Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2′-C-piperazino-UNA monomer

Article abstract of DOI:10.1016/j.bmc.2011.06.087

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2′-C-piperazino-UNA residue and UNA residu

Full text of DOI:10.1016/j.bmc.2011.06.087

Bioorganic & Medicinal Chemistry 19 (2011) 4739–4745
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thermodynamic and biological evaluation of a thrombin binding aptamer
modified with several unlocked nucleic acid (UNA) monomers and a
20-C-piperazino-UNA monomer
Troels B. Jensen a, Jonas R. Henriksen b, Bjarne E. Rasmussen c, Lars M. Rasmussen c, Thomas L. Andresen b,
Jesper Wengel a, Anna Pasternak a,
⇑
a Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
b Department of Micro and Nanotechnology, Ørsteds Plads Building 345 East, DK-2800 Kgs. Lyngby, Denmark
c Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, University of Southern Denmark, Sdr. Boulevard 29, 5000 Odense C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promis-
ing anticoagulant properties due to specific interactions with thrombin. Herein we present the influence
of a single 20-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermo-
dynamics, kinetics and biological properties of the aptamer. 20-C-Piperazino-UNA is characterized by
more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermo-
dynamic stability of TBA by 0.28–0.44 kcal/mol in a position depending manner with retained quadruplex
topology and molecularity. The presence ofꢀUNA-U in positions U3, U7, and U12 results in the highest sta-
bilization of G-quadruplex structure (DDG37 = ꢁ1.03 kcal/mol). On the contrary, the largest destabiliza-
tion mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and
U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants
and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased
affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as
potent as TBA in fibrin clotting inhibition.
Received 10 May 2011
Revised 27 June 2011
Accepted 30 June 2011
Available online 7 July 2011
Keywords:
Thrombin binding aptamer
Unlocked nucleic acid
Isothermal titration calorimetry
Thermodynamics
Thrombin time assay
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
facilitating reversal of its activity and excluding dose-adjusting
complications observed for other, commonly used anticoagulants.9
Thrombin is a serine protease involved in the blood coagulation
cascade.1 Inhibition of thrombin is applied in coronary surgery,
prevention and treatment of cardiovascular diseases, and cancer
therapy.2–5 Development of novel thrombin inhibitors is of interest
due to a number of side effects of anticoagulants used at present.
Recently, TBA progressed through preclinical to clinical develop-
ment (ARC183, Archemix).10
According to NMR and X-ray studies, TBA forms a complex with
two thrombin molecules inactivating only one of them.7,11,12 The
inhibitory properties of TBA are attributed to specific interaction
of the aptamer with the thrombin anion exosite I of one of the
thrombin molecules. The electropositive heparin exosite of the sec-
ond thrombin molecule is involved in neutralization of the nega-
Thrombin binding aptamer (TBA) is
a short DNA sequence
(50GGTTGGTGTGGTTGG30) which was found as a result of in vitro
selection targeted towards thrombin.6 This consensus 15-mer
forms an intramolecular, antiparallel G-quadruplex structure with
a chair-like conformation (Fig. 1).7,8 The core constitutes of two
G-quartets linked at one end by a TGT loop and by two TT loops
at the other end. TBA is characterized by strong anticoagulant
properties in vitro, short in vivo lifetime and rapid onset of action
Abbreviations: TBA, thrombin binding aptamer; UNA, unlocked nucleic acid; ITC,
isothermal titration calorimetry; SPR, surface plasmon resonance; CD, circular
dichroism; TDS, thermal difference spectra.
⇑
Corresponding author. Tel.: +45 65502510; fax: +45 66158780.
E-mail address: apa@ifk.sdu.dk (A. Pasternak).
Figure 1. Quadruplex structure of thrombin binding aptamer (TBA).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.087

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T. B. Jensen et al. / Bioorg. Med. Chem. 19 (2011) 4739–4745
tively charged backbone of the aptamer. It is still unresolved which
part of the aptamer interacts with thrombin anion exosite I since
X-ray studies indicate the middle TGT loop to be involved while
NMR results suggest that interactions responsible for inhibitory
process rather occur via the two TT loops.7,11,12
2. Materials and methods
2.1. Oligonucleotides
The 50-biotinylated UNA oligonucleotides (Table 1) were syn-
thesized on an automated RNA/DNA synthesizer using standard
phosphoramidite chemistry. The biotin phosphoramidite and O30-
phosphoramidites of UNA nucleotides were applied according to
the previously described procedures14 together with commercial
DNA phosphoramidites. The purity of all oligonucleotides was ver-
ified by ion-exchange HPLC and determined to be 80% or greater,
and the oligonucleotide compositions were confirmed by MALDI-
TOF mass spectrometry (Supplementary data).
Unlocked nucleic acid (UNA) is an acyclic RNA mimic (Fig. 2a).
