Primer extension reaction assays for incorporation of deoxynucleotide analogue into DNA

Article abstract of DOI:10.1002/cjoc.201400731

Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluo

Full text of DOI:10.1002/cjoc.201400731

FULL PAPER
DOI: 10.1002/cjoc.201400731
Primer Extension Reaction Assays for Incorporation of
Deoxynucleotide Analogue into DNA
Xilong Wang, Xun Chen, Yonghui Liu, and Jin Zhu*
Department of Polymer Science and Engineering, School of Chemistry and Chemical Engineering,
State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures,
Nanjing University, Nanjing, Jiangsu 210093, China
Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer
extension reactions are commonly used for the assay of such reaction events. However, current assay protocols
generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate
in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleo-
tide-terminated template strand and a phosphorothiolate-modified deoxynucleotide-terminated template strand. We
designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2-bromoisobutyryl group and
demonstrated that it could be well recognized by φ29DNA polymerase, E. coli DNA polymerase I Klenow Frag-
ment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo−), which trans-
lated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long
single-stranded DNA at high-density using φ29 DNA polymerase by rolling circle amplification.
Keywords primer extension reaction, enzymatic incorporation, deoxyuridine triphosphate analogue, nucleotides,
DNA
Introduction
lows the generation of an extremely long single-
stranded DNA with thousands of tandemly repeated
DNA sequences via an isothermal amplification on a
circular DNA template. As a simple, powerful, and ver-
satile DNA amplification strategy, RCA has become an
important tool for molecular diagnostics and nanotech-
nology.[29-32] The RCA products with chemically modi-
fied functional groups could offer significant application
advantages under various contexts. Herein we also re-
port on the design and synthesis of a C5-modified de-
oxyuridine triphosphate analogue (dU*TP) containing
2-bromoisobutyryl group, a versatile structural motif
that can undergo substitution or elimination reaction and
is also commonly used as an efficient initiator in atom
transfer radical polymerization (ATRP),[33-36] and its
recognition by φ29 DNA polymerase, E. coli DNA po-
lymerase I Klenow Fragment, Bst DNA polymerase
Large Fragment, and E. coli DNA polymerase I Klenow
Fragment (exo−) in three types of primer extension reac-
tions. The success of primer extension reactions has
been conveniently confirmed by denaturing polyacryla-
mide gel electrophoresis. Further, we have also been
able to incorporate dU*TP into extremely long DNA
strand at high-density using φ29 DNA polymerase by
rolling circle amplification.
The chemically modified oligonucleotides with
various functional groups have important applications in
nucleic acid detection,[1-3] fluorescent in situ hybridiza-
tion,[4,5] gene expression analysis,[6] gene therapy,[7,8]
RNA interference,[9,10] and nanobiotechnology.[11] These
oligonucleotides are typically generated by solid-phase
synthesis or enzymatic incorporation through primer
extension reaction,[12-17] polymerase chain reaction,[18,19]
nicking enzyme amplification reaction[20,21] or terminal
transferase incorporation.[22] The chemically modified
oligonucleotides with less than 100 nucleotides are ac-
cessible by solid-phase synthesis. To generate longer
chemically modified oligonucleotides, enzymatic in-
corporation is commonly utilized. The assay of enzy-
matic incorporation using primer extension reactions
currently suffers from the requirement of either radio-
labeling,[12,13,19,23,24] fluorescent reporter labeling,[14,25,26]
or removal of specific deoxynucleotide triphosphate in
the reaction mixture.[27,28] We have designed herein two
effective primer extension assay protocols that utilize a
dideoxynucleotide-terminated template strand and a
phosphorothiolate-modified deoxynucleotide-terminated
template strand. Rolling circle amplification (RCA) al-
*
E-mail: jinz@nju.edu.cn; Tel.: 0086-025-83686291; Fax: 0086-025-83317761
Received October 26, 2014; accepted December 5, 2014; published online January 14, 2015.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201400731 or from the author.
192
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Chin. J. Chem. 2015, 33, 192—198

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA
Experimental
8H), 6.90 (d, J＝8.8 Hz, 4H), 6.11 (t, J＝6.7 Hz, 1H),
5.31 (d, J＝4.5 Hz, 1H), 3.91 (s, 1H), 3.75 (s, 6H), 3.18
(qd, J＝10.6, 3.9 Hz, 2H), 2.35－2.07 (m, 2H). HRMS
(ESI) calcd for C30H29IN2NaO7: [M＋Na]＋, 679.0912;
found m/z: 679.0910.
