New Size-Expanded Fluorescent Thymine Analogue: Synthesis, Characterization, and Application

Article abstract of DOI:10.1002/chem.201900843

Here, the synthesis, photophysical characterization, and application of a new size-expanded thymine nucleoside, ^dioxT, is described. ^dioxT has desirable qualities as a T surrogate, including excellent quantum yield (0.36) and high environmental sensitivity. When incorporated into single- and double-stranded DNA, ^dioxT showed excellent photophysical characteristics including a high quantum yield (average 0.20), and unlike BgQ, demonstrated dependence on neighboring bases without significant destabilization of the duplex. Interestingly, the matched base pair of adenine (A) and ^dioxT has the unique property that it exhibits higher fluorescence than mismatched base pairs, and ^dioxT has self-quenching effects. As one example of the possible applications of these promising features, single nucleoside polymorphism typing is demonstrated for discrimination of A by using ^dioxT. The results suggest that ^dioxT can be used for a broad range of applications in chemical biology.

Full text of DOI:10.1002/chem.201900843

A Journal of
Accepted Article
Title: New Size-expanded Fluorescent Thymine Analogue: Synthesis,
Characterization, and Application
Authors: Shingo Hiroshima, Ji Hoon Han, Hitomi Tsuno, Yusuke
Tanigaki, Soyoung Park, and Hiroshi Sugiyama
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To be cited as: Chem. Eur. J. 10.1002/chem.201900843
Link to VoR: http://dx.doi.org/10.1002/chem.201900843
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New Size-expanded Fluorescent Thymine Analogue: Synthesis,
Characterization, and Application
Shingo Hirashima†,[a] Ji Hoon Han†,[a] Hitomi Tsuno,[a] Yusuke Tanigaki,[a] Soyoung Park*,[a] and Hiroshi
Sugiyama*[a, b]
Abstract: Here we describe the synthesis, photophysical
characterization, and application of a new size-expanded thymine
nucleoside, dioxT. dioxT has desirable qualities as a T surrogate,
including excellent quantum yield (0.36) and high environmental
sensitivity. When incorporated into single- and double-stranded DNA,
dioxT showed excellent photophysical characteristics including a high
quantum yield (average 0.20), and unlike BgQ, demonstrated
dependence on neighboring bases without significant destabilization
of the duplex. Interestingly, the matched base pair of adenine (A) and
dioxT has the unique property that it exhibits higher fluorescence than
mismatched base pairs, and dioxT has self-quenching effects. As one
example of the possible applications of these promising features, we
demonstrate single nucleoside polymorphism typing for discrimination
of A using dioxT. The results suggest that dioxT can be used for a broad
range of applications in chemical biology.
mechanistic investigations of RNA catalysis and RNA structural
analysis.14–17 We have focused on exploiting the potential of
isomorphic emissive DNA analogues based on a thieno[3,4-d]-
pyrimidine core.18–22 Quinazoline structure is also one of the
useful fluorescent cores. The quinazoline-derived fluorescent
nucleobases have been developed by fusion of electron-rich ring
into electron-deficient pyrimidine and used as fluorescent
probes.23-27,54,55 Tor and coworkers devised quinazoline-based
fluorescent nucleobases with electron donating amine group at
C5 or C7 position, 5- or 7-aminoquinazoline-2,4(1H,3H)-dione,
and used for not only developing FRET system with tryptophan in
native proteins but also discriminating probe as G mismatched
pair.23,24,55 Luedtke and his coworker synthesized nucleoside
analogue based on quinazoline structure with amine group at C6
position, and it was used for various studies such as kinetics of
metallo-base pair.25-27 In this study, we turned our attention to the
1,3-benzodioxole moiety as a heterocyclic core to endow the
nucleobases with emissive properties. The 1,3-benzodioxole
moiety is a well-known building block found in many naturally
occurring compounds that demonstrate anticancer and
Introduction
Since the first report of the emissive nucleobase 2AP by Stryer in
1969, the development and application of fluorescent nucleic
acids have provided very powerful tools to researchers and
greatly advanced our understanding of nucleic acid structures,
activities, and interactions with other biomolecules.1–3 Chemical
modifications are essential to generate fluorescent nucleobases
because of the nonemissive nature of natural nucleobases.4–7
Development of fluorescent nucleobases is also important to
expand the useful artificial genetic alphabet.8–10 The design and
synthesis of fluorescent isomorphic nucleobases are most
challenging and important. Chemical modifications are required to
retain their structural resemblance to natural nucleobases and
allow native Watson–Crick base pairing.11–13 For instance, Tor
and coworkers developed isomorphic fluorescent RNA
antioxidant
activities.28–32
Moreover,
dioxoloquinazoline
derivatives have been investigated because of their biological
activities as inhibitors of botulinum neurotoxin serotype A and
substrate-competitive inhibitors of G9a (histone lysine
methyltransferase).33,34 To our knowledge, fluorescent DNA
nucleoside analogues that include a dioxoloquinazoline core have
not been reported.
