Effect of 3′-end capping of aptamer with various 2′,4′-bridged nucleotides: Enzymatic post-modification toward a practical use of polyclonal aptamers

Article abstract of DOI:10.1016/j.bmcl.2010.01.028

The capping of the 3′-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not affected by the capping. The capping could be simply executed via a one step enzymatic process using 2′,4′-bridged nucleoside 5′-triphosphate and terminal deoxynucleotidyl transferase.

Full text of DOI:10.1016/j.bmcl.2010.01.028

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1626–1629
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effect of 30-end capping of aptamer with various 20,40-bridged nucleotides:
Enzymatic post-modification toward a practical use of polyclonal aptamers
a
Yuuya Kasahara a, Shunsuke Kitadume a, Kunihiko Morihiro b, Masayasu Kuwahara a,c, , Hiroaki Ozaki ,
*
Hiroaki Sawai a, Takeshi Imanishi b, Satoshi Obika b,c
a Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Gunma 376-8515, Japan
b Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
c PRESTO, Japan Science and Technology Agency (JST), Chiyodaku, Tokyo 102-0075, Japan
a r t i c l e i n f o
a b s t r a c t
The capping of the 30-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the
nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not
affected by the capping. The capping could be simply executed via a one step enzymatic process using
20,40-bridged nucleoside 50-triphosphate and terminal deoxynucleotidyl transferase.
Ó 2010 Elsevier Ltd. All rights reserved.
Article history:
Received 9 September 2009
Revised 18 December 2009
Accepted 13 January 2010
Available online 22 January 2010
Keywords:
20,40-Bridged nucleoside-50-triphosphate
Bridged nucleic acid (BNA)
Nuclease resistance
Post-modification
Terminal deoxynucleotidyl transferase
(TdT)
Researchers have created a variety of nucleic acid aptamers
with the systematic evolution of ligands by exponential enrich-
ment (SELEX) method1–3 and studied them in order to apply them
as research tools, biosensors, therapeutic agents and so on. With
this technique, nucleic acid aptamers can be selected from a library
of single-stranded oligonucleotides, differing in nucleotide se-
quence. In general, after being selected from an initial library, indi-
vidual aptamers are isolated from an enriched pool by cloning and
researchers determine their sequences. For practical use, nuclease
resistance is a necessary property of aptamers. Therefore, in order
to gain stability in cell cultures and in vivo, isolated monoclonal
aptamers should be modified with proper substituents, based on
their sequence information and the correlations between modifica-
tions and activities, which may involve costly and monotonous
processes. If an enriched pool, functioning as the polyclonal family,
could gain nuclease resistance without cloning, sequencing and the
aforementioned modifications, the enriched pool, that is, the poly-
clonal aptamer4, could be used as a polyclonal antibody.
we reported the convenient conferring of nuclease resistance on
nucleic acid by using 20,40-bridged nucleoside-50-triphosphates13,14
and terminal deoxynucleotidyl transferase (TdT).15 We were easily
able to add the 20,40-bridged nucleotides to the 30-ends of oligode-
oxy-ribonucleotides (ODNs) with an enzymatic reaction. After the
addition, the nuclease resistances of the ODNs were enhanced,
depending on the chemical structures of the bridged moieties. In
this work, we have designed and synthesized a new 20,40-bridged
nucleoside-50-triphosphate with a 20-CH(Ph)OCH2-40 bridged link-
age, QTP, expecting that the bulkiness of the phenyl group would
interfere with the nuclease degradation of the ODN. Furthermore,
the substrate properties of QTP for TdT and the characterizations
of thrombin binding aptamers (TBAs) capped with various 20,40-
bridged nucleotides for nuclease resistances, biostabilities in hu-
man serum and binding affinities to the target were investigated.
First, we studied the incorporation of the triphosphate QTP dur-
ing the enzyme reaction with TdT, using a 26-mer single-stranded
ODN with 50-FAM-GGC GTT GAG TGA GTG AAT GAG TGA GT-30
(ODN1); the 50-end of ODN1 was labelled with 6-carboxyfluorescein
(6-FAM) for detection. Syntheses and characterizations of QTP and
its intermediates are described in detail in Supplementary data. As
we have reported8, ODNs capped with K, L and M-type bridged
nucleotides were quantitatively produced using KTP, LTP and MTP
(see Figure 1), respectively, after incubating the reaction mixture
Until now, 20-O,40-C-methylene bridged/locked nucleic acid
(20,40-BNA5,6/LNA7) and its analogues8–12, have been developed,
and some of them showed excellent nuclease resistances. Recently,
* Corresponding author.
E-mail address: kuwahara@chem-bio.gunma-u.ac.jp (M. Kuwahara).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.028

