Click chemistry to construct fluorescent oligonucleotides for DNA sequencing

Article abstract of DOI:10.1021/jo026615r

"Click chemistry" 1,3-dipolar cycloaddition between alkynyl 6-carboxyfluorescein (FAM) and azido-labeled single-stranded (ss) DNA was carried out under aqueous conditions to produce FAM-labeled ssDNA in quantitative yield. The FAM-labeled ssDNA was succes

Full text of DOI:10.1021/jo026615r

Click Ch em istr y to Con str u ct F lu or escen t
Oligon u cleotid es for DNA Sequ en cin g
couple with the oligonucleotide in the solid-phase syn-
thesis. However, if the functional group is labile to the
9
basic deprotection conditions used in the solid-phase
DNA synthesis, the direct phosphoramidite approach
cannot be used. Thus, there is still a need to develop
coupling chemistry with high stability and high yield to
modify DNA and other biomolecules. To this end, chemo-
selective modification of proteins and cell surfaces by the
Staudinger ligation was developed.10 Diels-Alder reac-
tion was also explored for the selective immobilization
of proteins.11
Tae Seok Seo, Zengmin Li, Hameer Ruparel, and
J ingyue J u*
Columbia Genome Center, Columbia University College of
Physicians and Surgeons, New York, New York 10032, and
Department of Chemical Engineering, Columbia University,
New York, New York 10027
dj222@columbia.edu
Ideal coupling functional groups (one on the DNA and
the other on the molecule to be coupled) should be stable
under aqueous reaction conditions. The coupling reaction
should be highly chemoselective with a high yield, and
the resulting linkage should be stable under biological
conditions. Recently, Sharpless et al. defined “click
chemistry” as a set of powerful, highly reliable, and
selective reactions for the rapid synthesis of useful new
compounds and combinatorial libraries through hetero-
atom links.12 One of the click chemistry reactions involves
coupling between azides and alkynes to form the triazole
Received October 28, 2002
Abstr a ct: “Click chemistry” 1,3-dipolar cycloaddition be-
tween alkynyl 6-carboxyfluorescein (FAM) and azido-labeled
single-stranded (ss) DNA was carried out under aqueous
conditions to produce FAM-labeled ssDNA in quantitative
yield. The FAM-labeled ssDNA was successfully used as a
primer to produce DNA sequencing products with single-
base resolution in a capillary electrophoresis DNA sequencer
with laser-induced fluorescence detection.
1
3
version of Huisgen’s [2 + 3] cycloaddition family. Mock
et al.14 discovered that cucurbituril could catalyze this
1,3-dipolar cycloaddition. This coupling chemistry was
also used to form oligotriazoles and rotaxanes by Steinke
et al.15 The addition results in regioisomeric five-
Synthetic oligonucleotides are the most important
molecular tools for genomic research and biotechnology.
1
Modified oligonucleotides are widely used as primers for
2
3
DNA sequencing and polymerase chain reaction, anti-
1
6
4
membered heterocycles. This 1,3-dipolar cycloaddition
chemistry is very chemoselective, only occurring between
alkynyl and azido functional groups with high yield. In
addition, the resulting 1,2,3-triazoles are stable at aque-
ous conditions and high temperature.
sense agents for therapeutic applications, molecular
beacons for detecting genetic mutations, and probes for
measuring gene expression in DNA microarrays and gene
chips. The modification of either the 3′- and 5′-termini
5
6
or an internal position of the oligonucleotides with a
We recently explored the use of click chemistry 1,3-
dipolar cycloaddition reaction to couple a fluorophore to
DNA. We report here the synthesis of fluorescent single-
stranded DNA (ssDNA) using the click chemistry and the
application of the fluorescent ssDNA as a primer in the
Sanger dideoxy chain termination reaction17 to produce
DNA sequencing fragments. We synthesized an oligo-
nucleotide labeled by an azido group at the 5′-end as
shown in Scheme 1. 5-Azidovaleric acid was synthesized
primary alkylamine group is a widely used method for
introducing additional functional groups to the DNA.7
Introduction of these functionalities into DNA can be
achieved through the use of appropriate phosphoramidite
reagents in solid-phase synthesis. Once a unique func-
tional group is incorporated into the DNA, the functional
group can be subsequently conjugated to the desired
molecule by a selective chemical reaction. The succinim-
idyl ester of a fluorescent dye is widely used to couple
with a primary amine group introduced into an oligo-
nucleotide.8 However, the coupling reaction requires
(9) (a) Adamczyk, M.; Chan, C. M.; Fino, J . R.; Mattingly, P. G. J .