Here, we synthesized a new size-expanded fluorescent T-
mimic deoxyribonucleoside, dioxT, based on a dioxoloquinazoline
core, and explored its biophysical and the photophysical
properties. dioxT showed comparable biophysical properties and
displayed notable photophysical characteristics including high
brightness (Φε) as a fluorescent thymine surrogate. This suggest
s that dioxT would be a useful fluorescent probe for chemical
biology applications such as a molecular probe for detection of
single-nucleotide polymorphisms (SNPs).
nucleosides
derived from
thieno[3,4-d]-pyrimidine and
demonstrated their usefulness in various studies including
[a]
S. Hirashima, Dr. J. H. Han, Dr. S. Park, Prof. Dr. H. Sugiyama
Department of Chemistry, Graduate School of Science
Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku
Kyoto 606-8502 (Japan)
Results and Discussion
Synthesis of dioxT nucleoside
E-mail: oleesy@kuchem.kyoto-u.ac.jp
hs@kuchem.kyoto-u.ac.jp
[b]
Prof. Dr. H. Sugiyama
The new size-expanded fluorescent thymidine analogue was
synthesized using previously reported procedures with minor
modifications (Scheme 1).33,34 Direct oxidative methyl
esterification of commercially available 6-nitropiperonal 1 using
molecular iodine in methanol produced methyl-6-nitro-1,3-
benzodioxole-5-carboxylate 2. Methyl-6-amino-1,3-benzodioxole-
Institute for Integrated Cell-Material Science (iCeMS)
Kyoto University, Yoshida-ushinomiyacho Sakyo-ku
Kyoto 606-8501 (Japan)
Both authors contributed equally to this work. The authors wish it to
be known that the first two authors should be regarded as Joint First
Authors.
[†]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201900843
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5-carboxylate 3 was obtained by reduction with Fe powder and
NH4Cl. The fluorescent base moiety 5 was synthesized from 3 by
reaction with KOCN, followed by cyclization. Quinazolinedione 5
was glycosylated with Hoffer’s chlorosugar in the presence of
SnCl4 and N,O-bis(trimethylsilyl) trifluoroacetamide, and the
desired deoxyribonucleosides 6 were obtained in 32% yield as a
mixture of α- and β-anomer. Subsequent toluoyl deprotection of
the deoxyribonucleosides 6 with sodium methoxide in methanol
produced fluorescent T-mimic deoxyribonucleosides, dioxT, at a
yield of 99%. By using the conventional method, the 5’-hydroxy
group of the nucleoside analogue was protected by a 4,4’-
dimethoxytrityl (DMTr) group, and the desired β-anomer was
isolated at a yield of 26% by silica column chromatography. The
configuration at the C-1 carbon of each anomer was confirmed by
1D and 2D (NOESY) 1H NMR spectroscopy (Figure S1-4). The
3’-hydroxy group of the DMTr protected compound 8 was
converted into the phosphoramidite, and the product was used for
the automated solid-phase synthesis of oligonucleotides (details
in the supporting information).
energy absorbance of dioxT was slightly blue-shifted to 323 nm
and 322 nm, respectively. The emission intensity of dioxT, however,
dramatically decreased and the emission maxima were
significantly shifted toward shorter wavelengths of 377 nm and
361 nm, respectively. Furthermore, the quantum yield of dioxT
decreased to 0.21 and 0.09, respectively. The corresponding data
showed that the lifetime of dioxT also tended to decrease in
methanol (2.2 ns) and in dioxane (1.0 ns). The Stokes shift of diox
was reduced to 4435 cm–1 and 3403 cm–1, respectively.
