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13. Guieysse, A. L.; Praseuth, D.; Helen, C. J. Mol. Biol.
1997, 267, 289.
100
80
60
40
20
0
Concentration of TBP
100 nM
500 nM
1000 nM
´
14. Jimenez-Garacıa, E.; Vaquero, A.; Espinas, M. L.; Soliva,
R.; Orozco, M.; Bernues, J.; Azorın, F. J. Biol. Chem.
´
´
´
´
1998, 273, 24640.
15. Musso, M.; Nelson, L. D.; Van Dyke, M. W. Biochemistry
1998, 37, 3086.
`
16. Musso, M.; Bianchi-Scarra, G.; Van Dyke, M. W. Nucleic
Acids Res. 2000, 28, 4090.
17. Nelson, L. D.; Musso, M.; Van Dyke, M. W. J. Biol.
Chem. 2000, 275, 5573.
18. Guillonneau, F.; Guieysse, A. L.; Le Caer, J. P.; Rossier,
J.; Praseuth, D. Nucleic Acids Res. 2001, 29, 2427.
19. Rao, J. E.; Craig, N. C. J. Mol. Biol. 2001, 307, 1161.
mis
linked-
G3-G4-
G4-G3
mis linked-
G3-G4-
G4-G3
G3-G4-
G4-G3
G3-G4-
G4-G3
`
20. Ciotti, P.; Van Dyke, M. W.; Bianchi-Scarra, G.; Musso,
M. Eur. J. Biochem. 2001, 268, 225.
21. Li, G.; Tolstonog, G. V.; Traub, P. DNA Cell Biol. 2002,
21, 163.
Figure 6. Binding ratio of TBP to the triplexes, G3–G4–G4–G3, mis-G3–
G4–G4–G3, linked-G3–G4–G4–G3, and mis-linked-G3–G4–G4–G3.
22. Ueno, Y.; Takeba, M.; Mikawa, M.; Matsuda, A. J. Org.
Chem. 1999, 64, 1211.
23. Ueno, Y.; Mikawa, M.; Hoshika, S.; Matsuda, A.
Bioconjugate Chem. 2001, 12, 635.
24. Hoshika, S.; Ueno, Y.; Matsuda, A. Bioconjugate Chem.
2003, 14, 607.
25. Kim, S. J.; Bang, E.-K.; Kim, B. H. Synlett 2003, 1838.
26. Sinha, N. D.; Biernat, J.; Ko¨ster, H. Tetrahedron Lett.
1983, 24, 5843.
with the anthraquinonyl group. The binding ability of
the MBP-LOR3ARF protein to the triplexes was evaluat-
ed by the EMSA. It was found that the triplex, which
has an anthraquinonecarbonyl group at the 50-end of
the third strand and is connected with the pentaerythri-
tol linker, has greater affinity to the protein than an
unmodified triplex. Potaman and Sinden reported that
peptides composed of basic amino acids such as Lys
and Arg thermally stabilize triplexes.29,30 The MBP-
LOR3ARF protein also has clusters comprised of basic
amino acids at its C-terminus region. Thus, the MBP-
LOR3ARF protein will be a good stabilizer of triplexes
in antisense strategy utilizing the branched ONs.
27. The MBP-LOR3ARF protein was prepared as follows: The
cDNA encoding LOR3ARE fragment was PCR-amplified
from human keratinocyte cDNA library (Clontech) using
a primer set, 50-GGGGATCCGAGGGGGGTCGTCCG
GCG-30 and 50-GCGAATTCCATGAGAGCTCTAAG
CCCATC-30, as described by Ciotti et al.20 The PCR
product was cloned to pET-21a expression vector at the
BamHI and EcoRI restriction sites (pET-LOR3ARF). A
0.3 kb-DNA fragment was obtained from pET- LOR3ARF
by digesting with BamHI and HindIII, and was subcloned
to pEM vector (pEM- LOR3ARF). pEM vector was
constructed by inserting MBP cDNA in pET-22b. MBP
cDNA was obtained by PCR with a primer set, 50-
ATAGCATATGAAAATCGAAGAAGGT-30 and 50-
CGCTTCTGCGTTCTGATTTA-30, using pMAL-c2
vector (New England Biolabs) as a template. LOR3ARF
was expressed as MBP N-terminal and 6· His C-terminal
fusion protein in Escherichia coli strain JM109 (DE3).
Bacteria were grown in LB containing 50 lg/ml ampicillin
at 30 ꢁC. The expression was induced by adding 0.5 mM
IPTG to the culture at 25 ꢁC. The recombinant protein
was purified from E. coli using TALONTM Metal Affinity
Resins followed by Amylose ResinTM.
Acknowledgment
This research was supported in part by a Grant-in-Aid
for Scientific Research (C) (KAKENHI 16590082) from
Japan Society for the Promotion of Science (JSPS).
References and notes
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1. Praseuth, D.; Guieysse, A. L.; Helen, C. Biochim. Biophys.
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1993, 32, 666.
4. Giovannangeli, C.; Montenay-Garestier, T.; Rougee, C.
´ `
28. Electrophoretic mobility shift assays: Triplex (50 nM)
labeled at the 50-end of the 1st strand by 32P was incubated
in the presence of various amounts of the MBP-LOR3ARF
protein in a 15 mM Tris–HCl buffer (pH 7.0) containing
25 mM NaCl, 5 mM MgCl2, and 10% glycerol (total
20 lL) at room temperature for 1 h. The mixture was
analyzed by electrophoresis on 5% nondenaturing poly-
acrylamide gel (19:1 acrylamide/bisacrylamide) using a TB
buffer (22 mM Tris borate, pH 8, 0.5 mM EDTA) at 80–
100 mV for 2 h at 4 ꢁC. Density of radioactivity of the gel
was visualized by a Bio-imaging Analyzer (Bas 2500, Fuji,
Co. Ltd.).
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Sasisekharan, V. J. Am. Chem. Soc. 1995, 117, 6416.
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Nucleic Acids Res. 1995, 23, 1204.
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jugate Chem. 2003, 14, 684.
29. Potaman, V. N.; Sinden, R. R. Biochemistry 1995, 34,
14885.
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U.S.A. 1991, 88, 10450.
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