Metal ion-directed cooperative triple helix formation of glutamic acid-Oligonucleotide conjugate [7]

Full text of DOI:10.1021/ja0027362

1772
J. Am. Chem. Soc. 2001, 123, 1772-1773
Metal Ion-Directed Cooperative Triple Helix
Formation of Glutamic Acid-Oligonucleotide
Conjugate
Toshihiro Ihara,* Yuka Takeda, and Akinori Jyo
Department of Applied Chemistry and Biochemistry
Faculty of Engineering, Kumamoto UniVersity
Kurokami, Kumamoto 860-8555, Japan
ReceiVed July 25, 2000
In chromosome DNAs, there are many repetitive sequences,
such as the dyad symmetrical and tandem repeat sequences, which
play important roles in organisms. For example, many kinds of
1
Figure 1. Schematic illustration of metal ion-directed cooperative triple-
helix formation of the conjugates on the palindrome sequence.
transcription regulatory factors recognize the dyad symmetrical
sequences. Certain kinds of tandem repeat sequences are also
known to be associated with certain genetic diseases. Also, the
sequence of TTAGGG in humans is repeated dozens or even
thousands of times at each chromosome end, called telomeres.
Our attention is directed to the specific recognition of such
repetitive sequences on DNA in a smart manner, that is,
cooperative action between the units of the DNA binding ligands
2+
Now, the metal ion used here was Cu , which is the more
common ion in organisms and is related to several neurological
diseases such as Alzheimer’s disease and ALS (amyotrophic
7
lateral sclerosis). It is well-known that amino acids form a two-
2
+
8
to-one complex with Cu under the appropriate conditions.
2+
Therefore, Cu , as a cooperative factor, would gather two
GluODNs into a dimer on the complementary C2 symmetrical
sequence of ds-DNA. This is also regarded as a novel methodol-
2
3
4
such as small molecules, peptides, proteins, and oligonucleo-
5
tides (ODNs). We have already reported the specific recognition
9
ogy for recognizing the Pu-Py mixed-sequences (Figure 1).
of the C2 symmetrical sequence on double-stranded DNA (ds-
DNA) by ODN conjugated with iminodiacetic acid (IDA) through
the triple-helix formation, in which each IDA moiety of the two
conjugates coordinates to a rare earth metal to give the dyad
ODNs used in this study were prepared using a fully automated
DNA synthesizer (Beckman, Oligo 1000M). The synthesized
ODNs are indicated below.
6
symmetric dimer on the targeted palindrome sequence.
The aims of this study are the generalization of such a strategy
for the metal-assisted cooperative recognition of the C2 sym-
metrical sequences through triple-helix formation and the quan-
titative assessment of the cooperativity. Here, we synthesized the
conjugate (GluODN) between ODN and glutamic acid (Glu).
*
To whom any correspondence should be addressed. Phone:
81-96-342-3873. Fax: +81-96-342-3873 or 3679. E-mail: toshi@
chem.kumamoto-u.ac.jp.
1) For example: (a) Watson, J. D.; Hopkins, N. H.; Roberts, J. W.; Steitz,
GluODN was synthesized by the coupling between the activated
ester of Glu (N-R-Fmoc-L-glutamic acid γ-succimide ester) and
the 5′-aminohexyl-linked ODN (aminoODN) by following the
procedures previously published (Figure 2).10 f44 is the target
duplex that contains the quasi-dyad symmetrical sequence com-
posed of two 14-base purine tracts (shown in bold letters)
separated by 4 base pairs. On the other hand, h20 has an isolated
half-site for binding, so that the dimerization of GluODN on the
h20 duplex is impossible.
+
(
J. A.; Weiner, A. M. Molecular Biology of the Gene, 4th ed.; The Benjamine/
Cummings Publishing Company, Inc.: Menlo Park, CA, 1987. (b) Baba, Y.
J. Chromatogr. B 1996, 687, 271. (c) Cech, T. R. Angew. Chem., Int. Ed.
2
000, 39, 35.
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(
W.; Wilson, W. D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12. (b) Guelev,
V.; Harting, M. T.; Lokey, R. S.; Iverson, B. L. Chem. Biol. 2000, 7, 1. (c)
Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage, B. A. J.
