Modified DNA Aptamer
A R T I C L E S
DNA because modified DNA produced by PCR is limited due
2
0,21
to the low incorporation efficiency of modified bases.
To
overcome this obstacle, various modified nucleotides have been
synthesized, and their substrate specificities to several kinds of
22-28
DNA polymerases have been studied by a number of groups.
We reported that some triphosphates of thymidine analogues
bearing functional groups at the C5 position could be readily
accepted as a substrate for PCR, forming the corresponding
2
9-31
modified DNA when using KOD Dash DNA polymerase.
The modified DNA bears a cationic ammonium group via a
hydrophobic hexamethylene linker at the C5 of the thymidine
residue. We undertook in vitro selection of thalidomide-binding
aptamers using the modified DNA to prepare an aptamer with
high binding affinity and nuclease-resistance.
Thalidomide, which has significant physiological activity, has
one chiral center. It was used widely as a hypnotic drug, but
was withdrawn from the market in the 1960s because of its
strong teratogenic activity.32,33 Recently, thalidomide has come
into the limelight again as a potential drug for various diseases
Figure 1. Modified dUTP (1), thalidomide, and thalidomide derivatives,
2, 3, and 4, used for in vitro selection.
as a tool for the analysis and biological study of an enantiomer
of thalidomide. Herein, we report the selection of a modified
DNA aptamer that can recognize thalidomide with high enan-
tioselectivity. Although the selection was carried out using a
racemic thalidomide derivative, the selected aptamer clone
showed high binding affinity for the (R)-form thalidomide but
not for the (S)-form.
3
4-40
such as autoimmune disease, AIDS, and some cancers.
It
attracts great interest due to its unique biological activity and
its potential as a lead compound to develop a new biological
response modifier.4 The difference in the biological activity
between the enantiomers of thalidomide is not still clear,
although it has been reported that the biological actions of
1,42
4
3-45
thalidomide are different for the (S)- and (R)-isomers.
Results
Thalidomide is reported to racemize easily under physiological
conditions, and specification of the physiological activities of
the enantiomers of thalidomide is difficult.46-48 Thus, an aptamer
that binds thalidomide with high enantioselectivity will be useful
In Vitro Selection. A DNA aptamer recognizing thalidomide
was selected from a library of modified DNA in which a 60-
nucleotide random region was flanked by 5′- and 3′-constant
primer regions for PCR. The modified DNA library was
prepared by PCR with 5-N-(6-aminohexyl)carbamoylmethyl-
(
20) Latham, J. A.; Johnson, R.; Toole, J. J. Nucleic Acids Res. 1994, 22, 2817-
HM
2
′deoxyuridine triphosphate (T triphosphate, 1) in place of
2
822.
(
21) Battersby, T. R.; Ang, D. N.; Burgstaller, P.; Jurczyk, S. C.; Bowser,
TTP and using KOD Dash DNA polymerase, as 1 was found
M. T.; Buchanan, D. D.; Kennedy, R. T.; Benner, S. A. J. Am. Chem. Soc.
to be a good substrate for this polymerase, but not for Taq DNA
1
999, 121, 9781-9789.
(
22) Sakthivel, K.; Barbas, C. F. Angew. Chem., Int. Ed. 1998, 37, 2872-2875.
23) Lee, S. E.; Sidorov, A.; Gourlain, T.; Mignet, N.; Thorpe, S. J.; Brazier,
J. A.; Dickman, M. J.; Hornby, D. P.; Grasby, J. A.; Williams, D. M. Nucleic
Acids Res. 2001, 29, 1565-1573.
29
polymerase (Figure 1).
(
The terminal protonated ammonium ion, hydrophobic hex-
amethylene linker, and amido linkage at the thymidine residues
in a modified DNA aptamer could all contribute to form a
thalidomide-binding site. In vitro selection of thalidomide-
binding aptamer was carried out according to Scheme 1. A 110
mer DNA pool with a random region of 60 bases was amplified
by PCR with natural nucleotides as substrates.
(24) Gourlain, T.; Sidorov, A.; Mignet, N.; Thorpe, S. J.; Lee, S. E.; Grasby,
J. A.; Williams, D. M. Nucleic Acids Res. 2001, 29, 1898-1905.
(25) Thum, O.; J a¨ ger, S.; Famulok, M. Angew. Chem., Int. Ed. 2001, 40, 3990-
(
(
3
993.
