Signal turn-on probe for nucleic acid detection based on 19F nuclear magnetic resonance

Article abstract of DOI:10.1016/j.bmcl.2010.11.013

To image gene expression in vivo, we designed and synthesized a novel signal turn-on probe for ¹⁹F nuclear magnetic resonance (MR) imaging based on paramagnetic relaxation enhancement. The stem-loop structured oligodeoxyribonucleotide (ODN) hav

Full text of DOI:10.1016/j.bmcl.2010.11.013

Bioorganic & Medicinal Chemistry Letters 21 (2011) 303–306
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Signal turn-on probe for nucleic acid detection based on 19F nuclear
magnetic resonance
⇑
Takashi Sakamoto, Yu-ki Shimizu, Jun Sasaki, Hikaru Hayakawa, Kenzo Fujimoto
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan
a r t i c l e i n f o
a b s t r a c t
To image gene expression in vivo, we designed and synthesized a novel signal turn-on probe for 19F
nuclear magnetic resonance (MR) imaging based on paramagnetic relaxation enhancement. The
stem-loop structured oligodeoxyribonucleotide (ODN) having a molecular beacon sequence for point
mutated K-ras mRNA was doubly labeled with bis(trifluoromethyl)benzene moiety and Gd-1,4,7,10-tet-
raazacyclododecane-1,4,7,10-tetraacetic acid chelate moiety at the each termini of the ODN probe,
respectively. We found that the 19F MR signal of the bis(trifluoromethyl)benzene moiety tethered at
the 50 termini of the probe turned on by the addition of complementary ODN. The probe has the potential
to image gene expressions in vivo.
Article history:
Received 1 October 2010
Revised 27 October 2010
Accepted 1 November 2010
Available online 12 November 2010
Keywords:
19F magnetic resonance imaging
Paramagnetic relaxation enhancement
Gd-DOTA
Ó 2010 Elsevier Ltd. All rights reserved.
Nucleic acids
In vivo imaging technologies, such as X-ray computed tomogra-
phy, positron emission tomography, and nuclear magnetic resonance
imaging, are promising technologies for diagnosis of various disor-
ders and follow up after surgical treatments. To image biological
events in greater detail in vivo, molecular probes targeting biomole-
cules, such as disease marker enzymes1 or receptors,2 have been
developed. Imaging probes that can image enzyme activity,3–5 pro-
tein,6 or protein agrregate7 by 19F nuclear magnetic resonance (MR)
imaging technology have been reported. Due to the low background
signal of 19F MR in vivo, these 19F MR based imaging probes have a po-
tential for high contrast and specific imaging of target biomolecules.
However, no molecular probes that can image genome activity
in vivo have been reported. In this study, we tried to develop a novel
hybridization probe that can image gene expression in vivo based on
19FMRimagingtechnology. Such aprobewillenableusto image inva-
sively genomic activity related to various disorders and to investigate
directly the genome function in vivo.
We designed and synthesized a stem-loop structured oligonu-
cleotide probe having a fluorine and paramagnetic compound at
the 50 and 30 termini, respectively (Fig. 1). In the absence of target nu-
cleic acid, it is expected that the distance dependent paramagnetic
relaxation enhancement (PRE) effect8 of the paramagnetic com-
pound at the 30 termini quenches the 19F MR signal of the fluorine
compound at the 50 termini. The addition of target nucleic acid
causes change in the distance between 50 and 30 termini, and then
the PRE effect is reduced. The 19F MR signal is expected to be turned
on by the addition of target nucleic acid.
We adopted bis(trifluoromethyl)benzene moiety, as a fluorine
compound, and Gd-DOTA chelate, as a paramagnetic compound.
Firstly, we synthesized phosphoramidite unit of the fluorine com-
pound (3) according to Scheme 1. These compounds were introduced
to the 50 termini of oligonucleotide (CCTACGCCAACAGCTCCgtagg)
according to Scheme 2. As the use of standard phosphoramidite
chemistry, bis(trifluoromethyl)benzene moiety could be labeled
quantitatively with the 50 termini of the oligonucleotide. And the
phosphoramidite unit could be labeled in multiples with the 50 ter-
mini of the oligonucleotide as shown in Fig. S1 (See Supplementary
data). As the 19F MR signal of the multiply 3 labeled oligonucleotide
was linearly increased with the number of 3 introduced (Fig. S2), it
was suggested that the sensitivity of the probe could potentially
enhance by increase the number of 3 introduced to the probe. DOTA
was easily labeled with the 30 termini of the probe by standard
NHS-ester chemistry in aqueous solution. Gd was quantitatively che-
lated to DOTAat the 30 termini of theprobe. Astheconversions ofeach
labeling reaction were quantitative (Fig. S3), purification of all prod-
ucts except for the phosphoroamidite coupling of the fluorine com-
pound was easily performed by gel filtration using the NAP-5
column. These highly labeling yields and easypurificationprocedures
might contribute to mass production of the probe for practical usage.
To confirm the 19F MR signal turn-on manner of the mono-3
labeled probe, 19F NMR spectra were measured11 in the presence
or absence of the target oligonucleotide (Kras Mut; gtagttG-
GAGCTGTTGGCGTAGGcaag). As shown in Figure 2, the 19F MR sig-
nal of bis(trifluoromethyl)benzene moiety at the 50 termini of the
probe (62.9 and 63.0 ppm) appeared by the addition of Kras Mut
ODN having
a complementary sequence for the probe. The
⇑
Corresponding author. Tel./fax: +81 761 51 1671.
E-mail address: kenzo@jaist.ac.jp (K. Fujimoto).
appeared two peaks were assigned to each enantiomer of the
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.013

