Positively charged base surrogate for highly stable "base pairing" through electrostatic and stacking interactions

Article abstract of DOI:10.1021/ja9013002

(Figure Presented) "Base pairs" of cationic dyes (p-methylstilbazole) were incorporated into oligodeoxyribonucleotides (ODNs). This "base pair" greatly stabilized the duplex through electrostatic and stacking interactions. The melting temperature of modif

Full text of DOI:10.1021/ja9013002

Published on Web 07/07/2009
Positively Charged Base Surrogate for Highly Stable “Base Pairing” through
Electrostatic and Stacking Interactions
Hiromu Kashida,† Hidehiro Ito,† Taiga Fujii,† Takamitsu Hayashi,† and Hiroyuki Asanuma*,†,‡
Graduate School of Engineering, Nagoya UniVersity, Furocho, Chikusa-ku, Nagoya 464-8603, Japan, CREST,
Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Received February 28, 2009; E-mail: asanuma@mol.nagoya-u.ac.jp
Scheme 1a
The design of new artificial nucleobases is an attractive theme
in DNA chemistry. Broadly speaking, there are two trends in
nucleotide design, one of which aims at biocompatible artificial
nucleobases that can be substrates of enzymes, e.g., expansion of
genetic alphabets.1 The other is the design of material-oriented
artificial nucleotides that do not require such biocompatibility but
have excellent supramolecular properties.2 In this context, there is
no limitation on the components of the artificial nucleotides. Even
D-ribose is not necessary as long as a sufficiently stable duplex is
formed.2i
It is well-known that the addition of positively charged molecules,
such as spermine or polycation, strongly stabilize the duplex.3
Hence, the incorporation of positive charges into the oligodeoxy-
ribonucleotide (ODN) is an effective strategy for constructing stable
duplexes at low ionic strength. Recently, Behr et al. covalently
introduced oligospermine at the termini of ODNs to stabilize the
duplex and control its melting temperature.4 However, most artificial
base pairs reported so far utilize only stacking interactions and/or
hydrogen bonding, and direct incorporation of positive charge on
a pseudonucleobase has scarcely been investigated except for metal
a (a) Sequences of modified and native ODNs synthesized. (b) Schematic
representation of stabilization by cationic “base pairs”.
base pairs5 and oligoamine conjugates.4 One of the reasons is the
instability of the N-glycosidic linkage between cationic molecules
and D-ribose under acidic/basic conditions during DNA synthesis.6
Only a few quaternized nucleobases have been reported,7 although
base pairs of protonated bases were widely reported so far.8
Accordingly, this might be the first report on the “base pairing” of
quaternized molecules, which have positive charges irrespective
of pH.
Previously, we described highly stable modified DNA duplex
tethering neutral dyes.9 We found that D-threoninol is a facile but
excellent acyclic scaffold for functional molecules that is compatible
with natural nucleotides.10 In our design, dyes on D-threoninol were
located at the counterpart of each strand to form stacked “base
pairs”. These base surrogates, threoninol nucleotides, raised melting
temperatures through intermolecular stacking as the number of
nucleotides increased, demonstrating that dyes on D-threoninols
worked as artificial “base pairs”.
In the present study, we report a new artificial cationic base
surrogate with D-threoninol, which stabilizes duplexes through both
electrostatic and stacking interactions. D-Threoninol has an amino
residue, to which dyes can be conjugated through an amide bond.
Accordingly, this linker can avoid the above-mentioned synthetic
problems. Here, we incorporated p-methylstilbazole (Z in Scheme
1a) as a cationic pseudonucleobase into ODNs for further stabiliza-
tion of duplexes. Z has a quaternized pyridine ring and a positive
charge regardless of pH. Hence, a Z-Z pair should facilitate duplex
formation through electrostatic interactions between the dye cation
and phosphate anion. In addition, Z has strong SHG activity11 so
that dye aggregates of Z have the potential to be applied as optical
materials.