The missing bond between the C20 and C30 atoms of the ribose ring
results in increased flexibility relative to an RNA monomer. The
UNA thymine monomer was first synthesized in 1995 and shown
to significantly decrease thermal stability of DNA duplexes.13 Re-
cently, the synthesis of UNA-A, -C, -G, and -U phosphoramidites,
introduction of all UNA monomers into RNA and DNA duplexes
and thermodynamic stability analyses have been reported.14–16
UNA monomers are according to these studies potent in decreasing
or increasing base pairing specificity depending on positioning
within a duplex. The comprehensive thermodynamic and spectro-
scopic analysis made for UNA-modified RNA duplexes revealed
that stacking of nucleobases of a UNA monomer is less favorable
than of RNA monomers, that overall A-form of duplexes is pre-
served, that UNA pyrimidine nucleotides destabilize duplexes
more than UNA purine nucleotides, and that the destabilizing
properties of UNA nucleotides are slightly dependent on the nature
of the flanking bases.16 Moreover, it was shown that UNA-modified
antisense oligonucleotides are compatible with RNase H activ-
ity17,18 and are highly potent in gene silencing due to increased
specificity of action of siRNA duplexes.19–23
2.2. UV melting analysis of TBA variants
Oligonucleotides were dissolved in a buffer containing 100 mM
KCl and 10 mM sodium cacodylate, pH 7.0. Oligonucleotide single
strand concentrations were calculated using the HyTher program
from the absorbance measured above 80 °C and extinction coeffi-
cients which were approximated by a nearest-neighbor model.25,26
UNA-modified and unmodified DNA strands with identical
sequences were assumed to have identical extinction coefficients.
The samples were annealed for 5 min at 80 °C and then slowly
cooled to room temperature. The measurements were performed
for nine different concentrations of each quadruplex in the concen-
tration range 10ꢁ5–10ꢁ6 M using 10 mm (300
lL) quartz microcu-
The 20-C-piperazino-UNA-U monomer is
a UNA derivative
vettes. Absorbance versus temperature curves were obtained by
the UV melting method at 295 nm27–29 in the temperature range
5–95 °C on a Beckman DU 800 spectrophotometer equipped with
a six-position microcell holder and a thermoprogrammer. The
reversibility of transitions was ensured for all samples by measure-
ment of both heating and cooling profiles (data not shown). Three
different heating rates (0.5 °C/min, 0.3 °C/min, and 0.2 °C/min) were
tested to avoid hysteresis phenomena. The rate of 0.2 °C/min was
selected because here the melting and annealing curves were
reproducible and strictly superimposable. The lack of hysteresis
phenomena implied that the quadruplex dissociation–association
process was in a state of thermodynamic equilibrium.27,28 Melting
curves were analyzed and the thermodynamic parameters
determined by non-linear curve fitting with the program MeltWin
3.5 using an unimolecular structural transition approach.30 TM is
which possesses an additional positively charged piperazino moi-
ety ( Fig. 2b). It was suggested that this derivative destabilizes
DNA and DNA/RNA duplexes less than regular UNA monomers
and with retained mismatch discrimination.15
The influence of single substitution of DNA monomers of TBA
with UNA monomers on thermodynamic stability, binding affinity,
and biological activity of the quadruplex forming aptamer was re-
cently reported.24 It was shown that UNA monomers can be ap-
plied to modulate quadruplex thermodynamic stability in
a
position-depending manner. UNA introduced at positions U3, U7
or U12 (Fig. 1) makes quadruplex folding more energetically favor-
able in reference to the parent TBA with retained antiparallel,
intramolecular quadruplex topology. Biological studies revealed
that most of the UNA-modified TBAs possess anticoagulant proper-
ties, and that introduction of a UNA-U monomer in position 7 im-
proves inhibition of fibrin-clot formation relative to the
unmodified TBA reference aptamer.
attributed to melting temperatures determined for the 10ꢁ4
M
oligonucleotide concentration whereas Tm to melting points for
any other concentration of an oligonucleotide.
Herein we describe thermodynamic, kinetic and biological char-
acterization of TBA variants modified with UNA monomers in more
than one position. Moreover we report chemical synthesis of a 20-
C-piperazino-UNA-U monomer, its introduction into specific
positions of TBA, and the influence of the above modifications on
thermodynamic stability, binding affinity and biological activity
of the aptamer.
2.3. Circular dichroism spectra
CD spectra were recorded on a Jasco J-600A spectropolarimeter
using 1 mL quartz cuvettes with a 5 mm path length. The oligonu-
cleotides were dissolved to 3.1 lM concentration in 10 mM phos-
phate buffered saline (138 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.76 mM KH2PO4; pH 7.4). All samples were annealed
for 2 min at 100 °C and thereupon slowly cooled to room temper-
ature before data collection. The measurements were done at 20 °C
in the 200–300 nm wavelength range. The buffer spectrum was
subtracted from the sample spectra. The spectra were smoothed
in Microcal Origin 6.0 using a Savitzky-Golay filter.
a
b
U+
B
O
O
O
O
-
O
P
N
O
2.4. Thermal difference spectra
OH
O
O
P
O
-
O
The measurements were performed on a Beckman DU 800 spec-
trophotometer equipped with a six-position microcell holder and a
N
H
Figure 2. Structures of UNA (a) and 20-C-piperazino-UNA-U (b) monomers;
B = nucleobase; U = uracil-1-yl.
UNA phosphoramidite monomers and UNA-modified oligonucleotides are com-
mercially available from www.ribotask.dk.

