Fluorescence detection of single nucleotide polymorphisms using nucleic acid probe containing tricyclic base-linked acyclonucleoside

Article abstract of DOI:10.1016/j.bmcl.2011.11.022

A reliable and simple method for detecting nucleobase mutations is very important clinically because sequence variations in human DNA cause genetic diseases and genetically influenced traits. A majority of sequence variations are attributed to single nucl

Full text of DOI:10.1016/j.bmcl.2011.11.022

Bioorganic & Medicinal Chemistry Letters 22 (2012) 253–257
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorescence detection of single nucleotide polymorphisms using nucleic
acid probe containing tricyclic base-linked acyclonucleoside
Mayumi Hattori a, Tokimitsu Ohki a, Emiko Yanase b, Yoshihito Ueno b,
⇑
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2011
Revised 24 October 2011
Accepted 7 November 2011
Available online 12 November 2011
A reliable and simple method for detecting nucleobase mutations is very important clinically because
sequence variations in human DNA cause genetic diseases and genetically influenced traits. A majority
of sequence variations are attributed to single nucleotide polymorphisms (SNPs). Here, we developed a
method for SNP detection using DNA probes that contained a fluorescent tricyclic base-linked acyclonu-
cleoside N. The type of nucleobases involved in the SNP sites in an RNA target could be determined using
four DNA probes containing N. Further, we found that the SNP in the RNA target could be detected by a
visible color. Thus, this system would provide a novel and simple method for detecting SNPs in an RNA
target.
Keywords:
DNA
SNPs
Detection
Acyclonucleoside
Tricyclic base
Ó 2011 Elsevier Ltd. All rights reserved.
The development of a reliable and simple method for detecting
single nucleotide polymorphisms (SNPs), a common source of ge-
netic variations in the human genome, is currently an important
research area because SNPs are important for identifying disease-
causing genes and for pharmacogenetic studies.1–7 Recently, we
have developed a novel and simple method for SNP detection using
a nucleic acid probe that contains a fluorescent tricyclic base-
linked acyclonucleoside Q (Fig. 1).8 The design principle of SNP
detection using the nucleoside surrogate Q is outlined in Figure
2. We designed DNAs containing Q at the 50 (50-Q-probes) or 30
sides (30-Q-probes) of discriminating bases D to produce bulges.
The fluorescence intensity of the nucleoside surrogate Q changes
depending on solvent polarity. When the Ds match the sequences
of opposite bases, Ds form base pairs with complementary bases,
causing the nucleoside surrogate Q to flip outside the DNA helix
and strengthening the fluorescence intensity of Q. On the other
hand, when the mismatched bases have sites opposite the Ds, that
is, when the Ds cannot form base pairs with the opposite bases, Q
intercalates into the DNA helix because the tricyclic base-linked
nucleoside surrogate Q is more intercalative than natural mono-
or bicyclic natural nucleobases. This weakens the Q fluorescence
intensity. We can determine the type of nucleobase involved in
the SNP site by comparing the fluorescence intensities of duplexes
composed of each probe containing Q. However, since the fluores-
cence emission maximum of Q is around 390 nm, we could not
NH
NH
2
2
N
N
N
N
N
N
HO
HO
N
N
NH
N
N
2
HO
HO
1 (N)
2 (Q)
Figure 1. Structures of tricyclic base-linked nucleoside surrogates.
D = dA, dG, dT, dC
D
Probe
3’
5’
5’
3’
Nucleoside
Surrogate
Q or N
Target Sequence
Y
Y = rA, rG, rU, rC
Matched
Base Pair
Mismatched
Base Pair
Fluoresce
Quenched
D
3’
5’
5’
3’
D
3’
5’
5’
3’
Y
Y
Intercalation
Hydrogen Bonding
Figure 2. Design principle of SNP detection using the nucleoside surrogate Q or N.
D denotes discriminating bases. Y indicates SNP sites.
⇑
Corresponding author. Tel./fax: +81 58 293 2919.
E-mail address: uenoy@gifu-u.ac.jp (Y. Ueno).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.022

