, DNA-free Kit (Ambion) was used to remove genomic
DNA contamination. cDNA (1 μg RNA) was synthesized using the
ReverseAid First Strand cDNA Synthesis Kit (Fermentas) with oligo-
dT as a primer. Semiquantitative RT-PCR was performed using
Eppendorf Thermocycler amplification for 19−38 cycles (depending
on the saturation level of the genes amplified) using 1 min
denaturation at 94 °C, 1 min annealing at 55 °C for GAPDH and
62 °C for cyclin D1, 1 min extension at 72 °C, and 72 °C for 10 min
for final extension. Primers for GAPDH: sense, ACC ACA GTC CAT
GCC ATC AC; antisense, TCC ACC ACC CTG TTG CTG TA.
Primers for cyclin D1: sense, ATA CTC GAG CCA TGG AAC ACC
AGC TCC TGT GC; antisense, GCA ACG AAG GTC TGC GCG
TGT TTG C.
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