T
Table 1. Photophysical characteristic of dioxT nucleosides
λabs / nm
(ε / 103 M-1cm-1)
λem / nm
(Φ)
Φε /
M-1cm-1
τ /
ns
Stoke Shift /
Solvent
cm-1
325
(10.90)
323
(7.23)
322
388
(0.36)
377
(0.21)
361
Water
MeOH
3602
1519
624
3.5
2.2
1.0
4996
4435
3403
Dioxane
(6.93)
(0.09)
Fe, NH4Cl,
CHO
NO2
CO2Me
NO2
CO2Me
NH2
EtOH/H2O, 105 o
C
O
O
O
O
O
O
I2, KOH, MeOH
99%
These results suggest that the photophysical properties of dioxT
are affected by solvent polarity, indicating that there is a charge
transfer in the excited state of dioxT compared with its ground state.
56%
1
2
3
TolO
O
Cl
OTol
CO2Me
NH
NH2
O
O
Matched/mismatched base pairing of dioxT nucleobase inside
DNA
BSTFA, SnCl4
,
KOCN, AcOH (aq)
RT
NaOMe, MeOH
RT
MeCN, 0 oC to RT
O
NH
O
73%
73%
O
N
32%
O
O
H
4
5
O
N
To evaluate the biophysical properties of oligonucleotides
containing dioxT as a dT analogue, we used automated solid-
phase synthesis to produce the 18-mer DNA oligonucleotide 5’-
O
O
N
O
O
NH
NH
O
NH
DMTrCl, Pyridine
RT
NaOMe, MeOH
RT
O
O
N
O
O
DMTrO
TolO
O
O
HO
O
O
OTol
6
X =
dioxT. We also
O
CGTCCGTCXTACGCACGC-3’, where
99%
26%
OH
prepared complementary strands containing bases matched or
mismatched with dioxT and the corresponding natural DNA
duplexes containing T. As expected, in thermal denaturation
experiments the matched duplex (dioxT with A) showed higher
OH
7
8
O
N
O
O
NH
i-Pr2NEt, CH2Cl2
RT
O
o
DMTrO
thermal stability (Tm = 67.2 C) than did mismatched duplexes
O
containing T, G, and C, indicating that dioxT can form a Watson–
Crick base pair with A (Figure 1A and 1B). In addition, circular
dichroic (CD) spectra of oligomers containing dioxT matched with
A showed the shape of typical B-form DNA (Figure S8). Therefore,
this thermal stability, base pairing selectivity, and CD
spectroscopy suggest that dioxT can replace native T base without
significantly perturbing the DNA structure. Interestingly, as
compared with natural base T, dioxT shows the higher thermal
stability upon mismatched base pairing including abasic (AP) site.
Duplex containing dioxT and opposite AP site displays significantly
O
O
P
N
CN
iPr
iPr
9
Scheme 1. Synthesis of dioxT. Reagents and conditions: (a) I2, KOH, MeOH,
0 °C, 99%; (b) Fe, NH4Cl, EtOH/H2O, 105 °C, 56 %; (c) KOCN, AcOH (aq.), RT,
73%; (d) NaOMe, MeOH, RT, 68%; (e) 2-Deoxy-3,5-di-O-p-toluoyl-α-D-
ribofuranosyl chloride, N,O-Bis(trimethylsilyl)acetamide, SnCl4, MeCN, 0 °C to
RT, 32%; (f) NaOMe, MeOH, RT, 99%; (g) DMTrCl, pyridine, RT, 26%; (h) (i-
Pr)2NEt, DCM, RT.
o
higher melting temperature than that of natural T (ΔTm = 5 C).
Mismatched base pair with C or T show the slightly increased
thermal stability around 2 oC. This high Tm of dioxT could be
attributed to the increase stacking interactions by expanded
aromatic structure of dioxT in comparison to that of natural T base.