Materials and methods
All reagents and solvents were obtained from com-
mercial sources and used without further purification
unless otherwise stated. Trimethyl phosphate (98%) was
purchased from TCI Co. Ltd., distilled over calcium
hydride. φ29 DNA polymerase, E. coli DNA poly-
merase I Klenow Fragment, and E. coli DNA poly-
merase I Klenow Fragment (exo−) were purchased from
Thermo Scientific. Bst DNA polymerase Large Frag-
ment and Taq DNA ligase were purchased from New
England Biolabs (Beijing, China). All the DNA mole-
cules (Table S1 in ESI) were custom-synthesized by
Sangon Biotech (Shanghai) Co. Ltd. Nano-pure water
(18.2 MΩ•cm), purified by Sartorius Arium 611 system,
was used throughout the experiment. Ion exchange
HPLC purification was performed on Agilent Zorbax
Eclipse XDB-C18 (preparative) columns (250 mm×9
mm, 10 µm, Agilent, USA). The following eluents were
used: buffer A, 0.05 mol/L (triethylammonium bicar-
bonate, TEAB) buffer (pH 7.5); and buffer B, 60% (V/V)
0.05 mol/L TEAB buffer (pH 7.5) and 40% (V/V) ace-
tonitrile. A linear gradient of 0% B to 100% B within 45
min was utilized, and then 100% B for 15 min. Prepara-
tive thin-layer chromatography (TLC) was carried out
on glass plates (25 cm×15 cm×0.25 cm) coated with
Synthesis of compound 2
Compound 1 (8.64 g, 13.0 mmol) and CuI (0.49 g,
2.6 mmol) were added to a 500 mL Schlenk flask and
purged with nitrogen, N,N-dimethylformamide (150
mL), propargyl alcohol (2.21 g, 39.0 mmol), triethyl-
amine (3.94 g, 0.039 mol), and tetrakis(triphenylphos-
phine)palladium(0) (1.5 g, 1.3 mmol) were added in a
glove box to prevent oxidation of palladium catalyst.
The dark green solution was freeze-thaw 3 times under
N2 and stirred at 25 ℃ for 18 h, then subjected to a
short silica column chromatography using ethyl acetate
as eluent. The volatile solvent was removed under re-
duced pressure, and the resulting oil was dissolved in
chloroform (200 mL), washed with water (100 mL)
twice and with concentrated NaCl solution, dried over
anhydrous Na2SO4 and concentrated. The resulting vis-
cous oil was purified by silica gel chromatography (di-
chloromethane ∶ methanol ＝ 100 ∶ 1 to dichloro-
methane∶methanol＝100∶5; to all eluents triethyl-
amine 1% V/V was added) to afford 2 (3.90 g, 50.6%
1
yield) as a white foam. H NMR (500 MHz, DMSO) δ:
silica gel G (10－40 μm). H NMR, 13C NMR spectra
11.66 (s, 1H), 7.90 (s, 1H), 7.44－7.30 (m, 5H), 7.29－
7.21(m, 4H), 6.90 (dd, J＝8.8, 2.0 Hz, 4H), 6.11 (t, J＝
6.6 Hz, 1H), 5.32 (s, 1H), 5.22 (t, J＝5.8 Hz, 1H), 4.26
(s, 1H), 4.08 (d, J＝4.2 Hz, 2H), 3.93 (s, 1H), 3.74 (s,
6H), 3.25 (dd, J＝10.4, 5.5 Hz, 1H), 3.11 (dd, J＝10.3,
2.4 Hz, 1H), 2.33－2.12 (m, 2H). HRMS (ESI) calcd for
C33H32N2NaO8: [M ＋ Na] ＋ , 607.2051; found m/z:
607.2036.
1
were recorded in DMSO-d6 or D2O (with tetramethylsi-
lane as an internal reference) solution on a Bruker
AVANCE 500 or 600 MHz spectrometer. 31P NMR was
recorded in D2O on an Advance DPX-250 NMR spec-
trometer (Bruker, Rheinstetten, Germany) with an op-
eration frequency of 202 MHz for 31P nuclei. δ Values
were relative to external 85% H3PO4. High-resolution
MS (HRMS) spectra were obtained on a Waters Micro-
mass GCT Premier facility. Gel electrophoresis was
performed on a Bio-Rad system using 20% polyacryla-
mide or 1% agarose gel.
