A unique fluorescent base analogue for the expansion of the genetic alphabet

Article abstract of DOI:10.1021/ja100806c

Fluorescent nucleobase analogues are useful in a wide variety of biology and biotechnology tools as molecular probes and reporters for nucleic acids. Here we present a novel fluorescent purine analogue, 7-(2,2?-bithien-5-yl)- imidazo[4,5-b]pyridine (denot

Full text of DOI:10.1021/ja100806c

Published on Web 03/24/2010
A Unique Fluorescent Base Analogue for the Expansion of the Genetic
Alphabet
Michiko Kimoto,†,‡ Tsuneo Mitsui,‡ Shigeyuki Yokoyama,†,§ and Ichiro Hirao*,†,‡
RIKEN Systems and Structural Biology Center (SSBC) and TagCyx Biotechnologies, 1-7-22 Suehiro-cho,
Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan, and Department of Biophysics and Biochemistry, Graduate
School of Science, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received January 29, 2010; E-mail: ihirao@riken.jp
Fluorescent nucleobase analogues continue to be developed for
use in a wide variety of biology and biotechnology tools as
molecular probes and reporters for nucleic acids.1 Here we present
a novel fluorescent base analogue, 7-(2,2′-bithien-5-yl)-imidazo[4,5-
b]pyridine (denoted as Dss). Through the use of an unnatural base
pair system, the nucleoside triphosphates of Dss (dDssTP and
DssTP) can be site-specifically incorporated into DNA and RNA
by polymerases, opposite its pairing partner, pyrrole-2-carbaldehyde
(Pa),2 in DNA templates. Despite its high incorporation specificity
in replication and transcription, Dss in oligonucleotides functions
as a universal base that pairs with all four natural bases with nearly
equal thermal stabilities. This unique Dss base would thus be useful
for fluorescent base replacements at specific positions in DNA and
RNA molecules with minimal functional disruption.
We previously reported a fluorescent base analogue, 2-amino-6-
(2-thienyl)purine (s) (Figure 1A), for the expansion of the genetic
alphabet by unnatural base pair systems.3,4 The ribonucleoside triph-
osphate of s (sTP) is site-specifically incorporated into RNA opposite
its pairing partners Pa3 and imidazolin-2-one (z)4 by T7 RNA
polymerase. The fluorescent s base is useful for analyzing the local
structural conformation of RNA molecules by tracking of fluorescence
intensity changes.3 However, the s-Pa and s-z pairings in replication
are less specific than those in transcription. Therefore, we developed
two other base pairs, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and
Pa2b and Ds and 2-nitro-4-propynylpyrrole (Px),5 that exhibit high
specificity and efficiency in PCR amplification and transcription.
However, the fluorescence properties of Ds are significantly less
effective than those of s (Figure 1B).
To improve its fluorescence, we modified the Ds base by
attaching an extra thienyl group, affording the Dss nucleotides.
Oligothiophene substitutions are known to increase fluorescence
quantum yields and have been applied to oligonucleotide compo-
nents as fluorophores.6 Dss retained the same shape complemen-
tarity toward Pa as that of the Ds-Pa pair. We first synthesized
the Dss base moiety (56% in three steps) and then coupled it with
deoxyribose (45%) or ribose derivatives (23%), which were
converted to the triphosphates for replication and transcription
substrates or to the amidite for DNA chemical synthesis (see the
Supporting Information).
In comparison with s, the Dss nucleotides are strongly fluorescent
and slightly red-shifted (Figure 1B,C). The fluorescence of the Dss
deoxyribonucleoside is characterized by excitation at 370 nm and
emission at 442 nm with a quantum yield of 0.32 in ethanol. The
fluorescence of Dss in single- and double-stranded DNA fragments
was reduced slightly (see the Supporting Information), as shown
previously for s.3
Figure 1. (A) Unnatural base pair involving fluorescent Dss and its pairing
partner Pa. The Ds and fluorescent s bases also pair with Pa. (B)
Fluorescence of DssTP in comparison with that of sTP and DsTP upon
irradiation at 365 nm and (C) excitation and emission spectra of DssTP,
DsTP, and sTP. Each triphosphate (10 µM) was dissolved in 10 mM
phosphate buffer (pH 7.0).
