Published online September 27, 2004
Nucleic Acids Research, 2004, Vol. 32, No. 17 5113–5118
doi:10.1093/nar/gkh849
Method for direct discrimination of intra- and
intermolecular hydrogen bonds, and characterization
of the G(:A):G(:A):G(:A):G heptad, with scalar
couplings across hydrogen bonds
Hidetsugu Sotoya, Akimasa Matsugami, Tetsuro Ikeda, Kiyoshi Ouhashi, Seiichi Uesugi
and Masato Katahira*
Department of Environment and Natural Sciences, Graduate School of Environment and Information Sciences,
Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
Received August 17, 2004; Revised and Accepted September 8, 2004
ABSTRACT
Discrimination of intra- and intermolecular hydrogen
bonds in a symmetric multimer has not been accomplished yet, although such discrimination would provide a crucial basis for construction of the multimeric
architecture of nucleic acids by NMR. We have developed a direct and unambiguous method for such
discrimination involving the use of scalar couplings
across hydrogen bonds. The method has been validated with a symmetric dimer of d(GGGCTTTTGGGC),
for which the structure including both intra- and
intermolecular hydrogen bonds was already reported.
This has demonstrated that our method can clearly
discriminate these two kinds of hydrogen bonds.
Then, the method was applied to a symmetric dimer
of d(GGAGGAGGAGGA) and has provided
decisive information on its multimeric architecture.
Additionally, the values for scalar couplings across
hydrogen bonds for G:G and G:A base pairs in
the
G(:A):G(:A):G(:A):G
heptad
formed
by
d(GGAGGAGGAGGA) were determined for the first
time. This determination has provided an insight
into the nature of the heptad.
INTRODUCTION
GGA triplet repeats are widely dispersed throughout
eukaryotic genomes, and are frequently located within biologically important regions such as gene regulatory regions
and recombination hot spot sites (1–5). It was reported that
d(GGAGGAG) forms a unique quadruplex structure (6).
We determined the structure of d(GGAGGAGGAGGA)
(GGA 12mer) under physiological conditions, and found
the formation of an intramolecular parallel quadruplex for
the first time (7). Later, a similar intramolecular
parallel quadruplex was found for a telomere DNA in the
crystalline state (8). We also determined the structure of
d(GGAGGAGGAGGAGGAGGAGGAGGA) (GGA 24mer)
under physiological conditions, and demonstrated intramolecular higher-order packing of quadruplexes at atomic
resolution for the first time (9).
The structure of GGA 12mer is schematically shown in
Figure 1A. GGA 12-mer forms a dimer. We noticed, however,
that it is difficult to rule out the possibility of the other structure
shown in Figure 1B. The two structures have the common features of being a dimer, and having two G:G:G:G tetrads and two
G(:A):G(:A):G(:A):G heptads (Figure 1C) per dimer. A clear
difference exists in the mode of base pairing. For example, the
G4:G7 base pair of the heptad is formed within each monomer in
the case of Figure 1A, while it is formed between two monomers
in the case of Figure 1B. Therefore, one of the two possible
structures can be selected if we can determine whether the
G4:G7 base pair is intra- or intermolecular.
Discrimination of intra- and intermolecular hydrogen
bonds in a symmetric multimer has not been accomplished
yet. Isotope-editing/filtering techniques can tell us whether a
NOESY cross-peak is intra- or intermolecular. Therefore,
discrimination of the two kinds of hydrogen bonds may
be possible if information such as the proton pairs closest
to the hydrogen bond are gathered. This is, however, an
indirect method. Moreover, often there is no appropriate
proton pair close to a hydrogen bond. Here, we present a
method for direct discrimination of the two kinds of hydrogen bonds. The method utilizes the scalar couplings across
hydrogen bonds of nucleic acids (10–20). First, for its validation, the method has been applied to a dimer of
d(GGGCTTTTGGGC), the hydrogen bonding schemes for
which are already known. This has confirmed the usefulness
of the method. Next, the method has been applied to the
dimer structure of GGA 12mer. A decisive conclusion as to
the multimeric architecture has been obtained on the basis of
the unambiguous discrimination of intra- and intermolecular
hydrogen bonds.