Synthesis of compound 3
Compound 2 (3.9 g, 6.7 mmol) and triethylamine
(2.0 g, 20.0 mmol) were dissolved in chloroform (200
mL) and cooled in an ice water bath. 2-Bromoisobutyryl
bromide (0.83 mL, 6.7 mmol) was dissolved in chloro-
form (50 mL) and was added dropwise. The reaction
was monitored by TLC every 4 h. Triethylamine (0.5 g,
5.0 mmol) and 2-bromoisobutyryl bromide (0.21 mL,
1.68 mmol) was added if starting material 2 exists. The
reaction mixture was washed with water (150 mL) three
times and with concentrated NaCl solution, dried over
Na2SO4 and concentrated. The resulting viscous oil was
purified by silica gel chromatography (dichloromethane
to dichloromethane∶ethyl acetate＝3∶1; to all eluents
triethylamine 1% V∶V was added) to afford 3 (3.12 g,
Synthesis of compound 1
5-Iodo-2'-deoxyuridine (6 g, 16.9 mmol) was dis-
solved in pyridine (60 mL) and cooled in an ice-water
bath before 4,4'-dimethoxytrityl chloride (DMT-Cl) (6.8
g, 20.1 mmol) was added. The solution was stirred for 1
h under ice-water bath, warmed to 25 ℃ and stirred for
another 12 h. The reaction was quenched by adding ice
water (60 mL). Dichloromethane (180 mL) was added to
extract the product and the organic layer was washed
three times with brine (60 mL), dried over anhydrous
Na2SO4 and concentrated. Residual amount of pyridine
was removed by azeotropic distillation with petroleum
ether. The resulting viscous oil was purified by silica gel
chromatography (dichloromethane to dichloromethane/
ethyl acetate＝3/1, to all eluents triethylamine 1% V/V
was added) to afford 1 (8.65 g, 77.8% yield) as white
1
63.9% yield) as a white foam. H NMR (500 MHz,
DMSO) δ: 11.73 (s, 1H), 8.03 (s, 1H), 7.44－7.30 (m,
5H), 7.29－7.21 (m, 4H), 6.90 (d, J＝7.9 Hz, 4H), 6.10
(t, J＝6.6 Hz, 1H), 5.34 (d, J＝4.5 Hz, 1H), 4.80 (q, J＝
16.0 Hz, 2H), 4.29 (s, 1H), 3.91 (d, J＝26.6 Hz, 1H),
3.74 (s, 6H), 3.31－3.04 (m, 2H), 2.39－2.15 (m, 2H),
1.87 (s, 6H). HRMS (ESI) calcd for C37H37BrN2NaO9:
1
solid. H NMR (500 MHz, DMSO) δ: 11.74 (s, 1H),
8.02 (s, 1H), 7.41 (d, J＝7.6 Hz, 2H), 7.36－7.17 (m,
Chin. J. Chem. 2015, 33, 192—198
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193

Wang et al.
FULL PAPER
[M＋Na]＋, 755.1575; found m/z: 755.1570.
J＝6.7 Hz, 1H), 4.80 (d, J＝20.1 Hz, 2H), 4.39 (dd, J＝
10.3, 4.2 Hz, 1H), 3.97 (t, J＝5.2 Hz, 3H), 2.97 (q, J＝
7.3 Hz, 24H), 2.17 (t, J＝10.3 Hz, 2H), 1.73 (s, 6H),
Synthesis of compound 4
13
Compound 3 (1.0 g, 1.36 mmol) was dissolved in
dichloromethane (100 mL). Then a mixture of acetic
acid (18 mL) and water (2 mL) was added. The reaction
mixture was stirred vigorously at 40 ℃ for 16 h, then
volatile solvent was removed under reduced pressure.
The resulting oil was purified by silica gel chromatog-
raphy (dichloromethane∶ethyl acetate＝1∶1 to ethyl
acetate) to afford 4 (383 mg, 65.2% yield) as a white
1.05 (t, J＝7.3 Hz, 36H); C NMR (151 MHz, D2O) δ:
172.88 (s), 164.22 (s), 150.40 (s), 145.55 (s), 127.58 (s),
98.46 (s), 87.35 (s), 86.74－84.59 (m), 77.87 (s), 70.54
(s), 65.33 (s), 62.21 (s), 54.78 (s), 46.58 (s), 38.63 (s),
29.74 (s), 8.22 (s). 31P NMR (202 MHz, D2O) δ: −8.19
－ −13.57 (m), −23.35 (s). HRMS (ESI) calcd for
C34H68BrN5O16P3: [M−N(C2H5)3 ＋ H] , 974.3052;
＋
found m/z: 974.3018.