In contrast, thermal stability of dioxT mismatched pairing with G is
similar to the result from natural T probably due to G:T wobble
base pairing (Figure S16). To confirm the fluorescence changes
for matched and mismatched oligomers, we measured the
steady-state fluorescence. Interestingly, the fluorescence spectra
revealed that dioxT matched with A had higher fluorescence
intensity than did dioxT mismatched with G, C, T, and dS (Figure
1C). The fluorescence intensity of dioxT located at an AP site was
similar to that of a mismatched base pair and lower than that of a
matched base pair, suggesting that, consistent with its Tm, dioxT is
strongly bound in the duplex by π–π stacking interactions.
Furthermore, the lower fluorescence produced by dioxT with G and
Photophysical properties of dioxT nucleoside
The basic photophysical properties of dioxT mononucleoside were
examined in water, methanol, and dioxane (Table 1). In water,
dioxT showed absorption at 325 nm and fluorescence at 388 nm
with high quantum yield (0.36), resulting in very bright
fluorescence (Φε = 3602 M-1cm-1). This brightness value is higher
than previous reported fluorescent thymine/uridine analogues
such as DMAT (Φε = 87 M-1cm-1), 1PydU and 2PydU (Φε ≈ 500 M-
1cm-1 in MeOH), bT (Φε = 790 M-1cm-1), xT (Φε = 1020 M-1cm-1 in
MeOH), thdT (Φε = 1984 M-1cm-1) and TPAU (Φε = 2200 M-1cm-
1).27,52,53 The absorption and emission spectra of dioxT change
depending on the solvent. In methanol and dioxane, the lowest
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10.1002/chem.201900843
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duplexes were hybridized with matched complementary strand. All samples
contained 2.5 μM of each oligonucleotide strand, 20 mM Na cacodylate (pH 7.0)
and 100 mM NaCl. Tm experiments were performed in triplicate.
C as counter bases compared with dS (AP site) may be caused
by fluorescence quenching from G, as discussed below.
of strong stacking interactions between neighboring bases, even
though the Tm is a little lower than that of the native duplex
because of electronic repulsion between the phosphate group
and dioxole ring of dioxT (Figure S10). Duplexes with AT as the
neighboring nucleobases showed the highest thermal stability,
whereas those with GG showed the lowest thermal stability. As a
result, duplexes containing dioxT demonstrated better thermal
stability than the previously reported ring-fused fluorescent
nucleobase BgQ.35
It is generally known that fluorescent nucleobases are strongly
influenced by their nearest-neighbor bases.36–39 To select suitable
sequences for the application, the characteristics of dioxT-
containing
single-
and
double-stranded
DNA
(5’-
CGTCCGTNXNACGCACGC-3’, where X = dioxT, Table 3 and S1)
were investigated. The average wavelengths of the absorption
maxima of single- and double-stranded DNA containing dioxT were
325 nm and 327 nm, respectively. Such a red shift of the
absorption maximum is typically caused by stacking interactions
resulting from the formation of a duplex structure. Consistent with
its geometrical characteristics, the emission maximum of dioxT
within double-stranded DNA was slightly blue-shifted compared
with that within single-stranded DNA (388 nm and 379 nm,
respectively), suggesting that dioxT located within double-stranded
DNA is less polar than that in single-stranded DNA.49 Despite this,
the quantum yields for both single- and double-stranded DNA
decreased significantly when G was located next to dioxT. This
quenching effect can be explained by the electron transfer from
G. We calculated the order of the highest-occupied molecular
orbital (HOMO) energy of each nucleobase as G > dioxT > A > C
> T, which supports the concept that quenching was the result of
electron transfer from G (Figure S11). In single-stranded DNA,
combinations of dioxT with A, T, and C, but not G, as neighboring