Additionally, the values for 2hJNN scalar couplings across
hydrogen bonds have been obtained for the first time for G:G
and G:A base pairs in the G(:A):G(:A):G(:A):G heptad formed
in the dimer of GGA 12mer. The character of the
G(:A):G(:A):G(:A):G heptad has been addressed on the
basis of the 2hJNN values.
*To whom correspondence should be addressed. Tel: +81 45 339 4264; Fax: +81 45 339 4264; Email: [email protected]
Nucleic Acids Research, Vol. 32 No. 17 ª Oxford University Press 2004; all rights reserved
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Nucleic Acids Research, 2004, Vol. 32, No. 17
C-18 reverse phase column (Waters) (22). Corresponding
non-labeled DNAs were purchased and purified as described
previously (23). Each DNA was dissolved in 10 mM sodium
phosphate buffer (pH 6.5–6.7) containing 100–150 mM KCl
and 3 mM NaN3. The DNA concentrations were 0.7–1.0 mM.
DSS was used as an internal chemical shift reference. Samples
were heated at 95 C for 5 min, followed by gradual cooling
to room temperature prior to the measurements.
NMR spectroscopy
NMR spectra were recorded with a Bruker DRX600 spectrometer equipped with a quadruple-resonance probe with
x-, y- and z-gradients. HNN-COSY spectra (10,11) were
recorded with a reported pulse sequence (16) with the modification of replacement of rectangular 180 pulses with chirp
180 pulses for 15N. The 15N carrier was set at 155 p.p.m. The
HNN-COSY spectra were recorded with both a 13C, 15Nlabeled sample, and a 1:1 mixture of 13C, 15N-labeled and
non-labeled samples for each DNA. In both cases, the concentration of total DNA was the same. The 2hJNN value was
determined from the HNN-COSY spectra recorded with the
13
C, 15N-labeled sample on the basis of the following equation:
Ic =Id = tan2 2p2h JNN T ,
where Ic and Id are the intensities of cross and diagonal peaks,
and 2T is a constant time for the transfer of magnetization
between nitrogens (10). Errors were estimated from noise
levels. The 2hJNN value between AN6 and GN3 was also
determined from spin-echo difference constant time HSQC
spectra recorded with the 13C, 15N-labeled sample on the
basis of the following equation:
Iactive =Iinactive = cos 2p2h JNN T ,
where Iactive and Iinactive are the intensities of correlation peaks
for experiments in which the coupling between nitrogens is
active and inactive, respectively, and 2T is a constant time
during which the coupling is active (13). The 15N carrier was
set at 80 p.p.m. The selective 15N inversion pulses were phasemodulated at 75 p.p.m. so as to be centered at GN3
(155 p.p.m.). Spectra were processed with XWIN-NMR (Bruker), NMRPipe (24), and Capp/Pipp/Stapp (25).
Figure 1. (A and B) Schematic representation of two possible dimeric
architectures of d(GGAGGAGGAGGA). The monomers are colored black
and red, respectively. (C) The G(:A):G(:A):G(:A):G heptad.
MATERIALS AND METHODS
Sample preparation
Uniformly 13C, 15N-labeled DNAs, d(GGAGGAGGAGGA),
d(GGAGGAGGAGGAGGAGGAGGAGGA) and d(GGGCTTTTGGGC), were synthesized by the hairpin extension
method (21), using a Klenow fragment (30 –50 exo) (Daiichi
Kagaku Ltd) with 13C, 15N-labeled dNTPs (Nippon Sanso
Ltd). After alkaline hydrolysis, each labeled DNA was
separated from the template by denaturing polyacrylamide
gel electrophoresis. Each labeled DNA was extracted from
the corresponding gel by crushing and soaking, and then
desalted on either a DEAE anion-exchange column or a
RESULTS AND DISCUSSION
Principle of the discrimination of intra- and
intermolecular hydrogen bonds
For such discrimination, scalar coupling across a hydrogen
bond is utilized. In the present case, in particular, two-bond
scalar coupling between nitrogens across a hydrogen bond,
2h
JNN, is used. The discrimination relies on the fact that the
presence of 2hJNN gives a cross-peak in a HNN-COSY experiment (10,11). In the first experiment, the intensity of the crosspeak in the HNN-COSY experiment is recorded for a labeled
material. In the second experiment, the intensity is recorded
for a 1:1 mixture of labeled and non-labeled materials. In both
the experiments, the total amount of the material is kept
constant. It should be noted that for detection of the crosspeak, both the nitrogen atoms involved in a hydrogen bond
Nucleic Acids Research, 2004, Vol. 32, No. 17
should be 15N-labeled. Now, for simplicity, let us take the case
of a symmetric dimer structure. If a hydrogen bond is
intramolecular, the intensity of the cross-peak in the second
experiment should be half of that in the first experiment,
because the amount of the labeled monomer in the second
experiment is half of that in the first experiment. If a hydrogen
bond is intermolecular, on the other hand, the intensity of the
cross-peak in the second experiment should be a quarter of that
in the first experiment, because the amount of the dimer in
which both monomers are labeled in the second experiment is
a quarter of that in the first experiment. Thus, based on the
extent of the intensity reduction of the cross-peak, intra- and
intermolecular hydrogen bonds can be distinguished. It should
be added that this discrimination method does not need any
advance information on the structure.