Am. Chem. Soc. 1999, 121, 2987. (d) Hamdan, I. I.; Skellern, G. G.; Waigh,
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R. F.; Giannetto, A.; Pagano, P.; Gibbs, E. J. J. Am. Chem. Soc. 1991, 113,
The circular dichroism spectrum of the solution of GluODN/
f44 indicated the typical features of the triple-stranded complex
2+
(
ts-complex) of DNA in the presence of a half mole of Cu
toward GluODN under the appropriate conditions (data not
shown).11 The stability of the ts-complex containing GluODN
7
799. (i) Nord e´ n, B.; Tjerneld, F. Biophys. Chem. 1977, 6, 31.
3) (a) Aizawa, Y.; Sugiura, Y.; Ueno, M.; Mori, Y.; Imoto, K.; Makino,
2+
and the Cu effect on it were assessed by melting experiments
(
using a UV-vis spectrophotometer equipped with a program-
K.; Morii, T. Biochemistry 1999, 38, 4008. (b) Ranganathan, D.; Patel, B. K.;
Mishra, R. K. J. Chem. Soc., Chem. Commun. 1994, 107. (c) Palmer, C. R.;
Sloan, L. S.; Adrian, J. C., Jr.; Cuenoud, B.; Paolella, D. N.; Schepartz, A. J.
Am. Chem. Soc. 1995, 117, 8899. (d) Predki, P. F.; Sarkar, B. Biochem. J.
2
+
mable temperature controller (Figure 3). A half mole of Cu
(7) (a) Wadsworth, J. D. F.; Hill, A. F.; Joiner, S.; Jackson, G. S.; Clarke,
A. R.; Collinge, J. Nature Cell Biol. 1999, 1, 55. (b) Lamb, A. L.; Wernimont,
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1
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Giraldo, R.; Andreu, J. M.; D ´ı az-Orejas, R. EMBO J. 1998, 17, 4511. (c)
Walhout, A. J. M.; van der Vliet, P. C.; Timmers, H. Th. M. Biochim. Biophys.
Acta 1998, 1397, 189. (d) White, A.; Ding, X.; vanderSpek, J. C.; Murphy, J.
R.; Ringe, D. Nature 1998, 394, 502. (e) Terwilliger, T. C. Biochemistry 1996,
(8) Sill e´ n, L. G.; Martell, A. E.; H o¨ gfeldt, E.; Smith, R. M. Stability
Constants of Metal-Ion Complexes; Pergamon Press: Oxford, 1979.
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Marchand, C.; Sun, J.-S.; Bailly, C.; Waring, M. J.; Garestier, T.; H e´ l e` ne, C.
Biochemistry 1998, 37, 13322. (c) De Napoli, L.; Messere, A.; Montesarchio,
D.; Pepe, A.; Piccialli, G.; Varra, M. J. Org. Chem. 1997, 62, 9024.
(10) (a) Ihara, T.; Maruo, Y.; Takenaka, S.; Takagi, M. Nucleic Acids Res.
1996, 24, 4273. (b) Takenaka, S.; Uto, Y.; Kondo, H.; Ihara, T.; Takagi, M.
Anal. Biochem. 1994, 218, 436. (c) Bodanszky, M. Peptide Chemistry: A
Practical Textbook; Springer-Verlag: Berlin, 1988. (d) Hermanson, G. T.
Bioconjugate Techniques; Academic Press: San Diego, 1996.
3
5, 16652. (f) Kim, B.; Little, J. W. Science 1992, 255, 203.
5) (a) Koval, V. V.; Lokteva, N. A.; Karnaukhova, S. L.; Fedorova, O. S.
(
J. Biomol. Struct. Dyn. 1999, 17, 259. (b) Lokhov, S. G.; Koshkin, A. A.;
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1
0.1021/ja0027362 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/02/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 8, 2001 1773
Figure 2. Synthesis of GluODN: (a) NHS, DCC, DMAP, dioxane; (b)
TFA; (c) aminoODN, DMSO/water; and (d) 10% NEt H/DMF.
2
a
Table 1. Thermodynamic Parameters for the Triple-Helix
Formation of GluODN with a Binding Site on f44 in the Presence
2
+
and the Absence of Cu
∆
H/
∆S/
cal mol
∆G(298 K)/ K(298 K)/
-1 -1
-
1
-1
kcal mol
kcal mol
M
Tm/K
6
none
+
-89.5 ( 0.4 -271.8 ( 1.4
-71.0 ( 1.0 -204.8 ( 3.3
-8.50
-9.97
1.72 × 10 296.1
2
+
7
Cu
2.05 × 10 304.5
a
The parameters, which were for bimolecular interaction between
GluODN and a binding site on f44, were refined by fitting the
1
2
experimental data with theoretical curves, both of which were shown
in Figure 3.