26) J a¨ ger, S.; Rasched, G.; Kornreich-Leshem, H.; Engeser, M.; Thum, O.;
Famulok, M. J. Am. Chem. Soc. 2005, 127, 15071-15082.
27) Perrin, D. M.; Garestier, T.; H e´ l e` ne, C. Nucleosides Nucleotides 1999, 18,
3
77-391.
(
(
28) Held, H. A.; Benner, S. A. Nucleic Acids Res. 2002, 30, 3857-3869.
29) Sawai, H.; Ozaki, A. N.; Satoh, F.; Ohbayashi, T.; Masud, M. M.; Ozaki,
H. Chem. Commun. 2001, 2604-2605.
Each primer contains one ribonucleotide, and thus each strand
of the amplified dsDNA has one ribonucleotide residue (step
(
30) Kuwahara, M.; Takahata, Y.; Shoji, A.; Ozaki, A. N.; Ozaki, H.; Sawai,
H. Bioorg. Med. Chem. Lett. 2003, 13, 3735-3738.
1
). One primer PCR was conducted to prepare modified DNA,
(
31) Ohbayashi, T.; Kuwahara, M.; Hasegawa, M.; Kasamatsu, T.; Tamura, T.;
Sawai, H. Org. Biomol. Chem. 2005, 3, 2463-2468.
using 1 instead of TTP, a forward primer without a ribonucle-
otide portion and the amplified dsDNA (step 2). The one-primer
PCR mixture was treated with 100 mM NaOH at 95 °C for 5
min to hydrolyze the ribonucleotide part in the unmodified DNA
(
(
(
32) Burley, D. M.; Lenz, W. Lancet 1962, 279, 271-272.
33) Mcbride, W. G. Lancet 1961, 278, 1358.
34) Sampaio, E. P.; Kaplan, G.; Miranda, A.; Nery, J. A.; Miguel, C. P.; Viana,
S. M.; Sarno, E. N. J. Infect. Dis. 1993, 168, 408-414.
(
(
35) Parker, P. M.; et al. Blood 1995, 86, 3604-3609.
(step 3). Modified DNA was purified by denaturing PAGE (step
36) Makonkawkeyoon, S.; Limson-Pobre, R. N.; Moreira, A. L.; Schauf, V.;
Kaplan, G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5974-5978.
37) Franks, M. E.; Macpherson, G. R.; Figg, W. D. Lancet 2004, 363, 1802-
4) and applied to in vitro selection with thalidomide (step 5).
For positive selection, thalidomide bearing a linker (2) was
conjugated with biotin (3) and immobilized on a streptavidin
gel. Ethanolamine-biotin conjugate (4) immobilized on the gel
was used for negative selection. The modified DNA pool was
incubated with a negative selection gel to remove a fraction
(
1
811.
(38) Ribatti, D.; Vacca, A. Leukimea 2005, 19, 1525-1531.
(39) Calabrese, L.; Fleischer, A. B. Am. J. Med. 2000, 108, 487-495.
(40) Raja, N.; Anderson, K. N. Engl. J. Med. 1999, 341, 1606-1608.
(41) Hashimoto, Y. Bioorg. Med. Chem. 2002, 10, 461-479.
(42) Galustian, C.; Labarthe, M. C.; Bartlet, B. J.; Dalgleish, A. G. Expert Opin.
Biol. Ther. 2004, 4, 1963-1970.
(
(
(
43) Blaschke, G.; Kraft, H. P.; Fickentscher, K.; K o¨ hler, F. Arzneim.-Forsch.
1
979, 29, 1640-1642.
(46) Knoche, B.; Blaschke, G. J. Chromatogr., A 1994, 666, 235-240.
(47) Nishimura, K.; Hashimoto, Y.; Iwasaki, S. Biochem. Biophys. Res. Commun.
1994, 199, 455-460.
(48) Nishimura, K.; Hashimoto, Y.; Iwasaki, S. Chem. Pharm. Bull. 1994, 42,
1157-1159.
44) Wnendt, S.; Finkam, M.; Winter, W.; Ossig, J.; Raabe, G.; Zwingenberger,
K. Chirality 1996, 8, 390-396.
45) Eriksson, T.; Bjorkman, S.; Roth, B.; Hoglund, P. J. Pharm. Pharmacol.
2
000, 52, 807-817.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 5, 2007 1457