1
foam. H NMR (500 MHz, DMSO) δ: 11.66 (s, 1H),
Primer extension reaction assays
8.28 (s, 1H), 6.10 (s, 1H), 5.24 (s, 1H), 5.10 (s, 1H),
5.03 (s, 2H), 4.23 (s, 1H), 3.81 (s, 1H), 3.59 (d, J＝17.2
Hz, 2H), 2.14 (s, 2H), 1.92 (s, 6H). HRMS (ESI) calcd
for C16H19BrN2NaO7: [M＋Na]＋, 453.0268; found m/z:
453.0268.
To a PCR tube the following reactants were added: 1
μL of 10 μmol/L primer DNA solution, 1 μL of 10
μmol/L template DNA solution, 1 μL of 10×reaction
buffer of the corresponding DNA polymerase, 3 μL of
autoclaved distilled water, the resulting mixture was
heated to 90 ℃ and slowly cooled down to room tem-
perature in more than 30 min. Primer extension reaction
was carried out by adding 1 μL of dATP, dCTP, dGTP
mixture (1 mmol/L each) in primer extension reaction 1
and 2 or dCTP, dGTP mixture (1 mmol/L each) in
primer extension reaction 3, 1 μL of dU*TP (1 mmol/L)
or dTTP (1 mmol/L) as positive control or H2O as nega-
tive control, 0.5 μL of the corresponding DNA poly-
merase. For φ29 DNA polymerase 0.2 μL of BSA (10
mg/mL) was added. The reaction volume was made up
to 10 μL by adding autoclaved distilled water. The tube
was incubated for 3 h at 30 ℃ for φ29 DNA poly-
merase (0.5 U/μL), 37 ℃ for E. coli DNA polymerase
I Klenow Fragment (0.5 U/μL) and E. coli DNA poly-
merase I Klenow Fragment (exo−) (0.25 U/μL), and 65
℃ for Bst DNA polymerase Large Fragment (0.4 U/μL).
The reactions were stopped by adding 10 μL of 2×
denaturing PAGE loading buffer. Samples were heated
to 94 ℃ for 2 min, and immediately placed on ice, 10
μL of each sample was subjected to denaturing poly-
acrylamide gel electrophoresis containing 7 mol/L urea.
Gel electrophoresis was run at 90 V for 100 min in a 60
℃ water bath, and then the gel was stained with Gel-
Red for 30 min. After the gels were recorded, the im-
ages were inverted from gray bands on black back-
ground to gray bands on white background.
Synthesis of compound 5 (dU*TP)
All glasswares and syringes were heated and dried
under vacuum overnight. Compound 4 (100 mg, 0.23
mmol) and 1,8-bis(dimethylamino)naphthalene (proton
sponge, 116 mg, 0.54 mmol) were dried in vacuum in a
10 mL Schlenk flask overnight. Trimethyl phosphate
(1.5 mL, distilled and stored over 4 Å molecular sieves)
was added to dissolve the starting materials. The flask
was degassed and back-filled with N2 three times. Then
the mixture was cooled to −20 ℃, and freshly distilled
phosphorus oxychloride (35 μL, 0.38 mmol) was added
dropwise via a micro syringe over the course of 10 min.
The turbid solution was stirred at −20 ℃ for 2 h.
(HNn-Bu3)2H2P2O7 in N,N-dimethylformamide (0.5
mol/L, 2 mL, 1.0 mmol) and tri-n-butylamine (0.16 mL)
was premixed and added in portion via syringe over the
course of 10 min and further reacted at −20 ℃ for 45
min. Then the mixture was added to TEAB buffer (0.1
mol/L, 20 mL) and stirred at room temperature for 1 h.
The solution was washed three times with dichloro-
methane (20 mL) and three times with ethyl acetate (20
mL). The aqueous layer was lyophilized and dissolved
in about 5 mL of DI water. Possible cations formed by
tri-n-butylamine or proton sponge were removed by elu-
tion through a column packed with 10 mL of cation ex-
＋
change resin Dowex 50WX8® pretreated to HNEt3
form, the resin was washed with about 20 bed volumes
of DI water and the eluent was lyophilized. The result-
ing solid was further purified by HPLC (Zorbax Eclipse
XDB-C18, 250 mm×9 mm, 10 µm, Agilent, USA,
UV-detecting at 260 nm. Flow rate: 3 mL/min. Mobile
phase A: 0.05 mol/L TEAB buffer pH 7.5. Mobile phase
B: 60% 0.05 mol/L TEAB buffer and 40% acetonitrile.