bases showed good quantum yields (0.09–0.23). In double-
stranded DNA, all combinations of neighboring base sets
containing C (in the 5’ position), i.e., CA, CT, CG, and CC, showed
higher quenching compared with single-stranded DNA. Both
stronger stacking interactions upon formation of double-stranded
DNA and electron transfer from neighboring Gs paired with C may
have contributed to this quenching phenomenon. Interestingly,
neighboring base sets containing A and T showed an average 1.5
times higher quantum yield upon hybridization of double-stranded
DNA compared with that in single-stranded DNA. The comparable
thermal stability, hypochromic effect, and red shift of dioxT in
duplex DNA indicate that dioxT is well stacked between base pairs
and does not flip to the outside of the duplex. Furthermore, the
overall brightness of dioxT in ds DNA shows average 622 M-1cm-1
(Table S2), and this value is significantly higher than that of bT
(average Φε = 28 M-1cm-1 in duplex) and thdT (Φε = 90 M-1cm-1 in
ds DNA, 5’-GCGCGAthdTAthdTAthdTAGGAGC-3’),20,52 but less
than fluorescent adenine and guanine analogues such as tCo (Φε
= 2000 M-1cm-1) and 6MI (Φε = 1700 M-1cm-1).53
Figure 1. Matched/mismatched base pairs with dioxT. (A) Histogram for the
comparison of Tm values. (B) dioxT with Watson–Crick base pairing with A. (C)
Absorbance (dashed lines) and fluorescence properties (solid lines) of ODN1
hybridized with complementary strands containing matched or mismatched
bases. Fluorescence spectra were measured at an excitation of 328 nm. All
samples contained 2.5 μM of each oligonucleotide strand, 20 mM Na cacodylate
(pH 7.0) and 100 mM NaCl. dS indicates 5’-O-dimethoxytrityl-1’,2’-
dideoxyribose-3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
dioxT nucleobase affected by neighboring bases inside DNA
In general, the stability of a duplex containing an artificial
nucleobase is strongly affected by its neighboring bases. To
investigate the effect of dioxT incorporated into double-stranded
DNA, we measured the Tm for dioxT associated with all possible
neighboring bases (Table 2). The modified duplex containing diox
T
had a slightly lower Tm (average 1.9 oC) than the unmodified
duplex. Interestingly, sequences in which dioxT was flanked by two
purine bases (PuPu), a pyrimidine and a purine base (PyPu), a
purine and a pyrimidine base (PuPy), and two pyrimidine bases
(PyPy) all showed similar thermal stability (average 1.4‒2.5 oC).
This may be attributable to the good integration of dioxT because
Table 2. Thermal stability of dioxT effected by neighboring bases
Type of
Sequence
name b
Tmc (oC)
Tmd (oC)
ΔTm (oC)
nucleobases a
AA
AG
GA
GG
TT
TC
CT
CC
AT
AC
GT
GC
TA
TG
CA
CG
65.3 ± 0.2
67.4 ± 0.4
67.9 ± 0.3
69.8 ± 0.5
65.7 ± 0.3
69.7 ± 0.1
67.7 ± 0.2
70.6 ± 0.5
65.4 ± 0.4
68.8 ± 0.6
68.5 ± 0.1
71.7 ± 0.1
66.1 ± 0.4
69.4 ± 0.2
67.9 ± 0.2
71.1 ± 0.1
67.1 ± 0.03
70.0 ± 0.1
70.3 ± 0.1
73.2 ± 0.1
68.5 ± 0.2
71.1 ± 0.1
69.2 ± 0.1
73.3 ± 0.2
66.2 ± 0.2
70.4 ± 0.1
70.3 ±0.1
73.3 ± 0.1
68.3 ± 0.2
71.4 ± 0.2
70.2 ± 0.1
72.9 ± 0.2
-1.7 ± 0.1
-2.5 ± 0.2
-2.4 ± 0.2
-3.7 ± 0.3
-2.6 ± 0.2
-1.4 ± 0.1
-1.5 ± 0.1
-2.6 ± 0.3
-0.9 ± 0.2
-1.4 ± 0.3
-1.7 ± 0.1
-1.5 ± 0.1
-1.9 ± 0.2
-1.9 ± 0.1
-2.2 ± 0.1
-1.7 ± 0.1
PuPu
PyPy
PuPy
PyPu
To determine the emissive properties of dioxT, we analyzed the
HOMO and the lowest-unoccupied molecular orbital (LUMO)
(Figure S9). The charge density of the HOMO of dioxT showed that
the orbitals populated were those of two oxygens and the CH2
group of the dioxole ring as well as those of the carbon atoms in
the benzene ring. LUMO analysis showed that the orbitals were
located in the carbon atoms of the benzene ring and the ketone
group at the C4 position of the thymine scaffold, with a minor
population on the methoxy group of the dioxole ring. This
suggests that substantial charge transfer could occur from the
dioxole ring to the benzene ring fused with the thymine moiety.