T. B. Jensen et al. / Bioorg. Med. Chem. 19 (2011) 4739–4745
4741
Table 1
Thermodynamic parameters of DNA quadruplex formation with UNA (U or G) and 20-C-piperazino-UNA-U (X)a
Gꢀ37 kcal/mol)
DDGꢀ37 (kcal/mol)
Name
Sequence
ꢁ
D
H° (kcal/mol)
ꢁ
D
S° (eu)
TM (°C)
D
TM (°C)
D
TBA
ON1
GGTTGGTGTGGTTGG
GGXTGGTGTGGTTGG
GGTTGGXGTGGTTGG
GGTTGGTGTGGXTGG
GGUTGGTGTGGUTGG
GGTTGGUGTGGUTGG
GGUTGGUGTGGTTGG
GGUTGGUGTGGUTGG
GGTTGGUGUGGTTGG
GGUTGGUGUGGUTGG
36.6 0.4
36.0 0.9
116.1 1.3
113.1 2.8
ꢁ0.61 0.02
ꢁ0.96 0.03
42.2
45.4
0
0
3.2
ꢁ0.35
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
40.5 0.6
36.6 0.8
36.3 0.4
38.2 0.3
37.0 0.3
37.9 0.3
35.3 2.3
35.8 2.1
127.3 1.8
115.2 2.4
113.1 1.4
119.0 1.0
114.6 1.0
116.9 1.0
117.7 7.4
118.1 7.0
ꢁ1.05 0.04
ꢁ0.89 0.03
ꢁ1.18 0.01
ꢁ1.32 0.01
ꢁ1.50 0.02
ꢁ1.64 0.02
+1.18 0.06
+0.86 0.08
45.2
44.8
47.4
48.1
50.1
51.0
27.0
29.7
ꢁ0.44
ꢁ0.28
ꢁ0.57
ꢁ0.71
ꢁ0.89
ꢁ1.03
1.79
3.0
2.6
5.2
5.9
7.9
8.8
ꢁ15.2
ꢁ12.5
1.47
a
Buffer: 100 mM KCl, 10 mM sodium cacodylate, pH 7.0.
thermoprogrammer using 10 mm quartz microcuvettes. The oligo-
nucleotides were dissolved to 3.1 M concentration in a buffer
l
containing 100 mM KCl and 10 mM sodium cacodylate, pH 7.0.
Absorbance spectra were recorded at 13 °C and 85 °C in the
220–320 nm wavelength range. The scan speed was 1200 nm/
min with data collection of 1 pt/nm. Thermal difference spectra
were obtained by subtraction by the low temperature absorbance
spectra from the high temperature absorbance spectra with the
Origin 6.0 program. The difference spectra were normalized by
dividing the data by its maximum value.31
2.5. Isothermal titration calorimetry
The binding affinity between thrombin and aptamer was mea-
sured by isothermal titration calorimetry (iTC200, Microcal, GE).
The experiments were conducted at 37 °C by injecting 100 lM
aptamer in 10 mM phosphate buffered saline (138 mM NaCl,
2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4; pH 7.4) into a
5
l
M solution of thrombin. Examples of ITC heat traces are shown
in Figure 3 for the aptamers ON1, ON2 and ON3. The heat of dilu-
tion was measured by injecting 100 M aptamer into buffer and
was subsequently subtracted in the data analysis. All ITC experi-
ments include a single pre-injection of 0.2 L, which was dis-
carded from the analysis, followed by 19 ꢂ 2 L injections. The
l
l
l
ITC heat trace was processed by custom made software as de-
scribed previously32 and fitted to a single site binding model
based on the equation: AT ꢀ A þ T , which is governed by the
equilibrium dissociation constant, KD. The results are compiled
in Table 2.
2.6. Thrombin time assay
The inhibitory effect of TBA and the UNA-modified variants on
clotting time was measured by a thrombin time assay. The time
(in seconds) for clotting at 37 °C was measured on a STA-R EVOLU-
TION instrument (Stago) after mixing the identical volumes of cit-
rate plasma and Thrombin reagent (STA-THROMBIN 00611,
Diagnostica Stago). A pool of plasma from four healthy individuals
was used. The thrombin reagent was pre-incubated with TBA or
the TBA variants at 0.33 mM concentration for 5 min before addi-
tion to plasma and measurement of clotting time (= thrombin
time). The antithrombin effect is the additional time for clotting
in presence of the aptamers relative to a reference with water
added.
Figure 3. ITC binding affinity study of thrombin and aptamer conducted at 37 °C (a)
examples of ITC heat traces of 100 M aptamer (ON1, ON2 and ON3) titrated into
M thrombin (b) the corresponding heat of reaction. The data was analyzed using
l
5
l
a single site binding model and the best fits are given by the solid lines in (b).
except for CH2Cl2 which was distilled before use. Anhydrous
CH2Cl2 was dried over activated 4 Å molecular sieves. All reactions
(Fig. 4) were monitored by thin layer chromatography (TLC) using
precoated aluminum plates with indicator. Purification of the com-
pounds was carried out by silica gel column chromatography.