Gradient: form 0% B to 100% B within 45 min, 100% B
for 15 min. Desired product was collected at around 15
－19 min). Water and TEAB salt were removed by ly-
ophilization (addition of 10 mL of water and lyophiliza-
tion again twice for the complete removal of TEAB salt)
to afford 5 (50 mg, 20.0% yield) as a white deliquescent
foam. 1H NMR (600 MHz, D2O) δ: 7.95 (s, 1H), 6.02 (t,
Rolling circle amplification
A 20 μL ligation solution containing Ligation DNA
(1 μmol/L), Padlock DNA (1 μmol/L), Taq DNA ligase
(0.8 U/μL), and Taq DNA ligase reaction buffer was
placed at 45 ℃ for 4 h. The ligation product circular
Padlock DNA was used as a template for the rolling
circle amplification reaction with RCA-primer. A roll-
ing circle amplification reaction solution was prepared
through the mixing of following solutions: 2 μL of liga-
tion mixture (containing circular Padlock DNA), 1 μL
of RCA-primer (10 μmol/L), 1 μL of dATP, dCTP,
dGTP mixture (10 mmol/L each), 1 μL of dU*TP (10
mmol/L) or dTTP (10 mmol/L) as positive control or
194
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Chin. J. Chem. 2015, 33, 192—198

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA
Scheme 1 Synthesis of dU*TP
H2O as negative control, 0.5 μL of the corresponding
DNA polymerase, 1 μL of 10×reaction buffer of the
corresponding DNA polymerase. For φ29 DNA poly-
merase 0.2 μL of BSA (10 mg/mL) was added. The re-
action volume was made up to 10 μL by adding auto-
claved distilled water. The reaction solution was incu-
bated overnight at 30 ℃ for φ29 DNA polymerase (0.5
U/μL), 37 ℃ for E. coli DNA polymerase I Klenow
Fragment (0.5 U/μL) and E. coli DNA polymerase I
Klenow Fragment (exo−) (0.25 U/μL), and 45 ℃ for
Bst DNA polymerase Large Fragment (0.4 U/μL). And
then 2 μL of 6×loading buffer was subjected to each
dilution, the mixture was subjected to 1% native agarose
gel in 0.5×TBE buffer supplemented with 5 μg/mL
ethidium bromide. After the gels were recorded, the
images were inverted from gray bands on black back-
ground to gray bands on white background.
OCH3
O
I
O
N
HN
I
pyridine, r.t.
77.8%
HN
O
N
+
O
Cl
HO
O
DMT
O
O
OH
OH
1
OCH3
O
O
OH
Br
OH
Br
Pd(PPh3)4
CuI, NEt3
HN
NEt3
O
N
DMF
CHCl3
63.9%
50.6%
DMT
O
O
OH
2
O
O
Results and Discussion
O
Br
HN
Synthesis of deoxyuridine triphosphate analogue
(dU*TP)
CH3COOH
H2O
O
N
DMT
O
The chemical modifications can be introduced in the
base, sugar unit or phosphate moiety, and the C5 posi-
tion of the pyrimidine ring is widely used to introduce
new functional groups[37-40] because it is located in the
major groove of DNA double helix and therefore mini-
mizes any impact on the base pairing. For the synthesis
of dU*TP, the commercially available 5-iodo-2'-deoxy-
uridine was used as the starting material (Scheme 1).
We utilized 4,4'-dimethoxytrityl (DMT) to protect the
hydroxyl group on the C5 of the carbohydrate five-
membered ring.[41-43] Then compound 1 reacted with
propargyl alcohol via the Sonogashira coupling reaction
for the introduction of hydroxyl group, with 2 as the
product. Compound 3 was prepared by reacting com-
pound 2 with 2-bromoisobutyryl bromide through selec-
tive esterification. Then compound 4 was obtained by
removing the DMT protecting group under acidic condi-
tion. In order to prepare compound 5, dU*TP, the
triphosphorylation process is involved. Although there
were plenty of reports about triphosphorylation in recent
years,[25,27,44-46] challenges remain in the achievement of
this critical step. First of all, the phosphorylation reagent
is phosphorus oxychloride which is prone to hydrolysis,
so strictly anhydrous condition must be ensured in the
entire reaction process. And even if the C5 hydroxyl
group can be triphosphorylated, compound 5 is also
deliquescent. Second, compound 5 must be separated
and purified by high performance liquid chromatogra-
phy (HPLC) to remove 3'-phosphate and 3',5'-diphos-
phate byproducts, which could not be achieved by
commonly used anion exchange resin;[47] the reason is
that the introduced ester moiety cannot tolerate the high
ionic strength. With all these precautions taken, dU*TP
was successfully obtained after the triphosphorylation
process at the C5 hydroxyl group in compound 4 and
separation and purification by HPLC.