254
M. Hattori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 253–257
NH
2
NH
2
CN
N
N
N
N
N
N
N
N
NH
2
a
N
b
N
N
NH
2
N
N
NH
2
O
O
O
HO
3
4
1
O
OH
H
N
H
N
N
N
N(
n-Bu)
2
N
N
N(
n
-Bu)
2
N
N
N
N
H
H
c
e
N
N
N(n-Bu)
2
N
N
N(n
-Bu)
2
DMTrO
RO
d
O
O
OH
P
N
CN
5: R = H
6: R = DMTr
7
Scheme 1. Reagents and conditions: (a) NH@C(NH2)2, EtOH, reflux, 23 h, 76%; (b) 80% CH3CO2H, 60 °C, 8 h, 97%; (c) (MeO)2CHN(n-Bu)2, DMF, 60 °C, 24 h; (d) DMTrCl,
pyridine, rt, 3 h, 23% (two steps); and (e) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, i-Pr2NEt, THF, rt, 3 h, 93%.
SNP diagnosis rather than as DNA after RT-PCR. Furthermore, in
relation to discovery of a large number of noncoding RNAs, a meth-
600
od to detect intracellular RNAs in real-time is required. Thus, in
this study, we focused on detection of SNPs on RNA strands.
The synthesis of the tricyclic base-linked nucleoside surrogate N
is shown in Scheme 1. 5-Amino-6-cyano-3-[(2,2-dimethyl-1,
3-dioxolan-4-yl)methyl]imidazo[4,5-b]pyridine (3), which was
synthesized by a method reported previously,8 was treated with
guanidine to produce an isopropylidene derivative of N in 76%
yield. Deprotection of the isopropylidene group, protection of the
amino groups with a di(n-butyl)formamidine group,9 and protec-
tion of the primary hydroxy group with a 4,40-dimethoxytrityl
(DMTr) group afforded the properly protected nucleoside surrogate
6. Phosphitylation of the secondary hydroxy group of 6 gave
phosphoramidite unit 7 in 93% yield.
Typical emission spectra of the tricyclic base-linked nucleoside
surrogate N are shown in Figure 3. The nucleoside surrogate N
showed emission maxima at 427 nm under 334-nm excitation in
H2O, which was a wavelength approximately 35 nm longer than
that of Q. The fluorescence intensity of N was dependent on solvent
polarity; that is, the fluorescence intensity of N was greater in
more polar solvents such as methanol and water than in less polar
solvents such as chloroform (Table S1).
The sequences of oligodeoxynucleotides (ODNs) containing N
are listed in Table 1. CYP2C9 is one of the predominant cytochrome
P450 (CYP) enzymes expressed constitutively in the human li-
ver.10,11 It metabolizes a variety of therapeutically important
drugs.10,11 In the present study, we chose the sequence containing
an A1075(C) mutation in the CYP2C9 gene as a model sequence.
The probe ODNs were composed of sequences complementary to
the CYP2C9 gene. The probes (50-N-probes) termed NA, NG, NC,
and NT contained N at the 50 sides of Ds, whereas the probes
(30-N-probes) termed AN, GN, CN, and TN possessed N at the 30
sides of Ds. Target sequences are abbreviated as S. All the ODNs
containing N were synthesized using a DNA synthesizer. The ob-
tained ODNs were analyzed by MALDI-TOF/MS, and the observed
molecular weights were in agreement with their structures.
The Tm values of the duplexes between the probes and the RNA
targets are listed in Table S2. When the discriminating bases and
the target bases matched, the Tm values of the duplexes were the
greatest in all sequences except for the TN probe for the SrU target.
Thus, the discriminating bases seemed to have base selectivities in
the DNA/RNA duplexes containing the tricyclic nucleoside surro-
gate N.
H2O
450
300
150
0
MeOH
CHCl3
300
400
500
Wavelength (nm)
600
Figure 3. Fluorescence emission spectra of N in various solvents. Spectra were
obtained on a spectrofluorophotometer in quartz cuvettes with a path length of
1.0 cm and a 30 lM N concentration in an appropriate solvent. Spectra were
recorded by using an excitation slit of 1.5 nm and an emission slit of 1.5 nm.
detect the SNPs in the targets by visible colors. Here, we report SNP
detection by a visible color using DNAs that contain a fluorescent
tricyclic base-linked acyclonucleoside, 6,8-diamino-3-(2,3-dihydr-
oxypropyl)imidazo[40,50:5,6]pyrido[2,3-d]pyrimidine (N).
In general, amplified DNAs are used for the SNP typing.
However, the first product of gene expression is RNA, which is a
transcription product. Therefore, it is advantageous to use RNA in
Table 1
Sequences of oligonucleotides
Abbreviation
Sequence
50-d(GAA GGT CAA NAG TAT CTC T)-30
50-d(GAA GGT CAA NGG TAT CTC T)-30
50-d(GAA GGT CAA NCG TAT CTC T)-30
50-d(GAA GGT CAA NTG TAT CTC T)-30
50-d(GAA GGT CAA ANG TAT CTC T)-30
50-d(GAA GGT CAA GNG TAT CTC T)-30
50-d(GAA GGT CAA CNG TAT CTC T)-30
50-d(GAA GGT CAA TNG TAT CTC T)-30
30-r(CUU CCA GUU aCA UAG AGA)-50
30-r(CUU CCA GUU gCA UAG AGA)-50
30-r(CUU CCA GUU cCA UAG AGA)-50
30-r(CUU CCA GUU uCA UAG AGA)-50
NA
NG
NC
NT
AN
GN
CN
TN
SrA
SrG
SrC
SrU
The underlined letters indicate discriminating bases. The small italic letters rep-
resent target bases.