Afterwards, all fractions containing product were pooled, evapo-
rated to dryness and dried under vacuum for at least 12 h. NMR
spectra were recorded on a 400 MHz instrument. Chemical shifts
are reported in ppm relative to TMS (1H; internal standard, rH
2.7. Chemical synthesis of the uracil derivative of 20-C-
piperazino-UNA
All reagents and solvents were of analytical grade as obtained
form commercial suppliers and used without further purification
0.00 ppm), solvents peaks
(
13C, rC 77.0 ppm, DMSO-d6
31P; external standard, rP H3PO4
39.52 ppm) or 85% H3PO4
(

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T. B. Jensen et al. / Bioorg. Med. Chem. 19 (2011) 4739–4745
7.10 (m, 13H), 6.85 (m, 4H), 5.98 (t, J = 6.2 Hz, 1H, H10), 5.53 (d,
J = 8.0 Hz, 1H, H5), 4.37 (d, J = 6.4 Hz, 2H, CH2, Fmoc), 4.25 (t,
J = 6.3 Hz, 1H, CH, Fmoc), 3.73 (s, 6H, 2ꢂOMe), 3.67–3.52 (m, 3H,
H30, H40), 3.20 (s, 4H, piperazino), 3.05–2.90 (m, 2H, H50), 2.81–
2.55 (m, 2H, H20), 2.45–2.21 (m, 4H, piperazino), 0.78 (s, 9H,
3ꢂMe), ꢁ0.02 (s, 3H, Me), ꢁ0.03 (s, 3H, Me). 13C NMR (101 MHz,
DMSO) d 163.10, 157.94, 154.10, 151.24, 144.68, 143.75, 142.47,
140.69, 139.32, 137.33, 135.50, 135.31, 129.45, 128.82, 127.65,
127.46, 127.18, 126.98, 126.50, 124.84, 121.28, 119.93, 113.04,
113.01, 109.64, 101.79, 85.34, 81.16, 78.22, 66.24, 63.03, 62.01,
60.15, 59.28, 54.90, 54.34, 46.69, 45.54, 43.32, 25.56, 17.68,
ꢁ5.64, ꢁ5.70. Anal. for C55H64N4O9Siꢃ1/9 H2O: C, 68.77; H, 6.80;
N, 5.74 (calcd C, 69.16; H, 6,78; N, 5.87). ESI-HRMS: m/z
975.4323 ([M+Na]+; C55H64N4O9SiꢃNa calcd 975.4335).
Table 2
Binding affinity and biological effect of TBA modified TBA variants
b
Namea
KD (nM)
T-time/antithrombin effectc (s)
TBA
47.6
1600
52.6
1000
n.d.
300
1300
n.d.
32.6/13.6
21.3/2.3
31.2/12.2
22.5/3.5
19.4/0.4
28.7/9.7
31.2/12.2
19.5/0.5
20.5/1.5
19.0/0.0
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
1500
n.d.
a
50-Biotinylated oligonucleotides.
The relative errors for KD values are below 27%, KD value for
b
50GGTTGGUGTGGTTGG is 37.0 nM.
c
‘T-time’ is the time required for a clot formation in the plasma from a blood
2.7.2. 20-Deoxy-50-O-(4,40-dimethoxytrityl)-20-(4-(9-
sample; ‘Antithrombin effect’ is the T-time in the presence of an aptamer minus the
T-time in the absence of an aptamer; no oligo added resulted in 18.9 and 19.0 s
clotting times in two experimental sets and constitute reference values in the
antithrombin effect calculations. Non-biotin conjugated TBA results in 34.4 s
clotting time.
fluorenylmethoxycarbonyl)piperazino)-20,30-secouridine (3)
Nucleoside 2 (465 mg, 0.489 mmol) was dissolved in anhyd THF
(15 mL) and stirred at rt under an atmosphere of argon. Pyridinium
hydrochloride (565 mg, 4.89 mmol) and triethylamine trihydrof-
louride (1.18 mL, 7.24 mmol) were added and the resulting mix-
ture was stirred for 4 h. The reaction mixture was diluted with
CH2Cl2 (50 mL) and washed with satd aq NaHCO3 (2 ꢂ 50 mL).
The organic phase was dried (Na2SO4), filtrated and evaporated
to dryness under reduced pressure. The residue was purified by sil-
ica gel column chromatography using as eluent MeOH (0–10%) in
CH2Cl2 affording nucleoside 3 as a white foam (351 mg, 83%). 1H
NMR (400 MHz, CDCl3) d 8.92 (s, 1H, NH), 7.76 (d, J = 7.5 Hz, 2H),
7.55 (d, J = 7.4 Hz, 2H), 7.44–7.16 (m, 14H), 6.81 (d, J = 8.7 Hz,
4H), 5.90 (d, J = 6.6 Hz, 1H, H10), 5.58 (d, J = 8.0 Hz, 1H, H5), 4.97
(s, 1H, 30-OH), 4.44 (d, J = 6.6 Hz, 2H, CH2 Fmoc), 4.22 (t,
J = 6.6 Hz, 1H, CH Fmoc), 3.78 (s, 6H, 2ꢂOMe), 3.72–3.37 (m, 7H,
H30, H40 and piperazino), 3.15 (d, J = 5.1 Hz, 2H, H50), 2.56 (m, 6H,
H20 and piperazino). 13C NMR (101 MHz, CDCl3) d 162.90, 158.60,
154.97, 150.15, 144.45, 143.92, 141.34, 139.30, 135.61, 135.52,
129.91, 129.89, 127.94, 127.88, 127.72, 127.08, 126.98, 124.91,
120.00, 113.18, 102.56, 86.62, 84.19, 83.38, 77.34, 77.23, 77.03,
76.71, 67.30, 63.59, 63.05, 62.68, 55.25, 53.70, 53.43, 47.33,
43.34. Anal. for C49H50N4O9ꢃ1/2 H2O: C, 69.18; H, 5.91; N, 6.45
(calcd C, 69.41; H, 6.06; N, 6.61). ESI-HRMS: m/z 861.3473
([M+Na]+; C49H50N4O9ꢃNa calcd 861.3470).
n.d. stands for not determined.