aType of nucleobase indicates purine (Pu) and pyrimidine (Py) bases. bNN
denotes sequences named for the neighboring bases surrounding dioxT.
cModified sequence containing dioxT. dUnmodified sequence containing T. All
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Table 3. Photophysical properties of single and double stranded DNA containing dioxTa
Sequence
ssDNA
name
dsDNA
c
c
NN b
λAbs (nm)
λEm (nm)
Φf
〈 τ 〉(ns) d
λAbs (nm)
λEm (nm)
Φf
〈 τ 〉(ns) d
AA
AC
AG
AT
CA
CC
CG
CT
GA
GC
GG
GT
TA
TC
TG
TT
322
323
323
330
322
325
327
327
321
324
325
326
325
328
322
326
388
392
388
387
388
386
389
384
393
390
385
390
387
385
389
385
0.15
0.23
0.02
0.12
0.12
0.13
0.01
0.11
0.01
0.02
< 0.01
0.01
0.11
0.13
0.01
0.09
3.43
3.50
2.01
3.15
2.31
2.30
1.83
2.12
2.37
2.76
3.20
2.34
2.04
2.18
1.58
1.88
327
325
328
325
327
327
326
327
327
330
324
326
330
328
330
328
377
377
379
377
380
377
377
375
387
381
381
383
378
375
383
374
0.20
0.13
0.01
0.19
0.04
0.05
0.01
0.03
0.01
< 0.01
0.05
< 0.01
0.19
0.12
0.01
0.17
3.11
2.26
0.97
3.13
1.01
1.57
0.40
1.13
2.25
1.95
3.18
1.95
2.48
1.31
0.61
2.36
a Measurements were performed at room temperature. All duplexes were hybridized with matched complementary strand. All samples contained 20 mM Na
cacodylate (pH 7.0) and 100 mM NaCl. b NN denotes the sequences named for the neighboring bases surrounding dioxT. c,d For the measurement of lifetime and
quantum yield, all samples contained 5 μM of each oligonucleotide strand. Quantum yield measurements were performed in duplicate and the average value is
shown.
In previous reports, some fluorescent nucleobases such as DMAT,
2PyG, 4AP, and 8-DEA-tC showed enhanced fluorescence
depending on their location in double-stranded DNA, a unique
phenomenon that could be explained by a charge-separated
excited state.40–42 Therefore, the intramolecular charge transfer of
dioxT might contribute to the enhancement of quantum yield upon
the formation of double-stranded DNA. Both this enhancement
and the high quantum yield of dioxT-containing DNA duplexes will
allow the development of many useful tools.
electron donating groups on quinazoline ring. The present result
is very similar with DMAT,27 in which amino group attached at C6
position, while quinazoline-based nucleobase with amino group at
C7 position doesn’t show enhanced fluorescence upon
hybridizing with A.24 Although several fluorescent nucleobases
have been exploited for SNP typing,46-48 there are very few
examples of the use of fluorescent isomorphic T analogues. This
result suggests that dioxT can be utilized as a SNP typing detector
for A. 24-27
To utilize the advantages of the photophysical properties of
enhanced quantum yield upon conversion from single-stranded
DNA to double-stranded DNA and the strong fluorescence of
Watson–Crick base paring with A, we evaluated the use of dioxT
for SNP typing. This technique has received attention because
SNPs can act as biological markers of disease-causing genes and
predict individual responses to certain drugs.43–45 To confirm the
ability to discriminate SNPs using dioxT, we measured steady-
state fluorescence spectra and visualized samples by
photographing them under UV (302 nm) irradiation (Figure 2A).
We selected sequences containing dioxT flanked by AT and TA
from combinations of neighboring bases including A and T (AA,
AT, TA, and TT). In both instances, fluorescence of dioxT was
enhanced by approximately twofold upon hybridization with A
compared with that in single-stranded DNA (Figure 2B and 2C).