O
CH2Cl2, r.t.
65.2%
OH
3
O
O
O
Br
HN
POCl3
(1)
Proton Sponge
(CH3O)3PO
O
N
HO
O
(2) (HNn-Bu3)2H2P2O7
TEAB
20.0%
OH
4
O
O
N
O
Br
HN
O
O
P
O
O
O
-O
O
P
O P
O-
O
O-
O-
OH
5
DMT＝4,4'-dimethoxytrityl, Pd(PPh3)4＝tetrakis(triphenylphos-
phine)palladium(0), NEt3 ＝ triethylamine, Proton Sponge ＝
1,8-bis(dimethylamino)naphthalene, TEAB＝triethylammonium
bicarbonate.
Primer extension reaction assays
Primer extension reaction is one of the most classic
methods to evaluate the ability of nucleotide analogues
to function as substrates for DNA polymerases.[12-15,28,48]
Autoradiography is normally used to detect the product
of primer extension reaction because of its high sensi-
tivity and specificity,[19,23,28] but it is harmful and the
operation is complicated. So we chose denaturing poly-
acrylamide gel electrophoresis (PAGE) without radio-
labeling which is the relatively safe and simple way to
assay the product of primer extension reaction. In order
to distinguish the product and the template after the
Chin. J. Chem. 2015, 33, 192—198
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195

Wang et al.
FULL PAPER
primer extension reaction, the length of them should be
different. We designed two types of primer extension
strategies to assay the incorporation of dU*TP into
DNA by DNA polymerases. The first strategy is that the
same lengths of two DNA strands were hybridized to-
gether. Template-1 (30 nt, for information on all the
DNA sequences, see Table S1 in SEI) has a 3' dideoxy-
cytosine modification which prevents 3' extension by
DNA polymerases, so only the 5' non-hybridized section
of the Template-1 directs the DNA polymerase-
catalyzed addition of nucleotides to the 3' end of the
Primer-1 (30 nt). An extension product longer than
Template-1 and Primer-1 could be obtained when
dU*TP was successfully recognized by DNA poly-
merases (Figure 1A). A full-length (40 nt) single-
stranded DNA was observed when dU*TP substituted
dTTP as the substrate for four DNA polymerases (lanes
4 and 7, Figure 1B; lanes 4 and 7, Figure 1C) and the
quantity of extension product generated with dU*TP is
similar to the amount of DNA generated with dTTP,
which demonstrated dU*TP could be recognized as the
substrate effectively by the four DNA polymerases. The
mobility of the deoxyuridine analogue incorporated
DNA is slightly lower than the deoxythymidine incor-
porated DNA because of the difference in the molecular
weight. No full-length extension product was observed
in negative control experiments (lanes 3 and 6, Figures
1B and 1C).
The second strategy is that the 20 nt Primer-2 is hy-
bridized to the central region of a 40 nt Template-2,
with 10 nt non-hybridized region each located at the 5'
and 3' end of Template-2. Template-2 has three phos-
phorothioate-modified deoxyadenines at the 3' end,[49,50]
which is commonly used to prevent DNA degradation
by exonuclease. The phosphorothioate modification
prevents the cleavage of 3' end of Template-2 by φ29
DNA polymerase and E. coli DNA polymerase I
Klenow Fragment which possess a 3'→5' exonuclease
activity for single-stranded DNA in the primer extension
reaction and thus ensures that the extension product and
Template-2 can be distinguished (Figure 2A). A
full-length (30 nt) DNA was observed when dU*TP
Figure 1 Primer extension reaction 1 using DNA polymerase
with Primer-1 and Template-1. (A) Schematic illustration of
primer extension reaction using DNA polymerase with Primer-1
and Template-1. The Primer-1 and Template-1 form a DNA
duplex and the 5' non-hybridized section of the Template-1 di-
rects the DNA polymerase-catalyzed addition of nucleotides to
the 3' end of the Primer-1. (B) Denaturing PAGE analysis of
primer extension reactions with dU*TP using φ29 DNA poly-
merase and E. coli DNA polymerase I Klenow Fragment. Lane 1:
40 nt DNA (as the reference for the expected extension product);
lane 2: 30 nt DNA (as the reference for the starting material);
lanes 3－5: primer extension reactions using φ29 DNA poly-
merase; lane 3: dATP, dCTP, and dGTP (negative control); lane 4:
dATP, dCTP, dGTP, and dU*TP; lane 5: dATP, dCTP, dGTP,
and dTTP (positive control); lanes 6－8: primer extension reac-
tions using E. coli DNA polymerase I Klenow Fragment, lane 6:
dATP, dCTP, and dGTP (negative control), lane 7: dATP, dCTP,
dGTP, and dU*TP, lane 8: dATP, dCTP, dGTP, and dTTP; lane 9:
DNA marker. (C) Denaturing PAGE analysis of primer extension
reactions with dU*TP using Bst DNA polymerase Large Frag-
ment and E. coli DNA polymerase I Klenow Fragment (exo−).