The high selectivity of the Dss-Pa pairing in replication was
observed by single-nucleotide insertion experiments7 using the
exonuclease-deficient Klenow fragment (KF) of Escherichia coli
DNA polymerase I (Table 1). The incorporation efficiency of
dDssTP into DNA opposite Pa in the template was almost as high
as that of dATP opposite T and 18-49-fold higher than those of
Table 1. Steady-State Kinetics of Single-Nucleotide Insertion of
the Cognate Dss-Pa Pairing and Non-Cognate Pairings in
Replication by KF.a
template
base (N)
nucleoside
triphosphate
Vmax
(% min-1 b
Vmax/KM
(% min-1 M-1
)
entry
KM (µM)b
)
1
Dss
Dss
Dss
Dss
Dss
A
Pa
A
G
dPaTP
dATP
dGTP
dCTP
dTTP
150 (50)
n.d.
n.d.
n.d.
n.d.
0.7 (0.4)
1.3 (0.6)
5.0 (2.4)
3.6 (0.2)
7.5 (1.8)
6.2 (0.1)
0.8 (0.4)
5.9 (0.9)
n.d.
n.d.
n.d.
n.d.
2.8 (1.5)
8.3 (3.0)
0.7 (0.1)
0.9 (0.03)
1.0 (0.2)
2.2 (0.2)
3.3 (1.8)
3.9 × 104
2c
3c
4c
5c
6
-
-
-
-
dTTP
4.0 × 106
6.4 × 106
1.5 × 105
2.6 × 105
1.3 × 105
3.5 × 105
4.1 × 106
7
8
9
10
11
12
dDssTP
dDssTP
dDssTP
dDssTP
dDssTP
dATP
C
T
T
a See the Supporting Information for experimental details. b Standard
deviations are given in parentheses. c The reaction was too slow to
calculate the parameters (Vmax < 0.05).
† RIKEN Systems and Structural Biology Center.
‡ TagCyx Biotechnologies.
§ The University of Tokyo.
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4988 J. AM. CHEM. SOC. 2010, 132, 4988–4989
10.1021/ja100806c  2010 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Tm Values for DNA Duplexes Containing Dssa
N1-N2
Tm (°C)
N1-N2
Tm (°C)
Dss-Pa
Dss-G
Dss-T
44.8
45.9
43.9
Dss-A
Dss-C
A-T
45.3
44.9
48.6
a Determined in 100 mM NaCl, 10 mM sodium phosphate (pH 7.0),
and 0.1 mM EDTA with 5 µM DNA duplex.
that the Dss misincorporation opposite the natural bases (∼0.2%
misincorporation per base) was limited under these conditions.
Duplex formation involving Dss was thermodynamically unique.
In contrast to the enzymatic specificity of the Dss-Pa pairing, Dss
pairs with all four natural bases as well as Pa with equal thermal
stabilities (Tm ) 43.9-45.9 °C) in duplex DNAs (12-mers) (Table
2). The Tm values of the 12-mer duplexes containing these cognate
Dss-Pa and noncognate Dss-natural base pairs were 3-5 °C lower
than that containing the A-T pair (Tm ) 48.6 °C). Thus, Dss acts
as a universal base for duplex formation in addition to the specific
pairing between Dss and Pa in replication and transcription.
In summary, a strongly fluorescent base analogue, Dss, has been
developed. The triphosphates dDssTP and DssTP can be site-
specifically incorporated into DNA and RNA opposite Pa in
templates by replication and transcription. In addition, the Dss base
functions as a universal base and would be useful for fluorescent
labeling of the duplex and any region of nucleic acids by
replacement of the natural bases. As we previously demonstrated
with the fluorescent s base,3 Dss could also be applied to local
structural analyses of functional nucleic acids through detection of
fluorescence quenching by stacking with neighboring bases. Ap-
plications of the site-specific Dss labeling of DNA and RNA
molecules with long chains (>100-mer) are in progress.
Figure 2. Primer extension involving the Dss incorporation opposite Pa
by the exonuclease-proficient KF. Extension was performed at 37 °C for 5
min using the enzyme (1 unit) and 200 nM primer-template duplex.
the natural base substrates opposite Pa. In addition, misincorporation
of the natural base substrates opposite Dss rarely occurred.