Org. Chem. 2000, 65, 596. (b) Lyttle, M. H.; Walton, T. A.; Dick, D. J .;
Carter, T. G.; Beckman, J . H.; Cook, R. M. Bioconjugate Chem. 2002,
13, 1146. (c) Theisen, P.; McCollum, C.; Upadhya, K.; J acobson, K.;
Vu, H.; Andrus, A. Tetrahedron Lett. 1992, 33, 5033.
aqueous conditions that can hydrolyze the succinimidyl
ester moiety. To overcome this difficulty, phosphoramid-
ite derivatives of fluorescent dyes were used to directly
(10) (a) Saxon, K. E.; Bertozzi, C. R. Science 2000, 287, 2007. (b)
Kristi, L.; Saxon, K. E.; Tirrell, D. A.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 19.
(11) Yousaf, M. N.; Mrksich, M. J . Am. Chem. Soc. 1999, 121, 4286.
(12) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2005.
(13) (a) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier,
P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 1053. (b) Huisgen, R. Pure Appl. Chem. 1989, 61, 613.
(14) (a) Mock, W. L.; Irra, T. A.; Wepsiec, J . P.; Manimaran, T. L.
J . Org. Chem. 1983, 48, 3619. (b) Mock, W. L.; Irra, T. A.; Wepsiec, J .
P.; Adhya, M. J . Org. Chem. 1989, 54, 5302. (c) Mock, W. L. Top. Curr.
Chem. 1995, 175, 1.
(15) (a) Krasia, T. C.; Steinke, J . H. G. Chem. Commun. 2002, 22.
(b) Tuncel, D.; Steinke, J . H. G. Chem. Commun. 2002, 496. (c) Tuncel,
D.; Steinke, J . H. G. Chem. Commun. 2001, 253.
(16) Palacios, F.; Retana, A. M.; Ragalday, J . Heterocycles 1994, 38,
95.
*
To whom correspondence should be addressed.
(
(
1) Caruthers, M. H. Science 1985, 230, 281.
2) (a) Smith, L. M.; Sanders, J . Z.; Kaiser, R. J .; Hughes, P.; Dodd,
C.; Connell, C. R.; Heiner, C.; Kent, S. B. H.; Hood, L. E. Nature 1986,
21, 674. (b) J u, J .; Ruan, C.; Fuller, C. W.; Glazer, A. N.; Mathies, R.
A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4347.
3
(3) Mullis, K. B.; Faloona, F. A. Methods Enzymol. 1987, 155, 335.
(4) Verma, S.; Eckstein, F. Annu. Rev. Biochem. 1998, 67, 99.
(5) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303.
(6) (a) Fodor, S. P.; Read, J . L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.;
Solas, D. Science 1991, 251, 767. (b) Schena, M.; Shalon, D.; Davis, R.
W.; Brown, P. O. Science 1995, 270, 467.
(7) (a) Smith, L. M.; Fung, S.; Hunkapiller, M. W.; Hunkapiller, T.
J .; Hood, L. E. Nucleic Acids Res. 1985, 13, 2399. (b) Kahl, J . D.;
Greenberg, M. M. J . Am. Chem. Soc. 1999, 121, 597. (c) Dey, S.;
Sheppard, T. L. Org. Lett. 2001, 3, 3983.
(
8) Chehab, F. F.; Kan, Y. W. Proc. Natl. Acad. Sci. U.S.A. 1989,
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U.S.A. 1977, 74, 5463.
8
6, 9178.