Our sequence design is depicted in Scheme 1. One to three Z
were introduced into the counterpart of each strand. As a control,
p-stilbazole (B) and p-methylazobenzene (X),12 both of which have
structures similar to that of the Z residue and do not have a
positive charge, were introduced into ODNs.13 The Tm of N/C, the
native duplex without threoninol nucleotides, was 47.7 °C under
the conditions listed in Table 1.14 When one Z-Z pair was
introduced into the N/C duplex (Z1a/Z1b), however, the Tm became
as high as 57.7 °C. This increase with respect to the N/C duplex
(10.0 °C) was larger than that of the X-X pair (X1a/X1b).15 Thus,
the efficient stabilization of the duplex by incorporation of a positive
charge is conclusive. It should be noted that the Tm increase was
much larger than that of a natural A-T (A1a/T1b) or even G-C
(G1a/C1b) pair.
Table 1. Thermodynamic Parameters of Duplexes Synthesized in
This Study
∆H/kcal
∆S/cal
∆G°37/kcal
sequences Tm/°Ca
mol-1
K
-1mol-1
mol-1
∂Tm/∂ln[Na+]b ∆nNa+
Z1a/Z1b
X1a/X1b
N/C
A1a/T1b
G1a/C1b
57.7
53.3
47.7
49.4
53.4
-90.6
-86.2
-89.9
-94.0
-246
-238
-254
-266
-14.1
-12.3
-11.2
-11.6
-13.1
3.7
4.3
4.6
4.7
4.5
1.7
1.9
2.1
2.4
2.4
-100.5 -282
a [ODN] ) 5 µM, [NaCl] ) 100 mM, pH 7.0 (10 mM phosphate
buffer). b [ODN] ) 2 µM, pH 7.0 (10 mM phosphate buffer).
† Nagoya University.
‡ Japan Science and Technology Agency.
9
9928 J. AM. CHEM. SOC. 2009, 131, 9928–9930
10.1021/ja9013002 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Effect of Dye Number on Melting Temperatures
To evaluate the effect of positive charge on the Tm thermody-
namically, the Gibbs free energy, enthalpy, and entropy changes
upon duplex formation were determined from 1/Tm versus ln(CT/
4) plots as listed in Table 1.16 The -∆Go37 of X1a/X1b without a
positive charge was 12.3 kcal mol-1, which was 1.1 kcal mol-1
more stable than N/C.17 However, Z1a/Z1b was even more stable
Tm/°Ca
n
Zna/Znb
Bna/Bnb
Xna/Xnb
0
1
2
3
47.7
52.7
50.9
50.6
57.7
66.2
68.5
53.3
54.8
57.7
than X1a/X1b: -∆Go37 of Z1a/Z1b was as high as 14.1 kcal mol-1
.
-∆H of Z1a/Z1b was 4.4 kcal mol-1 larger than that of X1a/
X1b, indicating that a positive charge on the Z residue exothermi-
cally stabilized the duplex due to electrostatic interaction.18,19
a [ODN] ) 5 µM, [NaCl] ) 100 mM, pH 7.0 (10 mM phosphate
buffer).
-∆Go of Z1a/Z1b was even larger than those of A1a/T1b and
37
of X1a/X1b and N/C were 1.9 and 2.1, respectively: fewer cations
were released from Z1a/Z1b upon dissociation, indicating that the
electrostatic repulsion between phosphate anions was lessened by
cationic dyes.21
G1a/C1b, clearly demonstrating that the Z-Z pair stabilized the
duplex much more than native base pairs did (A-T and G-C).