The components of the lanes are identical to those of the lanes in
(B) except that the polymerases are Bst DNA polymerase Large
Fragment (lanes 3－5) and E. coli DNA polymerase I Klenow
Fragment (exo−) (lanes 6－8). The band marked in red represents
the deoxyuridine analogue-incorporated DNA and the band
marked in black represents the deoxythymidine-incorporated
DNA.
Figure 2 Primer extension reaction 2 using DNA polymerase
with Primer-2 and Template-2. (A) Schematic illustration of
primer extension reaction using DNA polymerase with Primer-2
and Template-2. The Primer-2 and Template-2 form a DNA
duplex and the 5' non-hybridized section of the Template-2 di-
rects the DNA polymerase-catalyzed addition of nucleotides to
the 3' end of the Primer-2. (B) Denaturing PAGE analysis of
primer extension reactions with dU*TP using φ29 DNA poly-
merase and E. coli DNA polymerase I Klenow Fragment. Lane 1:
30 nt DNA (as the reference for the expected extension product);
lane 2: 20 nt and 40 nt DNA (as the reference for the starting
material); lanes 3－5: primer extension reactions using φ29 DNA
polymerase, lane 3: dATP, dCTP, and dGTP (negative control),
lane 4: dATP, dCTP, dGTP, and dU*TP, lane 5: dATP, dCTP,
dGTP, and dTTP (positive control); lanes 6－8: primer extension
reactions using E. coli DNA polymerase I Klenow Fragment, lane
6: dATP, dCTP, and dGTP (negative control), lane 7: dATP,
dCTP, dGTP and dU*TP, lane 8: dATP, dCTP, dGTP and dTTP;
lane 9: DNA marker. (C) Denaturing PAGE analysis of primer
extension reactions with dU*TP using Bst DNA polymerase
Large Fragment and E. coli DNA polymerase I Klenow Fragment
(exo−). The components of the lanes are identical to those of the
lanes in (B) except that the polymerases are Bst DNA polymerase
Large Fragment (lanes 3－5) and E. coli DNA polymerase I
Klenow Fragment (exo-) (lanes 6－8). The band marked in blue
represents the deoxyuridine analogue-incorporated DNA and the
band marked in black represents the deoxythymidine-incorpo-
rated DNA.
196
www.cjc.wiley-vch.de
© 2015 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2015, 33, 192—198

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA
Rolling circle amplification
substituted dTTP as the substrate for four DNA poly-
merases (lanes 4 and 7, Figure 2B; lanes 4 and 7, Figure
2C), which demonstrated the successful incorporation of
dU*TP into DNA.
We then tested whether the four DNA polymerases
could incorporate dU*TP into single-stranded DNA at
high-density. First Padlock DNA is ligated to form cir-
cular Padlock DNA (Cir-Padlock DNA) by Taq DNA
ligase in the presence of Ligation DNA (Figure 4A and
Figure S8 in ESI). RCA-primer then hybridizes with
the Cir-Padlock DNA and RCA is initiated by using
Cir-Padlock DNA as the template and RCA-primer as
the primer in the presence of DNA polymerase and
dNTPs. The analysis of RCA reactions with four DNA
polymerases, as shown in Figure 4B, reveals that only
φ29 DNA polymerase could effectively recognize
dU*TP as the substrate. In contrast, the extension reac-
tions by E. coli DNA polymerase could effectively rec-
ognize dU*TP as the substrate. In contrast, the extension
reactions by E. coli DNA polymerase I Klenow Frag-
ment, E. coli DNA polymerase I Klenow Fragment
(exo−), and Bst DNA polymerase Large Fragment are
significantly slower. This is likely because φ29 DNA
To further confirm the ability to incorporate dU*TP,
a third strategy, which is analogous to the literature re-
ported one, is adopted. Primer-2 is hybridized to the 3'
end of Template-3 (40 nt) which has 10 deoxythymi-
dines named poly(T) at the 5' end. dATP was not added
in primer extension reactions and thus extension reac-
tions would stop at the T position of Template-3, which
allowed the differentiation of the extended Primer-2
and Template-3 (Figure 3A). With this primer exten-
sion reaction design, the successful incorporation of
dU*TP into DNA could also be readily demonstrated
(Figures 3B and 3C).