1
0.1021/jo026615r CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/21/2002
J . Org. Chem. 2003, 68, 609-612
609

SCHEME 1 a
a
Reagents and conditions: (i) NaN3, DMSO; (ii) NaOH, MeOH,
H2O; (iii) HCl; (iv) N-hydroxysuccinimide, EDC, CH2Cl2; (v)
Na2CO3/NaHCO3 buffer (pH 9.0), DMSO.
F IGURE 2. MALDI-TOF MS spectrum of 4 and 5.
SCHEME 2
F IGURE 1. MALDI-TOF mass spectrum of 2.
according to the literature18 and activated as N-succin-
imidyl ester 1 (87%). The oligonucleotide 5′-amino-GTT
TTC CCA GTC ACG ACG-3′ (M13-40 universal forward
sequencing primer) was reacted with excess succinimidyl
5
-azidovalerate 1 to produce the azido-labeled DNA 2.
After size-exclusion chromatography to remove excess
starting material 1 and desalting with an oligonucleotide
purification cartridge, the product was analyzed with
matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS). Figure 1 shows
the MALDI-TOF MS spectrum of the isolated product,
with a single major peak at 5757 Da that matched very
well with the calculated value of 5758 Da for the azido-
DNA 2. This indicates that the starting material amino-
DNA was quantitatively converted to the azido-DNA 2
TOF MS spectra. Characteristic peaks with maxima of
500 nm (FAM) and 260 nm (DNA) were obtained by UV-
vis measurement. The MALDI-TOF MS spectrum of 4
and 5 is shown in Figure 2. The mass peak of the azido-
labeled DNA (5758 Da) almost completely disappeared,
and a single major peak at 6170 Da corresponding to the
cycloaddition reaction product (4 and 5, theoretical mass
value of 6169 Da) was obtained with an isolated yield of
91%.
To demonstrate the utility of FAM-labeled oligonucle-
otides 4 and 5 constructed by the click chemistry for DNA
analysis, we used them in the Sanger dideoxy chain
termination method to produce DNA sequencing frag-
ments terminated by biotinylated dideoxyadenine triph-
osphate (ddATP-Biotin) using PCR-amplified DNA as
a template. Solid-phase capture using streptavidin-coated
magnetic beads allows the isolation of pure DNA exten-
sion fragments free from false terminations.19 These DNA
fragments were analyzed by a capillary array electro-
(
coupling yield ≈ 96%).
We then synthesized an alkynyl 6-carboxyfluorescein
FAM) 3 by reacting propargylamine with 6-carboxyfluo-
(
rescein-NHS ester (Scheme 2). Click chemistry 1,3-
dipolar cycloaddition between the alkynyl-FAM 3 and the
azido-labeled DNA 2 was carried out at 80 °C in aqueous
condition to produce FAM-labeled DNAs 4 and 5. After
the reaction, excess alkynyl-FAM 3 was removed by size-
exclusion chromatography and the resulting FAM-labeled
DNAs 4 and 5 were desalted with an oligonucleotide
purification cartridge. We characterized products 4 and
5
by measuring their UV-vis absorption and MALDI-
(18) McGeary, R. P. Tetrahedron Lett. 1998, 39, 3319.
(19) J u, J . Anal. Biochem. 2002, 309, 35.
6
10 J . Org. Chem., Vol. 68, No. 2, 2003

F IGURE 3. Electropherogram of DNA sequencing fragments generated with 4 and 5.
phoresis (CAE) system20 and resolved at single-base pair
bp) resolution to produce an electropherogram as shown
in Figure 3. The peaks represent the FAM fluorescence
emission from each DNA fragment that was extended
from 4 and 5 and terminated by ddATP. This “A”
sequencing ladder shown in Figure 3 matched exactly the
sequence of the DNA template.
Syn th esis of Azid o-La beled DNA (2). To incorporate the
azido group at the 5′-end of the oligonucleotide, 10 nmol of
amino-modified oligonucleotide in 40 µL of 0.25 M Na CO
NaHCO buffer (pH 9.0) was incubated for 12 h at room
temperature with 10 µmol of succinimidyl 5-azidovalerate 1 in
2 µL of dimethyl sulfoxide. Unreacted succinimidyl 5-azido-
valerate was removed by size-exclusion chromatography on a
PD-10 column, and the resulting azido-labeled DNA was de-
salted with an oligonucleotide purification cartridge. The con-
centration of the collected azido-labeled DNA was measured by
a UV-vis spectrophotometer, and the isolated yield was 96%.