The same principle can be applied for any symmetric multimer such as a trimer or tetramer. In all kinds of multimers, the
intensity of the cross-peak in the second experiment becomes
half for an intramolecular hydrogen bond due to the decrease
in the labeled monomer by 50%. On the other hand, in all kinds
of multimers, the intensity of the cross-peak in the second
experiment becomes a quarter for an intermolecular hydrogen
bond because the probability that two arbitrary monomers are
both labeled is 1/4 in the second experiment.
The methods of the discrimination of intra- and intermolecular NOESY cross-peaks were developed (26–29) by the
incorporation of the X-half-filter technique (30,31). The methods were successfully applied to a 1:1 mixture of 13C-labeled
and non-labeled proteins to identify intermolecular NOESY
cross-peaks for a symmetric dimer (27,28). The method was
also applied to a DNA–RNA hybrid triple helix to identify
intermolecular NOESY cross-peaks between DNA and RNA
(29,32). Our method has been developed in the context of these
previous approaches, particularly in the point of utilizing a
1:1 mixture of isotope-labeled and non-labeled materials. As
mentioned in Introduction, discrimination of intra- and intermolecular hydrogen bonds may be possible by accumulation
of pieces of information on discrimination of intra- and
intermolecular NOESY cross-peaks by previous approaches.
This is, however, an indirect method and applicable only if
an adequate proton pair close to a hydrogen bond is present.
Therefore, the development of a new method has been
awaited. The novelty of our method is that it directly
discriminates between the two kinds of hydrogen bonds. It
should also be noted that our method does not utilize the
X-half-filter technique. Incorporation of this technique to
the HNN-COSY experiment for the discrimination of intraand intermolecular hydrogen bonds may not be straightforward, if it is considered that both of the two nitrogen atoms
across a hydrogen bond should be 15N-labeled for successful
magnetization transfer in a COSY step. Additionally, it is also
known that careful inspection is needed to evaluate crosspeaks in the spectra made by addition and subtraction procedures with the filtering technique, in order to eliminate
artificial cross-peaks arising from imperfection in the pulse
sequence and from instrumental instability. Our discrimination method needs no incorporation of extra pulse sequences,
and is simply based on the intensity ratio of cross-peaks in the
HNN-COSY experiment without data manipulation. Our
method may be supposed to be comparatively tolerable against
possible experimental artifacts.
5115
Validation of the discrimination method
The new discrimination method has been validated with
d(GGGCTTTTGGGC). The structure of this DNA has already
been determined and reported (33). This DNA forms a
symmetric dimer structure comprising G:G:G:G tetrads and
G:C base pairs, as shown in Figure 2A. It is notable that the
G2:G11:G10:G3 tetrad involves both intra- and intermolecular
hydrogen bonds; the hydrogen bonds between G2 and G11,
and G3* and G10* are intramolecular, while those between G2
and G3*, and G11 and G10* are intermolecular, where ‘*’
denotes another monomer (Figure 2B). Therefore, the dimer
structure of d(GGGCTTTTGGGC) is ideal for validating our
method.
Figure 2. (A) Schematic representation of the dimer structure of
d(GGGCTTTTGGGC), cited from reference (26). The monomers are
colored black and red, respectively. (B) The G2:G11:G10*:G3* tetrad,
where ‘*’ denotes the other monomer.