Primer extension involving Dss incorporation opposite Pa was
examined by using the 3′ f 5′ exonuclease-proficient KF, a DNA
template (35-mer) containing Pa, and a 32P-labeled primer (Figure
2). Interestingly, primer extension including Dss incorporation
opposite Pa proceeded efficiently with a lower dDssTP concentra-
tion (0.5 µM dDssTP and 10 µM natural base dNTPs; Figure 2,
lane 5). However, equal amounts (10 µM) of dDssTP and the natural
dNTPs inhibited the extension after the incorporation of Dss at
position 29 (Figure 2, lane 2). Since the primer extension was
performed without dATP and dGTP, the elongation paused before
position 34C of the template and yielded the 33-mer product,
suggesting that no Dss misincorporation opposite C occurred. In
addition, since a lower dDssTP concentration (0.5 µM) relative to
those of the natural dNTPs (10 µM) is sufficient for efficient Dss
incorporation, Dss misincorporation opposite the natural bases can
be eliminated.
In transcription, Dss was also specifically incorporated into RNA
opposite Pa in DNA templates by T7 RNA polymerase. Transcrip-
tion was performed using a conventional kit (Ampliscribe T7-Flash,
Epicenter Biotechnologies) with a double-stranded DNA template
(69-mer) containing Pa in the template strand and a triphosphate
set (0.025 mM DssTP and 2 mM natural base NTPs) (Figure 3).
Total transcripts (52-mers) were detected by UV shadowing of the
gel, and the Dss incorporation within the transcripts was confirmed
by their fluorescence (excitation at 365 nm) on the gel. Very subtle
fluorescence was observed from the transcript using the natural base
template in the presence of DssTP (Figure 3, lane 3), indicating
Acknowledgment. This work was supported by Grants-in-Aid
for Scientific Research (KAKENHI 19201046 to I.H., 20710176
to M.K.) and by the Targeted Proteins Research Program and the
RIKEN Structural Genomics/Proteomics Initiative from the Ministry
of Education, Culture, Sports, Science and Technology of Japan.
Supporting Information Available: Details of Dss chemical
synthesis and physical and biochemical experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Asseline, U. Curr. Org. Chem. 2006, 10, 491. (b) Wilson, J. N.; Kool,
E. T. Org. Biomol. Chem. 2006, 4, 4265. (c) Zhao, Y.; Knee, J. L.; Baranger,
A. M. Biorg. Chem. 2008, 36, 271. (d) Krueger, A. T.; Kool, E. T. J. Am.
Chem. Soc. 2008, 130, 3989. (e) Bo¨rjesson, K.; Preus, S.; El-Sagheer, A. H.;
Brown, T.; Albinsson, B.; Wilhelmsson, L. M. J. Am. Chem. Soc. 2009,
131, 4288. (f) Xie, Y.; Dix, A. V.; Tor, Y. J. Am. Chem. Soc. 2009, 131,
17605. (g) Zhao, L.; Xia, T. Methods 2009, 49, 128.
(2) (a) Mitsui, T.; Kitamura, A.; Kimoto, M.; To, T.; Sato, A.; Hirao, I.;
Yokoyama, S. J. Am. Chem. Soc. 2003, 125, 5298. (b) Hirao, I.; Kimoto,
M.; Mitsui, T.; Fujiwara, T.; Kawai, R.; Sato, A.; Harada, Y.; Yokoyama,
S. Nat. Methods 2006, 3, 729.
(3) Kimoto, M.; Mitsui, T.; Harada, Y.; Sato, A.; Yokoyama, S.; Hirao, I. Nucleic
Acids Res. 2007, 35, 5360.
(4) Hirao, I.; Harada, Y.; Kimoto, M.; Mitsui, T.; Fujiwara, T.; Yokoyama, S.
J. Am. Chem. Soc. 2004, 126, 13298.
(5) Kimoto, M.; Kawai, R.; Mitsui, T.; Yokoyama, S.; Hirao, I. Nucleic Acids
Res. 2009, 37, e14.
(6) (a) Garcia, P.; Pernaut, J. M.; Hapiot, P.; Wintgens, V.; Valat, P.; Garmier,
F.; Delabouglise, D. J. Phys. Chem. 1993, 97, 513. (b) Stra¨ssler, C.; Davis,
N. E.; Kool, E. T. HelV. Chim. Acta 1999, 82, 2160.
(7) (a) Goodman, M. F.; Creighton, S.; Bloom, L. B.; Petruska, J. Crit. ReV.
Biochem. Mol. Biol. 1993, 28, 83. (b) Kimoto, M.; Yokoyama, S.; Hirao, I.
Biotechnol. Lett. 2004, 26, 999.
Figure 3. Dss incorporation into RNA by T7 transcription using the Pa-
containing DNA template. Transcription was performed at 37 °C for 2 h
using an Ampliscribe T7-Flash transcription kit with 200 nM DNA template.
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