M. Hattori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 253–257
255
= 420 nm
= 390 nm
λem
λem
100
75
50
25
0
100
75
50
25
0
NA-rA
NA-rC
NA-rU
NA-rG
QA- rA
QA- rC
QA- rU QA- rG
200
150
100
50
300
240
180
120
60
0
0
NC-rA NC-rC
NC-rU NC-rG
QC- rA QC- rC QC- rU QC- rG
200
150
100
50
200
150
100
50
0
0
QT- rA
QT- rC
QT- rU QT- rG
NT-rA
NT-rC
NT-rU
NT-rG
300
200
100
0
150
100
50
0
NG-rA NG-rC
NG-rU NG-rG
QG- rA QG- rC QG- rU QG- rG
Figure 4. Fluorescence intensities of duplexes. Left column: 50-Q-probe/RNA target. Right column: 50-N-probe/RNA target. Data of 50-Q-probe/RNA target were quoted from a
previous paper.8 Spectra were measured at 20 °C.
Fluorescence emission spectra of the duplexes between the
probes and the target RNAs are shown in Fig. S1. Fluorescence spec-
tra intensity of the duplexes correlated with the Tm values of the
duplexes. The fluorescence intensities were the greatest when
the discriminating bases were complementary to the target bases
except for the duplex comprising the NT probe and the SrG target.
Next, we compared the fluorescence intensities of the duplexes
containing N at 420 nm (Figs. 4 and 5, right columns) with those
containing Q at 390 nm (Figs. 4 and 5, left columns). The pattern
of the fluorescence intensities for the 50-N-probes containing the
nucleoside surrogate N was similar to that for the 50-Q-probes con-
taining Q (Fig. 4). The fluorescence intensities of the duplexes were
the greatest when the discriminating bases were complementary
to the target bases except for the QT/SrG and NT/SrG duplexes.
The fluorescence of the mismatched QT/SrG and NT/SrG duplexes
were attributed to the wobble-type base pairings between dT
and rG bases.12 The fluorescence intensities of the single-stranded
N-probes were almost equal to those of the mismatched duplexes
(data not shown).
The results for the probes containing the surrogate N or Q at the
30 side of the discriminating bases (30-N-probes and 30-Q-probes)
are shown in Figure 5. Fluorescence intensities of the duplexes
were the greatest when the discriminating bases were comple-
mentary to the target bases except for the CQ probe. No fluores-
cence was observed with the CQ probe for the target sequences.
On the other hand, when the 30-N-probes were used, fluorescence
intensities of the duplexes were the greatest when the discriminat-
ing bases were complementary to the target bases: the AN probe
for the SrU target, CN probe for the SrG target, TN probe for the
SrA target, and GN probe for the SrC target. The fluorescence inten-
sities of the duplexes increased 7–20-fold compared to those with
mismatched sequences. Thus, it was found that we could deter-
mine the type of nucleobases involved in the SNP site in the RNA
target using the 30-N-probes.
Finally, we assessed whether or not we could detect the SNP in
the RNA target by a visible color. Photographs of solutions contain-
ing the TN/SrU or TN/SrA duplexes, which were irradiated with a
handy-type UV lamp at 254, 302, or 365 nm, are shown in Figure