(
2
3
/
3
1
In summary, we have successfully demonstrated the
use of the click chemistry to synthesize fluorescent
oligonucleotides with high selectivity, high yield, and
high stability. Without further purification by gel elec-
trophoresis and HPLC that are required for conventional
fluorescent oligonucleotide synthesis, the primer synthe-
sized by the click chemistry can be used directly to
produce DNA sequencing products with single-base
resolution in a capillary electrophoresis DNA sequencer
with laser-induced fluorescence detection. Further im-
provement of the methodology will allow reduced reaction
Syn t h esis of 6-Ca r b oxyflu or escein -p r op a r gyla m id e
(
Alk yn yl F AM) (3). A solution of 3.4 µL (0.05 mmol) of
propargylamine in DMF (0.5 mL) was added to a solution of 11
mg (0.023 mmol) of 6-carboxyfluorescein-NHS ester in DMF (0.5
3
mL) and 0.1 M NaHCO solution (0.1 mL). After 5 h of stirring
at room temperature, the solvent was removed under vacuum
and the crude mixture was purified by a silica gel TLC plate
(
0
1:9 MeOH/CHCl
3
) to give 8.0 mg (85%) of alkynyl FAM (R
f
)
.45) as a red oil: 1H NMR (methanol-d
4
) δ 8.01 (s, 2H), 7.60 (s,
1H), 6.94 (d, 2H, J ) 9.1 Hz), 6.58-6.53 (m, 4H), 4.05 (d, 2H, J
) 2.4 Hz), 2.50 (t, 1H, J ) 2.2 Hz); 13C NMR (methanol-d
) δ
175. 3, 168.3, 158.5, 146.7, 136.9, 132.2, 129.9, 129.5, 128.7,
time by attaching an electron-withdrawing functional
4
group at the end of the triple bond.12 It is expected that
+
1
22.2, 121.0, 114.5, 104.0, 80.5, 72.2, 30.0; HRMS (FAB ) calcd
the optimized click chemistry will have potential applica-
tions in bioconjugation fields such as DNA covalent
attachment on a chip, chemoselective protein modifica-
tion, and immunoassays.
+
for C24
16 6
H O N, 414.0978 (M + 2H ); found, 414.0997.
Syn th esis of F lu or escen t DNA by Click Ch em istr y (4
a n d 5). Azido-oligonucleotide (3.93 nmol) in 120 µL of water was
reacted with a 150-fold excess of alkynyl FAM in 36 µL of DMSO
at 80 °C for 72 h. Unreacted dye was removed by size-exclusion
chromatography on a PD-10 column. The resulting fluorescent
DNA was then desalted with an oligonucleotide purification
cartridge, and the concentration was measured by a UV-vis
spectrophotometer. The isolated yield of 4 and 5 was 91%.
Exp er im en ta l Section
Ma ter ia ls a n d Gen er a l P r oced u r es. The amino-C6-M13
(-40) forward primer (18mer) and internal mass standard
Ma ss Sp ectr u m of DNA. Mass measurement of oligonucle-
otides was performed using a MALDI-TOF mass spectrometer.
DNA product (30 pmol) was mixed with 10 pmol of the internal
mass standard, and the mixture was suspended in 2 µL of a
oligonucleotides were commercially available and purified by
1
13
HPLC. The H and C NMR spectra were recorded on 400 and
5 MHz NMR spectroscopic instruments, respectively. The high-
7
resolution mass spectra (HRMS) were obtained under fast atom
bombardment (FAB) conditions. UV-vis spectra of DNA samples
were recorded in acetonitrile/water (1:1 volume ratio) at room
temperature using quartz cells with path lengths of 1.0 cm.