U
U
O
DMTrO
O
O
iv
-
O
P
O
N
O
N
N
O
R1
N
H
R2
Monomer X
1: R1 = TBDMS, R2 = H
i
ii
2: R1 = TBDMS, R2 = Fmoc
3: R1 = H, R2 = Fmoc
iii
4: R1 = PN(i-Pr)2O(CH2)2CN, R2 = Fmoc
Figure 4. Chemical synthesis of 20-C-piperazino-UNA-U monomer (Monomer X).
Reagents and conditions: (i) Fmoc-Cl, anhyd CH2Cl2:pyridine (4:1), rt, 2.5 h (89%);
(ii) triethylamine trihydrofluoride, pyridinium hydrochloride, anhyd THF, rt, 4 h,
(83%); (iii) diisopropylammonium tetrazolide, 2-cyanoethyl N,N,N0,N0-tetraisopro-
pylphosphorodiamidite, anhyd CH2Cl2, rt, 17 h (91%).
2.7.3. 30-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-20-
deoxy-50-O-(4,40-dimethoxytrityl)-20-(4-(9-
fluorenylmethoxycarbonyl)piperazino)-20,30-secouridine (4)
Nucleoside 3 (220 mg, 0.262 mmol) was dissolved in anhyd
CH2Cl2 (12 mL) and the solution was stirred at rt under an atmo-
sphere of argon. Diisopropyl ammonium tetrazolide (119 mg,
0.393 mmol) and bis(N,N-diisopropylamino)-2-cyanoethoxyphos-
0.00 ppm). Assignments of NMR signals are based on 2D correla-
tion experiments. High-resolution mass spectra (ESI-HRMS) were
recorded on a mass spectrometer in positive ion mode.
2.7.1. 30-O-tert-Butyldimethylsilyl-20-deoxy-50-O-(4,40-
dimethoxytrityl)-20-(4-(9-
phine (67 lL, 0.393 mmol) were added and the resulting solution
fluorenylmethoxycarbonyl)piperazino)-20,30-secouridine (2)
Nucleoside 133 (402 mg, 0.550 mmol) was dissolved in a 4:1
solution of anhyd CH2Cl2 and anhyd pyridine (20 mL) and the
resulting mixture was cooled to 0 °C and stirred under an atmo-
sphere of argon. 9-Fluorenylmethyl chloroformate (FmocCl,
171 mg, 0.660 mmol) was added to the reaction mixture and stir-
ring was continued for 2 h. EtOH (1 mL) was added and the reac-
tion mixture was stirred for additional 10 min. The solvent was
removed by evaporation under reduced pressure. The afforded res-
idue was redissolved in EtOAc (100 mL). The solution was washed
with satd aq NaHCO3 (2 ꢂ 50 mL). The organic phase was dried
(Na2SO4), filtrated and evaporated to dryness under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy using as eluent MeOH (0–5%) in CH2Cl2 affording nucleoside
2 as a white solid (470 mg, 89%). 1H NMR (400 MHz, DMSO) d
11.36 (br s, 1H, NH), 7.86 (d, J = 7.5 Hz, 2H), 7.62 (m, 3H), 7.46–
was stirred for 17 h and then diluted with satd aq NaHCO3 (25 mL)
and extracted with CH2Cl2 (2 ꢂ 50 mL). The organic phase was dried
(Na2SO4), filtrated and evaporated to dryness under reduced
pressure. The afforded residue was purified by silica gel column
chromatography using as eluent EtOAc (50–100%) in PE affording
phosphoramidite 4 as a white foam (249 mg, 91%). 31P NMR
(162 MHz, CDCl3) d 148.9, 148.5. Anal. for C58H67N6O10Pꢃ1/25 H2O:
C, 66.60; H, 6.55; N, 7.75 (calcd C, 66.99; H, 6.50; N, 8.08). ESI-HRMS:
m
/z 1061.4528 ([M+Na]+, C58H67N6O10PꢃNa calcd 1061.4548).
3. Results
3.1. Chemical synthesis of 20-C-piperazino UNA uridine
The startingmaterial (Fig. 4, compound 1) was prepared as earlier
described.33 Protection of the piperazino moiety was carried out

T. B. Jensen et al. / Bioorg. Med. Chem. 19 (2011) 4739–4745
4743
with Fmoc-Cl in anhyd CH2Cl2/pyridine (4:1) affording compound 2
in 89% yield. Desilylation using triethyl trihyrdofluoride and pyridi-
nium hydrochloride in THF (giving compound 3, 85% yield) followed
by O30-phosphitylation by reaction with 2-cyanoethyl N,N,N0,N0-
tetraisopropylphosphorodiamidite and diisopropylammonium
tetrazolide in CH2Cl2 afforded phosphoramidite 4 in 91% yield suit-
able for incorporation of monomer X into ONs on an automated
DNA synthesizer (ON1–ON3, Table 1). This was carried out using
1H-tetrazole as activator and 10 min coupling time. The stepwise
coupling yields for amidite 4 was ꢄ97% whereas the unmodified
DNA amidites displayed >97% coupling yield (2 min coupling time).