Furthermore, as we expected, a DNA duplex containing dioxT had
higher fluorescence intensity than did other duplexes in which
dioxT was mismatched with T, C, or G, while dioxT mismatched with
G as a counter base showed the lowest fluorescence intensity.
The photographs of samples under UV irradiation confirmed the
ability to discriminate A using changes in dioxT fluorescence
(Figure 2D and 2E). Interestingly, different base discriminating
ability was observed depending on the incorporation position of
Multiple dioxT nucleobases inside DNA
To investigate dioxT further, we also characterized the
photophysical behaviors of DNA oligonucleotides containing
multiple dioxTs (Figure 3A). We prepared DNA oligomers
containing three adjoining or alternating dioxTs in the center
positions, and measured the absorbance and fluorescence
spectra of single- and double-stranded DNA (Figure 3B and 3C).
Compared with the absorbance of duplex ODN1:ODN1’, duplex
ODN2:ODN2’ showed a blue-shifted band. This might be caused
by the degree of alignment of the transition dipole moment of
adjoining dioxTs.50,51 For the single-stranded DNAs, ODN1 showed
approximately 2.3-fold lower fluorescence intensity that did ODN2.
After hybridization with their complementary strands, the increase
in fluorescence intensity of duplex ODN1:ODN1’ was about 1.5-
fold lower than that of ODN2:2’, suggesting that multiple dioxTs
might interact and reduce fluorescence intensity by self-
quenching. This hypothesis is supported by the observation that
three adjoining dioxTs had similar intensity to a single dioxT (Figure
S12). As indicated in Table 3, it is not surprising that duplex
formation by both oligonucleotides resulted in enhanced
fluorescence because of the unique properties of dioxT. Upon
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located at adjoining and alternating positions. The excitation wavelength is 328
nm. All samples contained 2.5 μM of each oligomer, 20 mM Na cacodylate (pH
7.0) and 100 mM NaCl. ss- and ds- indicate single- and double-stranded DNA,
respectively. “con (XXX)” denotes the oligomer containing three adjoining dioxTs,
ODN1, and “sep (XAXAX)” denotes the oligomer containing three alternating
dioxTs, ODN2.
hybridization
of
single-stranded
to
double-stranded
DNA, the fluorescence of ODN1:1 was enhanced fourfold,
whereas that of ODN2:2’ increased 2.5 times and the
fluorescence of DNA containing three adjoining dioxTs increased
significantly more than that of DNA containing a single dioxT.
These results indicate that three adjoining dioxTs may be spatially
separated by a dioxT–dioxT interaction resulting from hydrogen
bonding with the counter base upon duplex formation. In addition,
DNA duplexes containing three adjoining dioxTs had higher
thermal stability by around 3.5 oC compared with those of
duplexes containing three alternating dioxTs (data not shown).
This higher stability is attributable to the strong stacking
interactions of dioxT itself. This property may be useful to the
development of versatile tools to probe conformational changes
in nucleic acids and of molecular beacons based on the self-
quenching characteristics of one fluorophore.
Conclusions
In conclusion, we have synthesized a new size-expanded
fluorescent T-mimic deoxynucleoside, dioxT, and demonstrated its
biophysical and photophysical properties as a T surrogate. dioxT
showed an excellent quantum yield (0.36) in water and its
fluorescence intensity was changeable depending on
environmental factors such as solvent polarity. When dioxT was
incorporated into DNA, it was noteworthy that the fluorescence of
the Watson–Crick pairing of dioxT and A was higher than that of
mismatched pairs. Furthermore, dioxT flanked by combinations of
neighboring bases such as AA, AT, TA, and TT showed enhanced
fluorescence with a significant quantum yield upon duplex
formation. These promising features suggest that dioxT may be a
useful tool for applications in chemical biology such as molecular
beacon assays, monitoring of structural changes in nucleic acids
and molecular imaging of living cells. We demonstrated one
example of the possible applications, visual discrimination of
SNPs for typing, by successfully distinguishing samples
containing a dioxT–A matched pair using the naked eye. In addition,
we identified a significant self-quenching effect with DNAs
containing multiple dioxTs. Investigation of further applications of
dioxT is underway in our laboratory.
Figure 2. Application of dioxT for single nucleotide polymorphism (SNP) typing.