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Nucleic Acids Research, 2004, Vol. 32, No. 17
In the HNN-COSY spectrum of d(GGGCTTTTGGGC), the
cross-peaks between G11NH2 and G2N7 are observed due
to the existence of scalar coupling between G11N2 and G2N7
across the intramolecular hydrogen bond (Figure 3A). Here,
the GNH2 assignment is taken from the previous report (33),
and the assignment of GN7 has been accomplished on the basis
of the intraresidue GH8–GN7 correlation peak in the HSQC
spectrum (data not shown). Slices along the 1H axis are shown
in Figure 3B. The slice for the first HNN-COSY experiment
with labeled d(GGGCTTTTGGGC) is shown in black and that
for the second HNN-COSY experiment with a 1:1 mixture of
labeled and non-labeled d(GGGCTTTTGGGC) in shown in
red. Clearly, the intensity of the cross-peaks in the second
experiment is half of that in the first experiment. The same
reduction by 50% was observed for the cross-peaks between
G3*NH2 and G10*N7 (data not shown). These results are what
we expected for the cross-peaks related to intramolecular
hydrogen bonds.
The cross-peaks in the HNN-COSY spectrum between
G2NH2 and G3*N7 are also observed due to the existence
of scalar coupling between G2N2 and G3*N7 across the
intermolecular hydrogen bond (Figure 3C). Slices are
shown in Figure 3D. In contrast to the previous case, the
intensity of the cross-peaks in the second experiment is quarter
of that in the first experiment. The same reduction by 75% was
observed for the cross-peaks between G10*NH2 and G11N7
(data not shown). These results are also what we expected for
the cross-peaks related to intermolecular hydrogen bonds.
Thus, it has been confirmed that our method can clearly discriminate between intra- and intermolecular hydrogen bonds.
Determination of the dimeric architecture formed by
GGA 12mer
The new discrimination method has been applied to the dimer
structure of GGA 12mer in order to select one of the two
possible architectures shown in Figure 1A and B. It should
be mentioned that only one set of resonances was observed for
the dimeric structure of GGA 12mer. The magnetic environment should be different between two architectures shown
in Figure 1A and B, particularly for A6 and G7 residues.
The observation of a single set of resonances strongly suggests
Figure 3. (A and C) Expansion of the HNN-COSY spectrum of d(GGGCTTTTGGGC), indicating cross-peaks between G11NH2 and G2N7, and G2NH2 and G3*N7,
respectively, where ‘*’ denotes the other monomer. (B and D) Slices of the HNN-COSY spectrum along the 1H axis at G2N7 and G3*N7, respectively. The slices of the
spectrum recorded with labeled d(GGGCTTTTGGGC) are shown in black, and those with a 1:1 mixture of labeled and non-labeled d(GGGGCTTTTGGGC) in red.
Nucleic Acids Research, 2004, Vol. 32, No. 17
that the solution contains a single dimeric species, either Figure 1A or B, not a mixture of the two species.
The cross-peaks between G4NH2 and G7N7 are observed
in the HNN-COSY spectrum due to the existence of scalar
coupling between G4N2 and G7N7 across a hydrogen bond
(Figure 4A). Here, the GNH2 assignments are taken from the
previous report (7), and the assignments of GN7 and AN7 have
been accomplished on the basis of the intraresidue GH8–GN7
and AH8–AN7 correlation peaks in the HSQC spectrum,
respectively (data not shown). The intensity of the crosspeaks between G4NH2 and G7N7 in the HNN-COSY experiment recorded with a 1:1 mixture of labeled and non-labeled
GGA 12mers is half of that with the labeled GGA 12mer
5117
(Figure 4C). This result indicates that the hydrogen bond
between G4NH2 and G7N7 is intramolecular. The G4NH2–
G7N7 hydrogen bond is intramolecular in the architecture
in Figure 1A, while it is intermolecular in that in Figure 1B.
Therefore, it turns out that the architecture in Figure 1A is
correct. Thus, the new method for the discrimination of intraand intermolecular hydrogen bonds could unambiguously
determine the dimeric architecture of GGA 12mer. In general,
this method provides a crucial basis for the construction of the
multimeric architecture of nucleic acids in a direct way.
