BIOCHEMICAL
Vol. 62, No. 4, 1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
NUCLEAR MAGNETIC RESONANCE STUDIES OF HYDROGEN BONDED COMPLEXES OF OLIGONUCLEOTIDES IN AQUEOUS SOLUTION. I. pdG-dC AND pdG-dT Thomas
R. Krugh* Department University Rochester,
Received
January
and Michael
A.
YOUng
of Chemistry of Rochester New York 14627
3,1975 Summary
The self-interaction of two deoxydinucleotides, pdG-dC and pdG-dT, was studied (in aqueous solution) by 100 MHz FT NMR spectroscopy. Concentration studies show that the self-complementary pdG-dC forms hydrogen bonded complexes. An analysis based on the concentration dependence of the chemical shift of the guanine amino protons strongly suggests that hydrogen bonded dimer formation occurs with a K for the dimerization equilibrium of 7.8 2 0.7 M-l. On the other hand, the non-self-complementary pdG-dT does not give evidence of similar complex formation in the same concentration range which thus illustrates the importance of complementarity in the formation of hydrogen bonded complexes of deoxydinucleotides. Further impetus is therefore provided for the use of dinucleotides in modeling interactions common to nucleic acids. Nuclear opportunity tons
of
in
proteins used
double
previous
complex other
the
Raszka protons
hydrogen
bonded
Cross the
association
(6) have
of mononucleotides
with
In
this
in
of the
Crothers
communication
while,
(4) observed protons. aqueous
for
solution
shifts the
we will
this
On the
(e.g.
downfield
is evidence
et al.
NH protons
d(T-T-G-T-T),
bases
the
NH and NH2 pro-
ring
and Crothers
observed
which
the
example,
nonexchangeable
stacking
(1) provide
exchangeable
of mononucleotides
vertical
Copyright Q 19 75 by Academic Press, Inc. All rights of reproduction in any form reserved.
the
of
For
of d(A-A-C-A-A)
by monitoring
base pairs.
(2).
observe
in D20 solution,
and Kaplan
H20 solutions
resonances
acids
to
complex
in the
in
important
approach
helical
results
although amino
this
work
hand,
the
studies
(1) and nucleic
formation
marily
resonance
of observing
(3) have of the
magnetic
pri-
see ref. of
5),
the
formation
of
show that
the
Vol. 62, No. 4, 1975
BIOCHEMICAL
G-m
C-H6
I
AND BIOPHYSJCAL RESEARCH COMMUNICATIONS
G-NH2
3
2 I wm Figure 1. The 100 MHz proton NMR spectra of pdG-dC in H20 at several concentrations; temperature 2.2+0.3OC, pH 7.15kO.15. Notes: Spectra were accumulated using a 180°-T-90' pulse sequence (WEFT method) with a supplemental homogeneity spoiling pulse applied to further reduce the H20 peak, as previously described (10). The sample, purchased from Collaborative Research Corp., was purified by passage through a Chelex-100 column and recovered by lyophilization. The dilutions were performed by adding weighed portions of glass distilled and filtered water. The molarity of the most concentrated sample was determined spectrophotometrically. Lower scale: ppm downfield from the H20 peak used as reference. concentration group
of
dependence the
of a hydrogen
nucleic
bonded
This
field
data
are as the
concentration
H5 and C-H6 protons
two of
of
the
guanine
2-amino
straightforward these
provides
as model
interactions
shown in
thus
shift provides
between
and ribo-dinucleotides acid
chemical pdG-dC
complex
The 100 MHz proton trations
the
deoxydinucleotide
deoxydinucleotides. deoxy
of
self-complementary
support
compounds
evidence
for
for
the
studying
use of the drug-
(7-9). nmr spectra Fig.
1. of
move upfield
of pdG-dC
The guanine the
2-amino
nucleotide with
1026
in
increasing
is
H20 at several protons increased, concentration.
concen-
move downwhile
the The
C-
Vol. 62, No. 4, 1975
G-H8 proton tion
chemical
range.
tainly
results
the
from
Pate1
pdG-dC
spectrum
performed
The cytosine titration.