(A) Schematic illustration of the SNP typing method using oligomers containing
dioxT (B) Fluorescence spectra of single- and double-stranded DNA containing
dioxT flanked by bases TA at an excitation of 328 nm. (C) Fluorescence spectra
of single- and double-stranded DNA containing dioxT flanked by bases AT at an
excitation of 330 nm. (D) and (E) Fluorescence images for sequences with the
bases opposite dioxT flanked by TA and AT under UV (302 nm) irradiation. ss
denotes single-stranded DNA. All samples contained 10 μM of each oligomer,
20 mM Na cacodylate (pH 7.0), and 100 mM NaCl.
Experimental Section
Materials used for synthesis of dioxdT
2-Cyanoethyl N,N-diisopropylchloro phosphoramidite and dimethoxytrityl
chloride were received from Wako Chemicals and used without further
purification. N,O-bis(trimethylsilyl)trifluoroacetamide was received from
TCI. All other chemicals and solvents were purchased from Sigma-Aldrich
Chemicals Co., Wako Pure Chemical Ind. Ltd., TCI, and used without
further purification. 2-Deoxy-3,5-di-O-p-toluoyl-α-D-ribofuranosyl chloride
was prepared by following the literature procedure.56 Synthetic
oligonucleotides were obtained from Sigma Genosys. Water was
Figure 3. Self-quenching analysis of dioxT. (A) Oligonucleotide sequence
containing dioxT. X denotes dioxT (B) Absorbance of single- and double-stranded
DNA containing dioxTs located at adjoining and alternating positions. (C)
Fluorescence of single- and double-stranded DNA containing three dioxTs
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10.1002/chem.201900843
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deionized (specific resistance of > 18.0 MΩ cm at 25 °C) by a Milli-Q
Calculation of energy level of dioxT and four natural bases
system (Millipore Corp.)
Calculations were carried out under B3LYP density functional method and
6-31G* bases set. HOMO energy of each nucleobase calculated by DFT
(B3LYP/6-31G*) at the optimized geometry by Spartan ’16 program. A =
9-methyladenine, T = 1-methylthymine, G = 9-methylguanine, C = 1-
methylcytosine.
Absorption and UV-melting measurement
Melting temperatures were determined by measuring changes in
absorbance at 260 nm as a function of temperature using a JASCO V-650
UV/VIS spectrophotometer. JASCO PAC-743R equipped with a high
performance temperature controller and micro auto eight-cell holder.
Absorbance was recorded in the forward and reverse direction at
temperatures from 5 to 95 °C at a rate of 1 °C/min. The melting samples
were denatured at 95 °C for 3 min and annealed slowly to RT then stored
at 5 °C until experiments were initiated. All melting samples were prepared
in a total volume of 110 μl containing 2.5 μM of each strand oligonucleotide,
20 mM Na cacodylate (pH 7.0) and 100 mM NaCl. Synthetic oligo
nucleotides were obtained from Sigma-Aldrich Chemicals Co.
Acknowledgements
The authors express sincere thanks for a Grant-in-Aid Priority
Research from Japan Society for the Promotion of Science
(JSPS). We also thank KAKENHI program (Grant-in-Aid for
scientific research C) and Toyota Riken Scholar program for
support to S. P. We like to thank Karin Nishimura (Graduate
School of Engineering, Kyoto University) for technical assistance
to measure mass spectra of synthetic compounds.
Steady state fluorescence measurement
Fluorescence measurements were conducted using fluorescence cells
with a 0.5-cm path length on a JASCO FP-6300 Spectrofluorometer
equipped with a JASCO EHC-573 temperature controller. The emission
spectra were recorded from 350 nm to 600 nm with an excitation
wavelength at 325 nm.
Keywords: DNA, Fluorescent nucleoside, Thymine, SNP typing
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We synthesize
a
new fluorescent
S. Hirashima, J. H. Han, H. Tsuno, Y.
Tanigaki, S. Park*,H. Sugiyama*
thymine analogue, dioxT, and explored
its photophysical characterization and
application.
Page No. – Page No.
New Size-expanded Fluorescent
Thymine Analogue: Synthesis,
Characterization, and Application
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