2). Only variant ON2 showed comparable binding relative to
unmodified TBA. Variants ON1, ON3, ON5, ON6, and ON8 showed
significantly decreased affinity to thrombin, whereas the KD value
for three remaining variants (ON4, ON7, and ON9) could not be
determined due to very low binding affinity.
3.4. Biological studies
The influence of modified TBA variants on enzymatic activity of
thrombin was investigated using a thrombin time assay (Table 2).
Three variants, namely ON2, ON5, and ON6 showed inhibitory ef-
fects slightly weaker but comparable to unmodified TBA. Signifi-
cantly reduced inhibition of fibrin-clot formation was observed for
aptamers ON1, ON3, ON8, whereas ON4, ON7, and ON9 were shown
not to have any significant impact on the coagulation process.
3.2. Thermodynamic and structural features
UNA and 20-C-piperazino-UNA residues were placed in specific
positions of TBA which were previously reported as stabilizing for
quadruplex structure upon single introduction of UNA monomers
(Table 1, ON1–ON7).24 The influence of radically increased flexibil-
ity of quadruplex structure was furthermore tested for two variants
with an entirely UNA-modified central loop (ON8, ON9). Most of
variants studied herein were found as more stable in reference to
unmodified TBA. A single introduction of the 20-C-piperazino-UNA
monomer in positions U3, U7 or U12 enhanced the thermodynamic
stability of the quadruplex structure by 0.28–0.44 kcal/mol. Two or
three UNA residues placed in various combinations in positions U3,
U7, and U12 increased thermodynamic stability by 0.57–1.03 kcal/
mol. Conversely, significant destabilization mounting to 1.79 kcal/
mol was observed for variants with an entirely UNA-modified cen-
tral loop. The Tm values found for each of the variants were concen-
tration independent (data not shown).
Circular dichroism (CD) and thermal difference (TDS) spectra
were recorded to evaluate possible changes in quadruplex confor-
mation and topology (Figs. 5 and 6). All thermodynamically stable
variants (ON1–ON7) show patterns similar to that of unmodified
TBA. CD spectra of ON1–ON7 were characterized by high-ampli-
tude, positive maxima at 293 nm with increased intensity in refer-
ence to TBA. The TDS patterns with two positive maxima near 240
and 270 nm, a moderately intense peak around 260 nm, and an in-
tense peak around 295 nm, were identical with that of the unmod-
ified aptamer. In contrast, unstable ON8 and ON9 showed CD
bands of strongly decreased intensity and TDS signatures untypical
for a quadruplex structure.
4. Discussion
Thermodynamic investigation of the stability of singly UNA-
modified TBA variants was recently described and three positions
(U3, U7, and U12) were found as energetically favorable for UNA
introduction.24 Substitution of the above three positions with the
20-C-piperazino-UNA uridine monomer X results in further in-
crease of quadruplex thermodynamic stability (Table 1). Monomer
X decreases quadruplex free energy by 0.35, 0.44, and 0.28 kcal/
mol (ON1, ON2 and ON3, respectively) what corresponds to
approximate 2-fold more favorable equilibrium constant for quad-
ruplex formation relative to that of the TBA reference. It was pre-
viously reported that a single UNA-U monomer stabilizes the TBA
structure by 0.23 (U3), 0.50 (U7) and 0.15 (U12) kcal/mol.24 The
energetically favorable change of Gibbs0 free energy (DDG3ꢀ 7) ob-
served for variants ON1 and ON3 is therefore attributed not only
to the presence of the flexible UNA monomer but also to the pres-
ence of the piperazino moiety. Similar behavior has been reported
previously for DNA/DNA and DNA/RNA duplexes modified with the
20-C-piperazino-UNA monomer.15 The increased stabilization of
duplexes modified with 20-C-piperazino-UNA monomer in refer-
ence to those containing UNA monomer was ascribed to partial
neutralization of the negatively charged backbone by the positively
charged piperazino moiety. Hence, analogous electrostatic effects
might be responsible for the additional stabilization of quadruplex
structure observed for variants modified with the 20-C-piperazino-
UNA uridine monomer.
3.3. Thrombin binding affinity of aptamers
The introduction of two or three UNA-U monomers in more
than one of the specific positions found previously as energetically
favorable resulted in all cases in an increase of quadruplex thermo-
dynamic stability (ON4–ON7). Notably, the energetic gain resulting
from the presence of two or three UNA-U residues is additive,
although even slightly higher than the sum of single substitution
effects.24 Accordingly, the highest thermodynamic stability was
achieved by incorporation of three UNA-U monomers in positions
U3, U7 and U12 (ON7, Table 1) leading to a decrease of Gibbs0 free
energy by about 1 kcal/mol. Such improvement can be translated
to a 5-fold more favorable equilibrium constant for quadruplex for-
mation in reference to that of unmodified TBA.