3
-hydroxypicolinic acid matrix solution. A small amount (0.5 µL)
of this mixture was spotted on a stainless steel sample plate,
air-dried, and analyzed. The measurement was taken using a
positive ion mode with a 25 kV accelerating voltage, a 94% grid
voltage, and a 350 ns delay time.
Syn th esis of Su ccin im id yl 5-Azid ova ler a te (1). 5-Azido-
valeric acid was synthesized according to the published proce-
1
8
P CR Am p lifica tion of Tem p la te. A PCR DNA product
amplified from a pBluescript II SK(+) phagemid vector was used
as a sequencing template because it has a binding site for the
M13-40 universal primer. Amplification was carried out using
the M13-40 universal forward and reverse primers in a 20 µL
reaction, which contained 1X Accutaq LA reaction buffer, 250
pmol of each dNTP, 40 pmol of each primer, 0.5 unit of
J umpstart Red Accutaq LA DNA Polymerase, and 100 ng of the
phagemid template. The reaction was performed in a DNA
thermal cycler using an initial activation step of 96 °C for 1 min.
This was followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s,
and 72 °C for 2 min. At the end of the PCR reaction, 20 µL of an
enzymatic mixture containing 5 units of shrimp alkaline phos-
phatase (SAP), 4 µL of 10X SAP buffer, 6 units of E. coli
exonuclease I, and 10 µL of water were added to the PCR
reaction to degrade the excess primers and dNTPs. The reaction
dure. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC; 500 mg, 2.61 mmol) was added to a suspension
of 358 mg (2.50 mmol) of 5-azidovaleric acid and 300 mg (2.61
mmol) of N-hydroxysuccinimide in CH
temperature and stirred for 7 h, followed by the addition of H
The separated CH Cl phase was washed with H O and brine
solution, dried over Na SO , and evaporated to yield 520 mg
87%) of succinimidyl 5-azidovalerate as a pale yellow liquid:
2
Cl
2
(20 mL) at room
2
O.
2
2
2
2
4
(
-
1 1
IR (thin film) ν 2100, 1640 cm ; H NMR (CDCl
3
) δ 3.31 (t, 2H,
J ) 6.6 Hz), 2.81 (s, 4 H), 2.63 (t, 2H, J ) 7.1 Hz), 1.86-1.68
1
3
(
2
3
m, 4H); C NMR (CDCl ) δ 169.1, 168.2, 50.8, 30.4, 27.8, 25.5,
+
+
1.8; HRMS (FAB ) calcd for C
9 13 4 4
H O N , 241.0937 (M + H );
found, 241.0948.
(20) Kheterpal, I.; Mathies, R. A. Anal. Chem. 1999, 71, 31A.
J . Org. Chem, Vol. 68, No. 2, 2003 611

mixture was incubated at 37 °C for 90 min before the enzymes
were heat inactivated at 72 °C for 30 min.
linear polyacrylamide (LPA) gel in a capillary array fluorescent
DNA sequencer and then separated at 8 kV in LPA buffer to
produce a fluorescence electropherogram.
Gen er a tion a n d Detection of Sa n ger DNA Sequ en cin g
F r a gm en ts. A primer extension reaction was performed using
the FAM-labeled primers 4 and 5 and the above PCR product.
A 30 µL reaction mixture was made, consisting of 2.22 nmol of
each dNTP, 37 pmol of Biotin-11-ddATP, 20 pmol of primer, 9
units of Thermo Sequenase DNA polymerase, 1X Thermo
Sequenase reaction buffer, and 20 µL of PCR product. The
reaction consisted of 30 cycles of 94 °C for 20 s, 50 °C for 20 s,
and 60 °C for 90 s. Correctly terminated DNA fragments by
Biotin-11-ddATP were purified from other reaction components
using solid-phase capture according to the published method.19
The fluorescent DNA fragments in 8 µL of formamide were
electrokinetically injected at 3 kV into a capillary filled with
Ack n ow led gm en t. This research is supported by
National Science Foundation (Sensing and Imaging
Initiative Grant 0097793).
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectral data for 1 and 3, IR spectrum of 1, and UV-vis
spectrum of 4 and 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O026615R
6
12 J . Org. Chem., Vol. 68, No. 2, 2003