Characterization of the G(:A):G(:A):G(:A):G heptad on
the basis of 2hJNN values for G:G and G:A base pairs
The unique G(:A):G(:A):G(:A):G heptad plane was first
found in the dimer structure of GGA 12mer (7), and later
also found in the structure of GGA 24mer (9). The formation
of a heptad plane in a wide range of eukaryotic genomes has
been implied (9). The values of scalar couplings across hydrogen bonds have not been reported for a heptad. It is expected
that the nature of the heptad can be examined on the basis of
the 2hJNN values for the involved hydrogen bonds.
For the portion comprising G1, A3 and G4 of the heptad
formed by GGA 12mer, cross-peaks in the HNN-COSY
spectrum are observed between G1NH2 and A3N7, G1NH2
and G4N7 (orange and green in Figure 4A), and A3NH2
and G1N3 (magenta in Figure 4B), due to the existence
of scalar couplings across hydrogen bonds between G1N2
and A3N7, G1N2 and G4N7, and A3N6 and G1N3, respectively. Cross-peaks indicating the formation of hydrogen
bonds are observed for the other portions of the heptad as
well (Figure 4A). Observation of these cross-peaks is consistent with the hydrogen bonding network in the heptad we
reported (7,9).
2h
JNN values were determined for the heptad for the first
time from the HNN-COSY experimental (10) results (Table 1).
For the scalar coupling involving AN6, the value was also
determined from the results of a spin-echo difference constant
time HSQC experiment (13). The values obtained in the two
experiments were about the same. The 2hJNN values between
GN2 and GN7 of G:G base pairs in the heptad formed by GGA
12mer are 6–7 Hz (Table 1). Slightly larger values of 7–8 Hz
are obtained for G:G base pairs in the heptad formed by GGA
24mer (data not shown). These values for the heptad are
comparable to those between GN2 and GN7 of the G:G
base pairs for a G:G:G:G tetrad, 6–8 Hz (17).
Table 1. 2hJNN values (Hz) for the heptad formed by d(GGAGGAGGAGGA)
at 288 K
Figure 4. Expansion of the HNN-COSY spectrum of d(GGAGGAGGAGGA),
indicating GNH2–GN7 and GNH2–AN7 cross-peaks (A), and ANH2–GN3 ones
(B), respectively. G1NH2–G4N7 cross-peaks are colored in green, G1NH2–
A3N7 ones in orange, and A3NH2–G1N3 ones in magenta. (C) Slices of the
HNN-COSY spectrum along the 1H axis at G7N7. The slice of the spectrum
recorded with labeled d(GGAGGAGGAGGA) is shown in black, and that with
a 1:1 mixture of labeled and non-labeled d(GGAGGAGGAGGA) in red.
G1N2–G4N7
G4N2–G7N7
G7N2–G10N7
G1N2–A3N7
G4N2–A6N7
G7N2–A9N7
A3N6–G1N3
a
Downfielda
Upfielda
Average
5.9
6.4
6.3
5.3
4.2
3.6
4.5
6.5
6.7
6.9
5.6
5.1
5.8
4.6
6.2
6.5
6.6
5.4
4.6
4.7
4.5
Cross-peaks involving either the downfield or upfield resonance of amino
protons were used to determine 2hJNN values.
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Nucleic Acids Research, 2004, Vol. 32, No. 17
As to G:A base pairs in the heptad formed by GGA 12mer,
the 2hJNN values between AN6 and GN3 are 3.8–3.9 Hz, and
those between GN2–AN7 are 4.5–6.0 Hz (Table 1). The values
are a little larger for the heptad formed by GGA 24mer, AN6–
GN3 and GN2–AN7 values being 4.5 and 6.5–7.0 Hz, respectively (data not shown). As to an G:A base pair in the A:G:C
triad, the 2hJNN values between AN6 and GN3 were reported to
be 3.2–3.4 Hz (20). Thus, the AN6–GN3 values for the heptad
are slightly larger than those for the triad. Moreover, the GN2–
AN7 values for the heptad are relatively large. Thus, the
observed 2hJNN values reveal that not only four G bases but
also three A bases in the heptad are tightly linked together
through hydrogen bonds.
17.
ACKNOWLEDGEMENTS
18.
M.K. was supported by Grants-in-Aid for Scientific Research
(Nos 12470487 and 15030214) and the Protein 3000 Project
from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
13.
14.
15.
16.
19.
20.
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