Significantly increased flexibility of the middle loop was
achieved by substituting all three loop residues for UNA monomers
(ON8, Table 1). This resulted in significant destabilization of the
quadruplex structure by 1.79 kcal/mol corresponding to an 18-fold
more unfavorable equilibrium constant for quadruplex formation
in reference to that of TBA. The positive value of Gibbs0 free energy
for ON8 indicates that under the conditions used herein the pre-
dominant species is the unfolded form. Additional introduction of
UNA monomers in positions U3 and U12 (ON9) increases quadru-
plex stability by 0.32 kcal/mol in reference to ON8, which is in
Isothermal titration calorimetry (ITC) was applied to determine
the thrombin binding affinity of the modified TBA variants (Table
10
9
8
TBA
ON1
7
6
5
4
3
2
1
0
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
-1
-2
-3
-4
-5
220
240
260
280
300
320
wavelength [nm]
Figure 5. CD spectra of TBA and modified variants.

4744
T. B. Jensen et al. / Bioorg. Med. Chem. 19 (2011) 4739–4745
a
1.1
0.8
0.7
b
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
-0.8
-0.9
-1.0
-1.1
220
240
260
280
300
320
220
240
260
280
300
320
wavelength [nm]
wavelength [nm]
Figure 6. Thermal difference spectra (green) of (a) ON1 (characteristic also for TBA, ON2-ON7) and (b) ON8 (characteristic also for ON9). High- and low-temperature spectra
are marked in black and red, respectively.
agreement with the previously reported thermodynamic effect of
UNA-U placed in positions U3 and U12.24 The overall stability of
ON9 is however reduced (a 10-fold decrease in the equilibrium
constant for quadruplex formation) relative to that of unmodified
TBA.
The molecularity of folding of all variants studied herein was
verified by monitoring the concentration dependence of Tm val-
ues29 to be intramolecular as all studied quadruplexes revealed
no linear increase in the function of Tm versus aptamers concentra-
tion (data not shown). The results confirm a previous report show-
ing that UNA monomers do not impact the folding molecularity of
TBA.24
with thrombin. The negative influence of piperazino moiety on
aptamers binding affinity might originate from the presence of
the positively charged piperazino moiety interacting electrostati-
cally with the positively charged protein, but possibly also from
steric hindrance. According to NMR studies, T3 and T12 are situ-
ated in close proximity to the thrombin anion exosite I,7,12 where-
fore the presence of the bulky piperazino moiety might inhibit an
optimal fit between aptamer and thrombin.
It was suggested that high stability of quadruplex structure is
essential for proper and specific interactions between aptamer
and thrombin.35 The results described herein reveal that the most
thermodynamically stable aptamer ON7 is a very poor binder (Ta-
ble 2, KD not determined) as is ON9. Notably, the latter display a
positive Gibbs0 free energy (ON9) upon denaturation indicating
predominance of unfolded states. Similarly, also ON4, ON6, and
ON8 posses at least 27-fold decreased binding affinity towards
thrombin in reference to TBA. Moreover, moderate binding affinity
decreased by about 6-fold in reference to TBA was found for ON5
which is characterized by improved thermodynamic stability.
The significantly reduced binding affinity towards thrombin for
the majority of variants studied herein suggests that thermody-
namic stability of quadruplexes, or even predominant quadruplex
structure, is not the excusive determinant of specific interactions
between aptamer and thrombin.
The thrombin time assay partially confirmed results derived
from ITC studies. Biological activity of most of the studied vari-
ants (ON1, ON3, ON4, ON7, ON8, and ON9, Table 2) which posses
poor thrombin binding affinity was significantly decreased in ref-
erence to that of unmodified TBA. In contrast, ON5 characterized
by only a moderate KD value shows considerable ability to inhibit
fibrin clot formation. Surprisingly ON6 seems to have biological
activity comparable to that of TBA despite strongly decreased
binding affinity towards thrombin. However, incorporation of 20-
C-piperazino-UNA-U monomer X at position 7 of the aptamer
results not only in improved thermodynamic stability and a KD
value similar to that of TBA, but also in inhibition of fibrin
clotting as for TBA.
Quadruplexes posses various folding topologies which can be
easily distinguished by different CD profiles.34 CD spectra for all
thermodynamically stable variants (TBA and ON1–ON7) show a
high-amplitude band near 293 nm characteristic for an antiparallel
folding topology (Fig. 5). Moreover, the intensity of the band in CD
profiles of ON1–ON7 is increased in reference toꢀthat of unmodified
TBA. Two modified aptamers with positive
DG37 value (ON8 and
ON9) show significant decrease of characteristic bands intensity
suggesting a disruption of quadruplex structure. TDS profiles sup-
port the results obtained from CD measurements. The TDS shapes
of TBA and ON1–ON7 are specific for intramolecular quadruplex
structures with two maxima at 240 and 273 nm and two minima
at 260 and 295 nm (Fig. 6).31 The irregular TDS profiles observed
for ON8 and ON9 indicate more unstructured states for these
constructs.24
The isothermal titration calorimetry studies revealed that only
the aptamer modified with 20-C-piperazino-UNA-U monomer (X)
in position 7 posses comparable thrombin binding affinity as
unmodified TBA (ON2, Table 2). X-ray studies of a TBA-thrombin
complex revealed that T7 is plunged into a hydrophobic cluster
within the fibrinogen recognition exosite.11,12 Moreover, it was
suggested that a UNA-U monomer introduced in position U7 in-
duces favorable structural changes and in consequence improves
aptamer affinity towards thrombin (KD determination using sur-
face plasmon resonance (SPR)).24 ITC studies show similar trend
for KD change of UNA-modified aptamer in position 7 (variant
U7, Table 2). Slightly worse affinity of ON2 towards thrombin in
reference to TBA and variant U7 suggests that the presence of the
positively charged 20-C-piperazino-UNA-U monomer is decreasing
thrombin affinity. The two remaining aptamers modified by 20-C-
piperazino-UNA-U in position 3 or 12 (ON1 and ON3, respectively)
show more than 20-fold higher KD value, whereas their UNA-mod-
ified counterparts showed only moderate changes in reference to
that of TBA (less than 2-fold change of KD)24 what confirms an
unfavorable impact of the piperazino moiety on the interaction
5. Conclusions
The results described herein confirm that UNA monomers are
suitable tools to achieve extraordinarily stabilized quadruplexes.