UV-vis and CD spectra of Z1a/Z1b revealed the stacked
structure of the Z-Z pair in the duplex (see Figure 2).22 At 0 °C
where duplexes were completely formed, a peak at ∼340 nm in
the UV-vis spectrum shifted to a shorter wavelength and became
narrower (see Figure 2a). This hypsochromic shift and narrowing
of the band demonstrated that two Z residues were stacked
antiparallel along the helical axis in the DNA double helix.23 The
coincidence of the Tm at 360 nm (58.8 °C) with that at 260 nm
also supports the hypothesis that Z-Z stacking was associated with
duplex formation (see Supporting Information for melting profiles).
As expected, CD was also induced at ∼360 nm by lowering the
temperature (see Figure 2b). However, its intensity was rather weak
indicating that the Z-Z pair did not wind strongly, because the
threoninol scaffold allowed a firmly stacked, unwound ladder-like
structure due to its flexibility.9
Figure 1. Dependence of Tms of Z1a/Z1b, X1a/X1b, N/C, A1a/T1b, and
G1a/C1b on salt concentration. [ODN] ) 2 µM, pH 7.0 (10 mM phosphate
buffer).
Further introduction of cationic dyes greatly stabilized the duplex.
Table 2 shows the melting temperatures of Zna/Znb, Bna/Bnb,
and Xna/Xnb. Tm monotonically increased when the number of
Z-Z pairs in the duplex increased from one to three. In particular,
the Tm of Z3a/Z3b was 68.5 °C, which was as much as 20 °C
higher compared with N/C (see Figure 3). The melting temperature
of Z3a/Z3b increased as the concentration of the duplex increased
(see Supporting Information). In addition, single-stranded Z3a and
Z3b did not show such hypochromicity, demonstrating that the
sigmoid curve of Z3a/Z3b was unambiguously derived from their
hybridization.24 These results clearly show that multiple incorpora-
tion of Z-Z pairs did not destabilize but instead largely stabilized
the duplex. It should be noted that accumulated positive charges
did not act repulsively but facilitated assembly.25 On the other hand,
incorporation of multiple X-X pairs showed less stabilization. The
Tm of X3a/X3b was ∼10 °C higher than that of the N/C duplex
Figure 1 shows linear plots of Tm with respect to logarithmic
salt concentrations, whose slope gives the number of cations
released upon duplex dissociation (∆nNa+).20 As summarized in
Table 1, ∆nNa+ of Z1a/Z1b was 1.7 (per duplex) whereas ∆nNa+s
Figure 3. Melting profiles of Z3a/Z3b (solid line), single-stranded Z3a
(broken line), and Z3b (dotted line) by monitoring absorbance at 260 nm.
Melting profiles were normalized by the absorbance at 90 °C. [ODN] ) 5
µM, [NaCl] ) 100 mM, pH 7.0 (10 mM phosphate buffer).
Figure 2. (a) UV-vis and (b) CD spectra of Z1a/Z1b at 80, 40, and 0 °C.
[ODN] ) 5 µM, [NaCl] ) 100 mM, pH 7.0 (10 mM phosphate buffer).
9
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C O M M U N I C A T I O N S
(12) Nishioka, H.; Liang, X. G.; Kashida, H.; Asanuma, H. Chem. Commun.
2007, 4354–4356.
but 10 °C lower than that of Z3a/Z3b. Furthermore, the multiple
introduction of p-stilbazole (B) residues did not raise the Tm at all.
Taken together, these results clearly demonstrate that a positive
charge on the dye crucially contributes to the enhanced stabilization
of the duplex.