Y. Kasahara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1626–1629
1627
Figure 4. The time course of the degradation of thrombin binding aptamers (TBAs)
by phosphodiesterase I; reaction using TBA1 (open squares), TBA-K (closed
diamonds), TBA-L (closed squares), TBA-M (open triangles), and TBA-Q (open
circles). The x-axis indicates the reaction time (min), and the y-axis represents the
relative amount of intact TBAs (%). Total quantities of the products were set at 100%
in each reaction mixture.
ODN capped with a single Q-type bridged nucleotide under the same
conditions. If we increased the enzyme concentration to 10 U/lL and
Figure 1. Chemical structures of 20,40-bridged nucleotides used in this experiment.
extended the incubation time for a few hours, the addition reaction
was almost completed (see Fig. 2). We then assessed the nuclease
resistance of the capped ODN for snake venom phosphodiesterase
I, whichhas a strong30–50 exonucleaseactivity. It wasabout736-fold
morestablethanuncappedODN1and51-, 19-and2.3-foldmoresta-
ble than the other capped ODNs with K, L and M-type bridged nucle-
otides, respectively, under the above reaction conditions (see
Supplementary data Table S1). These results indicated that the bulky
phenyl group on the bridged linkage affected the addition reaction
with TdT but improved resistance for phosphodiesterase I.
The addition of the Q-type bridged nucleotide at the 30-end
greatly enhanced the nuclease resistance; however, ODN1 is not
an oligodeoxyribonucleotide with a special function, such as anti-
sense ODNs16, decoy ODNs17 or DNA aptamers.18 Therefore, we
then performed experiments with the same conditions using a
thrombin binding aptamer19, 50-FAM-AGT CCG TGG TAG GGC
AGG TTG GGG TGA CT-30 (TBA1) and its capped aptamers, TBA-K,
TBA-L, TBA-M and TBA-Q, instead of ODN1 (see Fig. 1). As shown
in Figure 3, the band of the intact TBA1 disappeared after 120 min-
utes of incubation (lane 6, image A) while the bands of the intact
TBA-M and TBA-Q were thick (lanes 6, images D and E). Compared
to the decay curves of intact aptamers in Figure 4, the capped apta-
mers were most stable in the order TBA-Q > TBA-M > TBA-L > TBA-
K in the solution containing the nuclease. The initial reaction rates
Figure 2. Representative gel images of reactions using the 20,40-bridged nucleoside
triphosphates with TdT. The reaction mixtures for the positive control with LTP and
TdT (0.2 U/
incubated for 1 h (lane 3), QTP and TdT (10 U/
QTP and TdT (10 U/ L) incubated for 3 h (lane 5). ODN1 migrated only in lane 1.
lL) incubated for 1 h (lane 2), those containing QTP and TdT (0.2 U/
lL)
l
L) incubated for 2 h (lane 4), and
l
containing 0.4 lM of ODN1, 200 lM of KTP, LTP or MTP, 0.2 U/lL of
TdT and the reaction buffer (an enzyme at 1 ꢀ concentration) for 1 h
at 37 °C. However, the use of QTP did not provide the corresponding
Figure 3. Representative gel images of degradation of TBAs with phosphodiesterase I. Reactions using TBA1 (image A), TBA-K (image B), TBA-L (image C), TBA-M (image D),
and TBA-Q (image E). The reaction mixtures at 0, 10, 20, 30, 60, and 120 min are in lanes 1–6, respectively.