Additional functionalization of the UNA-U monomer by a piperaz-
ino moiety decreases free energy of quadruplex formation even
further. The presence of 20-C-piperazino-UNA monomer or several
UNA monomers within the aptamer has a negative effect on bind-
ing affinity and biological activity for the majority of studied

T. B. Jensen et al. / Bioorg. Med. Chem. 19 (2011) 4739–4745
4745
variants. Nevertheless, one of the aptamers, modified by the 20-C-
piperazino-UNA-U monomer at position 7, possess improved ther-
modynamic stability and binding affinity and inhibitory properties
comparable to those of unmodified TBA. Our results provide
insights into specific interactions between TBA and thrombin and
constitute a new alternative of improving the thermodynamic
stability of quadruplexes.
12. Padmanabhan, K.; Tulinsky, A. Acta Crystallogr. Sect. D 1996, D52, 272.
13. Nielsen, P.; Dreiøe, L. H.; Wengel, J. Bioorg. Med. Chem. Lett. 1995, 3, 19.
14. Langkjær, N.; Pasternak, A.; Wengel, J. Bioorg. Med. Chem. 2009, 17, 5420.
15. Jensen, T. B.; Langkjær, N.; Wengel, J. Nucleic Acids Symp. Ser. 2008, 52,
133.
16. Pasternak, A.; Wengel, J. Nucleic Acids Res. 2010, 38, 6697.
17. Mangos, M. M.; Min, K.-L.; Viazovkina, E.; Galarneau, A.; Elzagheid, M. I.;
Parniak, M. A.; Damha, M. J. J. Am. Chem. Soc. 2002, 125, 654.
18. Fluiter, K.; Mook, O. R. F.; Vreijling, J.; Langkjær, N.; Højland, T.; Wengel, J.;
Baas, F. Mol.Biosyst. 2009, 5, 838.
19. Pasternak, A.; Wengel, J. Org. Biomol. Chem. 2011, 9, 3591.
20. Bramsen, J. B.; Laursen, M. B.; Nielsen, A. F.; Hansen, T. B.; Bus, C.; Langkjær, N.;
Babu, B. R.; Højland, T.; Abramov, M.; Van Aerschot, A.; Odadzic, D.; Smicius, R.;
Haas, J.; Andree, C.; Barman, J.; Wenska, M.; Srivastava, P.; Zhou, C.;
Honcharenko, D.; Hess, S.; Muller, E.; Bobkov, G. V.; Mikhailov, S. N.; Fava, E.;
Meyer, T. F.; Chattopadhyaya, J.; Zerial, M.; Engels, J. W.; Herdewijn, P.; Wengel,
J.; Kjems, J. Nucleic Acids Res. 2009, 37, 2867.
21. Bramsen, J. B.; Pakula, M. M.; Hansen, T. B.; Bus, C.; Langkjær, N.; Odadzic, D.;
Smicius, R.; Wengel, S. L.; Chattopadhyaya, J.; Engels, J. W.; Herdewijn, P.;
Wengel, J.; Kjems, J. Nucleic Acids Res. 2010, 38, 5761.
Acknowledgments
The Nucleic Acid Center is a research center of excellence
funded by The Danish National Research Foundation for studies
on nucleic acid chemical biology. We gratefully acknowledge
financial support by The Danish National Research Foundation.
22. Kenski, D. M.; Cooper, A. J.; Li, J. J.; Willingham, A. T.; Haringsma, H. J.; Young, T.
A.; Kuklin, N. A.; Jones, J. J.; Cancilla, M. T.; McMasters, D. R.; Mathur, M.; Sachs,
A. B.; Flanagan, W. M. Nucleic Acids Res. 2010, 38, 660.
23. Vaish, N.; Chen, F.; Seth, S.; Fosnaugh, K.; Liu, Y.; Adami, R.; Brown, T.; Chen, Y.;
Harvie, P.; Johns, R.; Severson, G.; Granger, B.; Charmley, P.; Houston, M.;
Templin, M. V.; Polisky, B. Nucleic Acids Res. 2011, 39, 1823.
24. Pasternak, A.; Hernandez, F. J.; Rasmussen, L. M.; Vester, B.; Wengel, J. Nucleic
Acids Res. 2011, 39, 1155.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.087.
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