Taken together, we have demonstrated that dU*TP
could be well recognized as the substrate by φ29 DNA
polymerase, E. coli DNA polymerase I Klenow Frag-
ment, Bst DNA polymerase Large Fragment, and E. coli
DNA polymerase I Klenow Fragment (exo−) in three
types of primer extension reactions.
Figure 3 Primer extension reaction 3 using DNA polymerase
with Primer-2 and Template-3. (A) Schematic illustration of
primer extension reaction using DNA polymerase with Primer-2
and Template-3. The Primer-2 and Template-3 form a DNA
duplex and the 5' non-hybridized section of the Template-3 di-
rects the DNA polymerase-catalyzed addition of nucleotides to
the 3' end of the Primer-2. poly(T) denotes 10 deoxythymidines.
(B) Denaturing PAGE analysis of primer extension reactions with
dU*TP using φ29 DNA polymerase and E. coli DNA polymerase
I Klenow Fragment. Lane 1: 30 nt DNA (as the reference for the
expected extension product); lane 2: 20 nt and 40 nt DNA (as the
reference for the starting material); lanes 3－5: primer extension
reactions using φ29 DNA polymerase, lane 3: dCTP and dGTP
(negative control), lane 4: dCTP, dGTP and dU*TP, lane 5: dCTP,
dGTP and dTTP (positive control); lane 6－8: primer extension
reactions using E. coli DNA polymerase I Klenow Fragment, lane
6: dCTP and dGTP (negative control), lane 7: dCTP, dGTP, and
dU*TP, lane 8: dCTP, dGTP, and dTTP; lane 9: DNA marker. (C)
Denaturing PAGE analysis of primer extension reactions with
dU*TP using Bst DNA polymerase Large Fragment and E. coli
DNA polymerase I Klenow Fragment (exo−). The components of
the lanes are identical to those of the lanes in (B) except that the
polymerases are Bst DNA polymerase Large Fragment (lanes 3－
5) and E. coli DNA polymerase I Klenow Fragment (exo−) (lanes
6－8). The band marked in blue represents the deoxyuridine ana-
logue-incorporated DNA and the band marked in black represents
the deoxythymidine-incorporated DNA.
Figure 4 Incorporation of dU*TP into DNA strand by rolling
circle amplification using DNA polymerase. (A) Schematic illus-
tration of the ligation-rolling circle amplification (Ligation-RCA)
protocol. Padlock DNA is ligated to form circular Padlock DNA
(Cir-Padlock DNA) by Taq DNA ligase in the presence of Liga-
tion DNA. RCA-primer hybridizes with the Cir-Padlock DNA
and then RCA is initiated by using Cir-Padlock DNA as the
template and RCA-primer as the primer and a large number of
tandem DNA products are generated. (B) Agarose gel electro-
phoresis diagram of incorporation of dU*TP into DNA strand by
rolling circle amplification using DNA polymerases. Lane 1:
rolling circle amplification using φ29 DNA polymerase in the
presence of dATP, dCTP, and dGTP (negative control); lanes 2－
5: rolling circle amplification using φ29 DNA polymerase, E. coli
DNA polymerase I Klenow Fragment, E. coli DNA polymerase I
Klenow Fragment (exo−), and Bst DNA polymerase Large Frag-
ment in the presence of dATP, dCTP, dGTP, and dU*TP, respec-
tively; lane 6: rolling circle amplification using φ29 DNA poly-
merase in the presence of dATP, dCTP, dGTP, and dTTP (posi-
tive control); lane 7: DNA marker. The band marked in red
represents the deoxyuridine analogue-incorporated DNA and the
band marked in black represents the deoxythymidine-incorpo-
rated DNA.
Chin. J. Chem. 2015, 33, 192—198
© 2015 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
197

Wang et al.
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oxynucleotide analogue into DNA. Starting from com-
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(dU*TP) has been successfully synthesized andcharac-
1
terized by H NMR, 13C NMR, 31P NMR and HRMS.
dU*TP could be effectively recognized as the substrate
of φ29 DNA polymerase, E. coli DNA polymerase I
Klenow Fragment, Bst DNA polymerase Large Frag-
ment and E. coli DNA polymerase I Klenow Fragment
(exo–) in three types of primer extension reactions,
which translated to effective incorporation of dU*TP
into DNA. The dU*TP incorporated DNA, containing
2-bromoisobutyryl group, is novel and could serve as an
initiator in surface ATRP. Surface-initiated ATRP from
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© 2015 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2015, 33, 192—198