304
T. Sakamoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 303–306
Figure 1. Schematic drawing of the mechanism of target nucleic acid detection using stem-loop typed oligonucleotide probe having 19F and Gd-DOTA at the each termini of
the probe.
Scheme 1. Synthetic method for compounds 1–3;9 (a) 2-amino-1,3-propanediol,
HOBt, WSC, DMF; (b) 4,40-dimethoxytritylchloride, N,N-dimethylaminopyridine,
pyridine; (c) 2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphordiamidite, benzylthio-
1H-tetrazole, acetonitrile.
fluorine compound tethered to 50 termini. The addition of GAPDH
ODN having an uncomplimentary sequence (aactttggtatcgtggaaga-
actcatgacc) did not affect the 19F MR signal. These results indicate
that the probe could discriminate the sequence of the target ODNs.
In the case of the addition of Kras Wt ODN, which has one base
mismatch sequence for the probe (gtagttGGAGCTGgTGGCGTAGG-
caag), the 19F MR signal was increased to the same intensity as in
the case of the addition of Kras Mut ODN. This indicates that the
probe could not discriminate one base mismatch sequence in the
present condition, and that the concentration of the probe and tar-
get ODN was too high to discriminate one base mismatch. Indeed,
the melting temperature of each duplex was not so different at the
present condition (57 °C for probe/Kras Mut and 52 °C for probe/
Kras Wt). These results suggest that the probe has the potential
to target DNA or RNA by 19F MR imaging technology, although fur-
ther improvement of the sensitivity of the probe may be required.
Change in the chemical shift of the probe was observed by the
addition of Kras Mut ODN. As the chemical shift of 19F compound
was changed dependent on the environment around 19F
Scheme 2. Synthetic method for the probe;10 (a) compound 3, benzylthio-1H-
tetrazole, acetonitrile, 999 s; (b) 28% NH3 aq, 55 °C, 18 h; (c) DOTA-NHS, 50 mM
Na–borate buffer (pH 9.0); (d) Gd(Cit)2, 50 mM Tris–HCl buffer (pH 7.6).

T. Sakamoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 303–306
305
tional change of the probe caused by the hybridization with the tar-
get DNA strand.
In summary, we developed a novel sensor DNA molecule that
can detect complementary nucleic acid by 19F MR signal based
on the PRE effect. This molecular beacon type probe can detect
target nucleic acid by its 19F MR signal turn-on manner and has
the potential to become a nucleic acids imaging probe by 19F MR
imaging technology. To improve the selectivity and sensitivity of
the probe, fine-tuning of the number and structure of fluorine
compound for 50 label is now underway.
Acknowledgment
This study was supported by a Grant in-Aid for Scientific Re-
search by the Ministry of Education, Science, Sports and Culture
of Japan.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.11.013.
References and notes
1. Scherer, R. L.; McIntyre, J. O.; Matrisian, L. M. Cancer Metastasis Rev. 2008, 27,
679.
2. Bouchelouche, K.; Capala, J.; Oehr, P. Curr. Opin. Oncol. 2009, 21, 469.
3. Mizukami, S.; Takikawa, R.; Sugihara, F.; Hori, Y.; Tochio, H.; Wälchli, M.;
Shirakawa, M.; Kikuchi, K. J. Am. Chem. Soc. 2008, 130, 794.
Figure 2. 19F NMR spectra and signal intensity of the probe in the presence of
various oligonucleotides. (a) Probe alone; (b) + Kras Mut; (c) + Kras Wt; (d) +
GAPDH; (e) relative signal intensity of the probe. [probe] = [ODN] = 10
lM in
4. Mizukami, S.; Takikawa, R.; Sugihara, F.; Shirakawa, M.; Kikuchi, K. Angew.
Chem., Int. Ed. 2009, 48, 3641.
50 mM Tris–HCl (pH 7.6), 10% (v/v) D2O, 10 M TFA (as internal standard), 25 °C.
l
5. Tanabe, K.; Harada, H.; Narazaki, M.; Tanaka, K.; Inafuku, K.; Komatsu, H.; Ito,
T.; Yamada, H.; Chujo, Y.; Matsuda, T.; Hiraoka, M.; Nishimoto, S. J. Am. Chem.
Soc. 2009, 131, 15982.
6. Takaoka, Y.; Sakamoto, T.; Tsukiji, S.; Narazaki, M.; Matsuda, T.; Tochio, H.;
Shirakawa, M.; Hamachi, I. Nat. Chem. 2009, 1, 557.
7. Higuchi, M.; Iwata, N.; Matsuba, Y.; Sato, K.; Sasamoto, K.; Saido, T. C. Nat.
Neurosci. 2005, 8, 527.
8. Helm, L. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 49, 45.
compound,12 this result also might cause by the change in the
environment around the 19F compound, for example, the 19F com-
pound possessed blunt end of the stem in the stem-loop structured
state (probe alone) or sticky end in the hybridized state (+ Kras
Mut). This result suggests that the probe has a potential to become
a chemical shift ratio-imaging sensor for hybridization detection.
A titration study of the target ODN, Kras Mut, was performed to
demonstrate the concentration dependency of the signal turn-on
manner of the probe. As shown in Figure 3, the 19F MR signal was
increased in a concentration dependent manner of the target ODN
and reached a plateau at the equimolar concentration of the probe,
indicating that the probe can quantitatively detect a target nucleic
acid strand. As the addition of the excess amount of DOTA (2 mM)
9. 2-Amino-1,3-propanediol (0.18 g, 1.95 mmol) and bis(trifluoromethyl)benzoic
acid (0.50 g, 1.94 mmol) were added in dry DMF (30 mL) containing WSC (0.45 g,
2.33 mmol) and 1-hydroxybenzotriazole (0.36 g, 2.33 mmol). After the reaction
mixture was stirred at room temperature for 24 h, the solvent was removed, and
the remaining oil was subjected to silica gel column chromatography (0–5%
MeOH/CHCl3) to afford 1 (0.48 g, 75%). 1H NMR (400 MHz, [D6]DMSO): d = 8.61 (d
(8.4 Hz), 1H, –NHCO–), 8.53 (s, 2H, o-ArH), 8.30 (s, 1H, p-ArH), 4.72 (t (6 Hz), 2H, –
OH), 4.01 (m, 1H, –NHCH(CH2OH)2) and 3.53 (m, 4H, –NHCH(CH2OH)2). MALDI-
TOF-MS: calcd 354.05 ([(M+Na)+]), found 354.71. To a solution of 1 (0.33 g,
1.00 mmol) in dry pyridine (5 mL) was added
a
solution of 4,40-
dimethoxytritylchloride (0.41 g, 1.20 mmol) and N,N-dimethylaminopyridine
(24 mg, 0.2 mmol) in dry pyridine (5 mL) on ice bath. After the reaction mixture
was stirred at room temperature for 20 h, the solvent was removed, and the
remainingoilwas subjected to silica gel column chromatography (CHCl3) to afford
2 (0.15 g, 23%). 1H NMR (400 MHz, CDCl3): d = 8.17 (s, 2H, o-ArH), 8.02 (s, 1H,
to the 1:1 mixture of the probe (10 lM) and Kras Mut ODN
(10 l
M) did not cause any change in the 19F MR signal (Fig. S4),
the quenched 19F MR signal that was caused by the stem-loop
structure of the probe was completely recovered by the conforma-
p-ArH), 7.40–6.80 (m, 13H, ArH), 4.29 (s, 1H, –OH), 3.99 (m, 1H,
–
NHCH(CH2OH)(CH2O-DMTr)), 3.79 (dd (11.2 Hz), 2H, –CH2O-DMTr), 3.77 (s, 6H,
(ArOCH3)2) and 3.49 (m, 2H, –CH2OH). MALDI-TOF-MS: calcd 656.18 [(M+Na)+],
found656.90. The residual trivial amount of waterin 2 was removedbyazeotropic
distillation with dry acetonitrile (twice). Then, 2 (0.10 g, 0.16 mmol), 0.1 M
solution of 5-benzylthio-1H-tetrazole in dry MeCN (0.63 mL, 0.16 mmol) and 2-
cyanoethyl N,N,N0,N0-tetraisopropylphosphordiamidite (50 mL, 0.16 mmol) were
mixed in dry acetonitrile (0.8 mL) under nitrogen for 2 h. The crude mixture was
dissolved in ethyl acetate. The solution containing 3 was washed with water, a
saturated solution of NaHCO3, and a saturated solution of NaCl. The solution was
dried overMgSO4, andthenfiltered andthe ethylacetate was removed. The yellow
foam (0.14 g, quant.) was obtained and immediately used for DNA synthesis
without further purification.
10. Bis(trifluoromethyl)benzene moiety and Gd-DOTA labeled oligonucleotide
probe were synthesized according to Scheme 2. Oligonucleotide was
synthesized by standard cyanoethyl phosphoroamidite chemistry with an
automated DNA synthesizer (3400 DNA Synthesizer, Applied Biosystems)
using 30-PT-amino-modifier C6 CPG (Glen Research). The phosphoramidite
monomer of fluorine compound 3 was coupled with the 50 termini of the
oligonucleotide by the DNA synthesizer with a coupling time of 999 s. After the
cleavage with 28% aqueous ammonia (1 h, rt), the solution was incubated for
18 h at 55 °C and dried. The residual was solved in deionized water and
purified by a reversed-phase HPLC system (JASCO) equipped with a column
(COSMOSIL 5C18-AR-II, nacalai tesque, 4.6 ꢀ 150 mm); elution was with
Figure 3. 19F NMR signal intensity of the probe as a function of Kras Mut ODN.
[probe] = 10
lM in 50 mM Tris–HCl (pH 7.6), 10% (v/v) D2O, 10 lM TFA (for an
internal standard), 25 °C.