(
0.5 µΜ) toward GluODN (1.0 µΜ) substantially stabilized the
Figure 3. UV melting curves recorded at 260 nm for the complexes of
ts-structure; the difference between the melting temperatures in
2
+
the presence and in the absence of Cu2+ was ca. 8 °C. On the
GluODN with f44 in the presence and the absence of Cu . Melting
experiments were carried out in a buffer solution containing 50 mM MgCl
and 1.0 mM HEPES (pH 7.0), and the solutions were heated at a rate of
2
other hand, the melting temperatures obtained by the control
systems, c14/f44 and GluODN/h20, were scarcely changed by
the addition of Cu . The ionic strength under all conditions is
essentially the same, because the salts (1.0 mM HEPES and 50
mM MgCl
-
1
2+
0
0
.5 deg min . The concentrations of GluODN, f44, and Cu were 1.0,
.5, and 0.5 µM, respectively. (a) Both curves showed biphasic features.
2+
The transitions at lower temperatures are the meltings of the triplex to
2
) exist in large excess. Increasing concentration of
the duplex. The solid and broken curves indicate the melting behavior in
2+
Cu up to 1.0 and 1.5 µM hardly affects the melting curve
2+
the presence and the absence of Cu , respectively. (b) Curve fitting to
2
+
obtained from the GluODN/f44 system at 0.5 µM Cu .
Therefore, these results clearly indicate that the stabilization of
the ts-complex observed here is due to the cooperative action of
the two molecules of GluODNs for binding to f44 in the presence
the theoretical equation. The points and the curves are the experimental
data and theoretical curves, respectively.
constant for a kind of disproportionation reaction, is defined as
follows:
2+
of Cu , which plays a critical role as an allosteric effector.
The thermodynamic parameters for the binding of GluODN
with each binding site on target f44 could be estimated by curve
fitting to the theoretical equations assuming the two-state model
where the melting processes should be an all-or-none transition.12
The parameters refined by the nonlinear least squares are
summarized in Table 1. The ts-complex seems to be stabilized
2+
by an entropic factor in the presence of Cu . Considering the
binding constant between Cu2 and the amino acids in the
literature, a quantitative one-to-two complexation is not conceiv-
able under the experimental conditions (especially, pH and the
+
where K1R and K1L are equilibrium constants for binding of the
first GluODN on the right and on the left binding sites on f44,
respectively. K2R and K2L are similar constants for the second
ones on the residual half sites. According to a previously proposed
procedure, the ω value was estimated to be ca. 165 at 298 K.
This means that the first binding of GluODN on f44 magnifies
8
concentration of each component). Therefore, dimerization of
GluODNs in the bulk solution (prior to binding with f44) is not
likely. Cupric ion presumably binds to the Glu moiety in the one-
to-one complex of GluODN/f44 with the assistance of a negative
electrostatic field in the microenvironment in the vicinity of the
ODN’s backbone and then facilitates the subsequent accommoda-
tion of the second GluODN to the neighboring residual half-
binding site on f44. As a result, the addition of Cu2 made the
apparent binding constant of GluODN at 298 K increase more
than 10 times.
5b
the binding constant of the second one by 165 times in the
2+
presence of a half mole of Cu and the difference in the free
+
-1
energy of the first and the second binding is ca. 3.0 kcal mol .
Cooperative binding by the units having a DNA binding ability
should result in a higher sequence specificity as well as a greater
sensitivity to concentration changes. When using the conjugates
modified at both ends, the strategy presented here, metal-directed
cooperative recognition, would be extendable to other repetitive
sequences such as tandem repeats and the telomere.
However, the thermodynamic parameters estimated from the
UV-melting experiments, ∆H, ∆S, and K, represent a combination
of parameters corresponding to the first binding of GluODN to
an isolated complementary site on f44 and those corresponding
to subsequent binding to a neighboring site in the cooperative
mode. To assess the cooperativity, we carried out the quantitative
treatment of the results according to Weber’s method.13 The
cooperativity, ω, which is also regarded as the equilibrium
Acknowledgment. We thank Professor Y. Katayama for advice
concerned with the synthesis of GluODN.
1
Supporting Information Available: Synthesis scheme, 400 MHz H
NMR data, and the MALDI-TOF mass spectrum for GluODN as well
as the procedure of UV-melting experiments (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
(
12) Petersheim, M.; Turner, D. H. Biochemistry 1983, 22, 256.
(
13) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry III: The
BehaVior of Biological Macromolecules; W. H. Freeman and Company: New
York, 1980.
JA0027362