1628
Y. Kasahara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1626–1629
Table 1
Table 2
Degradation of thrombin binding aptamers with snake venom phosphodiesterase I
Degradation of thrombin binding aptamers in 80% v/v human serum
Thrombin binding aptamers
v0 (%/min)a
Relative rate
Thrombin binding aptamers
v0 (%/min)a
Relative rate
TBA1
8.5
2.4
1.6
0.46
0.32
27
7.5
5.0
1.5
1
TBA1
3.5
2.4
1.3
0.88
1.1
4.0
2.7
1.5
1
TBA-K
TBA-L
TBA-M
TBA-Q
TBA-K
TBA-L
TBA-M
TBA-Q
1.2
a
a
Initial rates of degradation v0 were calculated by the fitting of their decay
Initial rates of degradation v0 were calculated by the fitting of their decay
curves, respectively.
curves, respectively.
estimated from the decay curves of TBA1, TBA-K, TBA-L, TBA-M and
TBA-Q were 8.5, 2.4, 1.6, 0.46 and 0.32, respectively (see Table 1).
The capping effect of TBA1 was smaller than that of ODN1 presum-
ably because the aptamer formed a specific conformation, that is,
G-quadruplex,20,21 which would have decreased the accessibility
of the nuclease to the aptamer. However, the capping effect with
bridged nucleotides clearly reflected in the nuclease resistances
and the correlations between the chemical structures of the
bridged linkages and the nuclease resistances were extremely con-
sistent with the case of ODN1.
Next, we examined the biostabilities of these capped TBAs in
human serum containing various types of nucleases. As shown in
Figure 5, TBA-M and TBA-Q were apparently more stable than
the others; more than 20% of the intact TBA remained in the cases
of these two aptamers while the intact TBA was completely gone in
the case of the uncapped TBA1 (see Supplementary data Figure S1)
after incubating in 80% v/v human serum for 8 hours at 37 °C. The
relative rates of the degradation showed that TBA-M and TBA-Q
were about 4.0 and 3.3-fold more stable than TBA1 (see Table 2),
but on the other hand, TBA-Q was more stable than TBA-M in solu-
tion containing phosphodiesterase I. The difference in the stabili-
ties was small, but we observed this tendency had good
reproducibility, indicating that it may be due to the difference of
the substrate specificities between phosphodiesterase I and 30–50
exonuclease contained in human serum and not to any measure-
ment errors. The capping effect for the stability in human serum
compared to that in phosphodiesterase I solution was significantly
smaller because of the action of endonuclease contained in the ser-
um. However, the effect on the nuclease resistance was still clearly
observed, indicating that the action of 30 to 50 exonuclease would
be significantly dominant compared to that of any other types of
nuclease in human serum. Recently, Peng et al. reported an effort
of enhancing the nuclease resistance of a thrombin binding apt-
amer in the sequence d(G2T2G2TGTG2T2G2) by modification with
Figure 6. Representative capillary electropherograms for individual thrombin-TBA
complexes. The x-axis indicates the migration time (min), and the y-axis represents
the relative intensity of signals from emission of 50-FAM detected at 520 nm
(excited at 488 nm). The peak with the asterisk (*) corresponds to TBA-M
dissociated from thrombin during CE, which might conform a particular structure.
The concentrations of TBAs and thrombin were 1 nM and 4 nM. The sample buffer
and running buffer were 20 mM Tris–HCl (1 mM MgCl2) at pH 7.4 and 100 mM
sodium borate at pH 8.35, respectively. Experimental conditions: separation
voltage, +12 kV; injection 0.5 psi ꢀ 7.8 s.
20-deoxy-20-fluoro- -arabinonucleotide.22 Some modified apta-
D
mers exhibited increased nuclease resistances (4–7-fold) in 10%
v/v fetal bovine serum at 37 °C without losing their binding abili-
ties. However, gaining stability in the serum required at least four
replacements with the modified nucleotide. Furthermore, some
modified aptamers almost lost their binding abilities completely
depending on the numbers and positions of the replacement
points. Thus, conferring nuclease resistance on aptamers by post-
modification23 is not very easy in general.
Finally, we measured the binding affinities of capped TBAs to
human thrombin using non-equilibrium capillary electrophoresis
of equilibrium mixtures (NECEEM)24 in order to assess the capping
effect on the binding ability of the aptamer25 (Fig. 6). The Kd values
for the target-TBA interactions were determined from the biding
saturation curves of TBA with different concentrations of thrombin
as seen in Figure S3. The Kd values of TBA1, TBA-K, TBA-L, TBA-M
and TBA-Q for the target were 0.25 0.06, 0.18 0.04,
0.27 0.05, 0.21 0.06 and 0.28 0.09 nM, respectively; those
affinities were found to be almost the same level.
In conclusion, the capping of the 30-ends of thrombin binding
aptamers with bridged nucleotides increased the nuclease resis-
tances 3.6–27-fold and the stabilities in human serum 1.5–4.0-fold.
Also, the binding abilities of the aptamers were not affected by the
capping. The capping could be simply executed via a one step enzy-
matic process using 20,40-bridged nucleoside 50-triphosphate and
TdT. The merit of this method is in developing a method that will
enable us to modify and use aptamers without isolating them from
the polyclonal family. The chemical structure of the bridged linker
should be improved further. Particularly, it is interesting that
Figure 5. The time course of degradation of thrombin binding aptamers (TBAs) in
80% v/v human serum; reaction using TBA1 (open squares), TBA-K (closed
diamonds), TBA-L (closed squares), TBA-M (open triangles), and TBA-Q (open
circles). The x-axis indicates the reaction time (min), and the y-axis represents the
relative amount of intact TBAs (%). Total quantities of the products were set at 100%
in each reaction mixture.

Y. Kasahara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1626–1629
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