In conclusion, cationic “base pairs” were successfully incorpo-
rated into an ODN. This “base pair” strongly stabilized duplexes
by electrostatic interactions as well as stacking interactions. Further
stabilization was observed by multiplying the dye number. Due to
this large stabilization, the preparation of dye aggregates without
the assistance of natural bases is promising. This design might allow
for new double helical motifs that are completely different from
the natural duplex.26
(13) Schemes for synthesis of phosphoramidite monomer containing these dyes
are depicted in the Supporting Information. All modified ODNs listed in
Scheme 1a were purified by reversed-phase HPLC and characterized by
MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometry: Z1a:
Obsd m/z 4033 (Calcd for [Z1a+H+]: m/z 4034); Z1b: Obsd m/z 4033
(Calcd for [Z1b+H+]: m/z 4034); Z2a: Obsd m/z 4423 (Calcd for
[Z2a+H+]: m/z 4423); Z2b: Obsd m/z 4423 (Calcd for [Z2b+H+]: m/z
4423); Z3a: Obsd m/z 4813 (Calcd for [Z3a+H+]: m/z 4812); Z3b: Obsd
m/z 4810 (Calcd for [Z3b+H+]: m/z 4812); B1a: Obsd m/z 4019 (Calcd
for [B1a+H+]: m/z 4019); B1b: Obsd m/z 4019 (Calcd for [B1b+H+]:
m/z 4019); B2a: Obsd m/z 4392 (Calcd for [B2a+H+]: m/z 4393); B2b:
Obsd m/z 4393 (Calcd for [B2b+H+]: m/z 4393); B3a: Obsd m/z 4767
(Calcd for [B3a+H+]: m/z 4767); B3b: Obsd m/z 4768 (Calcd for
[B3b+H+]: m/z 4767). X1a: Obsd m/z 4034 (Calcd for [X1a+H+]: m/z
4034); X1b: Obsd m/z 4034 (Calcd for [X1b+H+]: m/z 4034); X2a: Obsd
m/z 4424 (Calcd for [X2a+H+]: m/z 4423); X2b: Obsd m/z 4423 (Calcd
for [X2b+H+]: m/z 4423); X3a: Obsd m/z 4813 (Calcd for [X3a+H+]:
m/z 4812); X3b: Obsd m/z 4813 (Calcd for [X3b+H+]: m/z 4812).
(14) Melting curves of duplex DNA were obtained with a Shimadzu UV-1800
by measuring the change of absorbance at 260 nm versus temperature
(unless otherwise noted). The melting temperature (Tm) was determined
from the maximum in the first derivative of the melting curve. Both the
heating and cooling curves were measured, and the Tm measurements
obtained from them coincided with each other within 2.0 °C. The
Acknowledgment. This work was supported by Core Research
for Evolution Science and Technology (CREST), Japan Science
and Technology Agency (JST). Partial support by a Grant-in-Aid
for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan and The Mitsubishi Founda-
tion (for H.A.) is also acknowledged.
temperature ramp was 0.5 °C min-1
.
(15) The size of X is virtually identical to that of Z. See Supporting Information
for energy-minimized structures of Z and X.
(16) Thermodynamic parameters of duplex (∆H, ∆S) were determined from
1/Tm versus ln(CT/4) plots by the following equation: 1/Tm ) R/∆H ln(CT/
Supporting Information Available: Experimental procedures of
the preparation of modified ODNs and spectroscopic measurements,
UV and CD spectra and melting profiles of duplexes, energy-minimized
structures of dyes and X1a/X1b. This material is available free of charge
via the Internet at http://pubs.acs.org.
4) + ∆S/∆H, where CT is the total concentration of ODNs. ∆Go was
37
calculated from the ∆H and ∆S values. The number of sodium ions released
upon duplex dissociation (∆nNa+) was determined from a Tm versus ln[Na+]
plot by the following equation: ∆nNa+ )-1.11(∆H/RTm2)∂Tm/∂ln[Na+],
where ∂Tm/∂ln[Na+] is the slope of the line in Figure 1. The Tm value at a
total oligomer strand concentration of 4 µM in the presence of 100 mM
NaCl was used. ∆H was determined from a 1/Tm versus ln(CT/4) plot. Errors
of ∆H, ∆S, ∆Go37, and ∆nNa+ were estimated to be 4%, 4%, 1%, and 7%,
respectively.
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minimized structure of X1a/X1b calculated by InsightII/Discover 3.
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