306
T. Sakamoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 303–306
0.05 M ammonium formate containing 2–32% CH3CN, linear gradient (30 min)
manufacturer’s procedure, and the purity was checked by a reversed-phase
HPLC. All ODN derivatives were identified by MALDI-TOF-MS analysis. 19F-
ODN-NH2; calcd 7235.25 [(M+H)+], found 7235.89, 19F-ODN-DOTA; calcd
7621.43 [(M+H)+], found 7621.79, 19F-ODN-Gd-DOTA; calcd 7776.33 [(M+H)+],
found 7776.52.
at a flow rate of 1.0 mL/min. To a solution of 50 fluorine compound and 30
amino modified oligonucleotide (19F-ODN-NH2) in 50 mM Na–Borate buffer
(pH 9.0) (400 lM, 125 lL), the solution of DOTA-NHS in DMSO (40 mM, 50 lL)
was added slowly on ice bath, and then incubated for 12 h at rt. The fluorine
and DOTA labeled ODN (19F-ODN-DOTA) was purified by gel filtration (NAP-5,
GE Healthcare) according to the manufacturer’s instructions, and the purity
was checked by a reversed-phase HPLC. Fluorine and DOTA labeled ODN
11. 19F NMR spectra were recorded on a Bruker AVANCE III 500 instrument with a
5 mm probe head (PA BBO 500S2 BBF-H-D-05 Z) at 470 MHz for 19F. The
solvent for 19F NMR measurement was 50 mM Tris–HCl buffer (pH 7.6)
(300
lM, 40 nmol) was incubated with Gd citrate (3 mM, 400 nmol) in 50 mM
containing 10 lM trifluoroacetic acid (for an internal standard, 75.6 ppm) and
Tris–HCl (pH 7.6) for 24 h at rt. The resultant fluorine and Gd-DOTA labeled
10% (v/v) D2O. Acquisition; 4096 times.
12. Tanabe, K.; Sugiura, M.; Nishimoto, S. Bioorg. Med. Chem. 2010, 18, 6690.
ODN probe (19F-ODN-Gd-DOTA) were purified by gel filtration according to the