256
M. Hattori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 253–257
= 420 nm
= 390 nm
λem
λem
150
100
50
250
200
150
100
50
0
0
AN- rA
AN- rC
AN- rU
AN- rG
AQ- rA
AQ- rC
AQ- rU AQ- rG
150
100
50
150
100
50
0
0
CN-rA CN-rC
CN-rU CN-rG
CQ-rA CQ-rC CQ-rU CQ-rG
200
150
100
50
200
150
100
50
0
0
TQ-rA
TQ-rC TQ-rU
TQ-rG
TN-rA
TN-rC
TN-rU
TN-rG
150
100
50
300
240
180
120
60
0
0
GN-rA GN-rC
GN-rU GN-rG
GQ- rA GQ- rC GQ- rU GQ- rG
Figure 5. Fluorescence intensities of duplexes. Left column: 30-Q-probe/RNA target. Right column: 30-N-probe/RNA target. Data of 30-Q-probe/RNA target were quoted from a
previous paper.8 Spectra were measured at 20 °C.
TN-rU
TN-rA
In conclusion, we demonstrated the synthesis of DNA contain-
ing a fluorescent tricyclic base-linked acyclonucleoside N. We
examined properties of the DNA containing N as an SNP-detecting
probe. It was found that we can determine the type of nucleobases
involved in the SNP sites in an RNA target with high sensitivities
using four probes containing N. Further, it turned out that the
SNP in the RNA target could be detected by a visible color. Thus,
this system would provide a reliable and simple method for detect-
ing SNPs in an RNA target.
λex =
254 nm 302 nm 365 nm
254 nm 302 nm 365 nm
Acknowledgments
Figure 6. Photographs of solutions containing the TN/SrU or TN/SrA duplexes
irradiated with a handy-type UV lamp at 254, 302, or 365 nm. Duplex concentra-
tions: 3.0 lM in a Tm buffer.
This study was supported in part by a Grant-in-Aid from Precur-
sory Research for Embryonic Science and Technology (PRESTO) of
the Japan Science and Technology Agency (JST), a Grant-in-Aid
from the Koshiyama Research Grand and a Grant-in-Aid for
Scientific Research (C) from the Japan Society for the Promotion
of Science (JSPS) to Y.U.
6. No fluorescence was observed for the TN/SrU duplex, whereas
blue colors were observed for the TN/SrA duplex when the solu-
tions were irradiated with UV light of 254 or 302 nm.

M. Hattori et al. / Bioorg. Med. Chem. Lett. 22 (2012) 253–257
257
6. (a) Okamoto, A.; Tanaka, K.; Saito, I. J. Am. Chem. Soc. 2003, 125, 4972; (b)
Okamoto, A.; Tanaka, K.; Fukuta, T.; Saito, I. J. Am. Chem. Soc. 2003, 125, 9296;
(c) Okamoto, A.; Kanatani, K.; Saito, I. J. Am. Chem. Soc. 2004, 126, 4820; (d)
Tainaka, K.; Tanaka, K.; Ikeda, S.; Nishiza, K.; Unzai, T.; Fujiwara, Y.; Saito, I.;
Okamoto, A. J. Am. Chem. Soc. 2007, 129, 4776.
7. (a) Köhler, O.; Seitz, O. Chem. Commun. 2003, 2938; (b) Köhler, O.; Jarikote, D.
V.; Seitz, O. ChemBioChem 2005, 6, 69; (c) Jarikote, D. V.; Krebs, N.; Tannert, S.;
Röder, B.; Seitz, O. Chem. Eur. J. 2007, 13, 300; (d) Bethge, L.; Singh, I.; Seitz, O.
Org. Biomol. Chem. 2010, 8, 2439.
8. Furukawa, K.; Hattori, M.; Ohki, T.; Kitamura, Y.; Kitade, Y.; Ueno, Y. Bioorg.
Med. Chem. submitted for publication.
9. Froehler, B. C.; Matteucci, M. D. Nucleic Acids Res. 1983, 11, 8031.
10. Rettie, A. E.; Wienkers, L. C.; Gonzalez, F. J.; Trager, W. F.; Korzekwa, K. R.
Pharmacogenetics 1994, 4, 39.
Supplementary data
Supplementary data (experimental procedure, Tm values, fluo-
rescence emission spectra) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.11.022.
References and notes
1. Sachidanandam, R.; Weissman, D.; Schmidt, S. C.; Kakol, J. M.; Stein, L. D.;
Marth, G.; Sherry, S.; Mullikin, J. C.; Mortimore, B. J.; Willey, D. L., et al Nature
2001, 409, 928.
2. Venter, J. C.; Adams, M. D.; Myers, E. W.; Li, P. W.; Mural, R. J.; Sutton, G. G.;
Smith, H. O.; Yandell, M.; Evans, C. A.; Holt, R. A., et al Science 2001, 291, 1304.
3. McCarthy, J. J.; Hilfiker, R. Nat. Biotechnol. 2000, 18, 505.
4. Wang, D. G.; Fan, J.-B.; Siao, C.-J.; Berno, A.; Young, P.; Sapolsky, R.; Ghandour,
G.; Perkins, N.; Winchester, E.; Spencer, J., et al Science 1998, 280, 1077.
5. Hoheisel, J. D. Nat. Rev. Genet. 2006, 7, 200.
11. Sullivan-Klose, T. H.; Ghanayem, B. I.; Bell, D. A.; Zhang, Z.-Y.; Kaminsky, L. S.;
Shenfield, G. M.; Miners, J. O.; Birkett, D. J.; Goldstein, J. A. Pharmacogenetics
1996, 6, 341.
12. Hunter, W. N.; Kneale, G.; Brown, T.; Rabinovich, D.; Kennard, O. J. Mol. Biol.
1986, 190, 605.