These
restricted
protons
have
rotation
addition,
the
can also
help
resonances
will
transform
of
exchange
in Fig.
of the for
broaden be discussed
spectroscopy
pdG-dT
been
cytosine
this
shift (Fig.
of
aqueous the
(2~4,6,
and
300 MHz proton in
connection dependence
(11)
assumed
to monitor
(6,12)
in
This
3) changes
Figure
with
of the
cytosine
may not
a separate
paper
the
of
2. solvent
enough
saturation
on proton
fourier
(13).
2-amino less
than
protons 5 Hz over
of
the the
deoxyconcentra-
W
3
2
I
wm
Figure 2. The 100 MHz proton NMR spectrum 87 mM, temperature O.O°C, pH 7.00. Lower H20 used as reference.
1027
In
amino
be fast
transfer
the
due to
protons
exchange
solutions
guanine
that
during
as shown in
4-amino
resonances.
in detail in
cer-
(50 mM, pH 5.8).
low intensity
though
almost
shown to be nonequivalent
group
the
the
Pate1
are difficult
amino
1, even
The chemical dinucleotide
the
to account
to substantially effect
protons
the
a concentration
discussion
concentra-
protons
H20 solution,
However,
4-amino
this
formation
reported in
as monomers
over
amino
bond
recently
D.
exist
the
hydrogen
and in his
molecules
of
of pdG-dC
RESEARCH COMMUNICATIONS
constant
shift
(11)
on actinomycin
was not
essentially
intermolecular
resonance
a study
is
AND BIOPHYSICAL
downfield
therein).
magnetic
study
shift
The large
references
with
BIOCHEMICAL
of pdC in H20; concentration scale: ppm downfield from
Vol. 62, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
T-l-6 G-H8
-
r
4
G-NH,
i\.;ui[
20
+\1-
Difference
3
2
mM D,O
Spectwn
I
PPm
Figure 3. The 100 MHz proton NMR spectra temperature 1.7t0.3'C; pH (meter reading) apply here also.
tion
range
pdG-dT this
of
are
5 mM to 80 mM.
not
involved
concentration
complementary mentary
not
expect
base protons this
concentration
5 mM to
80 mM.
pdG-dC
vertical
chemical
dinucleotide base
concentration
are
stacking
in
dependence
(Fig.
Although
the
1) must the
observed
less
result
the
from
chemical
(14)
shifts
of not
the
formation
of the the
the
non-comple-
et al.
(15)),
non-exchanging in
non-exchanging
concentration
range
non-exchanging
protons
of of
by intermolecular then
shifts
of
of the
hydrogen
are
self-
concentration
range,
changes
1028
the
on ribodinucleo-
the
influenced
concentration chemical
for
or TS'O
of
shifts
in
of complementarity
dinucleotide
4 Hz over
shift
for
of
formation
experiments
shifts
likewise
this of
than
spectra
effect
and Chan
increasing
protons
bond
spectra
of previous
The chemical
changed If
the
significant of
range.
of pdG-dT
tons
to observe
the
the
Bangerter
as a function
protons
the
(e.g.
of
with
basis
a-amino
hydrogen
illustrates
On the
monophosphates
we did
pdG-dC
pdG-dT
9).
guanine
A comparison
dinucleotide
see ref.
the
intermolecular
range.
dinucleotide
(e.g. side
in
Thus
of pdG-dT in H20 and D20; 7.0 The notes of Figure 1
in
the
the
observed
C-H5 and C-H6 pro-
accord
bonded with
complex. the
Vol. 62, No. 4, 1975
formation
BIOCHEMICAL
of a double
further
experiments
helical are
The concentration 2-amino
protons
may
hydrogen
bond
formation.
sults
from
the
AND BIOPHYSICAL
complex
RESEARCH COMMUNICATIONS
containing
Watson-Crick
required
before
a detailed
dependence
of
chemical
be used
to determine
Assuming
formation
the
of
the
that
a dimer
P 'dG
is warranted.
of
equilibrium
the
(i.e.
analysis shift
the
guanine
constant
hydrogen
bond
a double
pairs,
base
of
formation
re-
helix)
P
K\
2
Vol. 62, No. 4, 1975
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
NUCLEAR MAGNETIC RESONANCE STUDIES OF HYDROGEN BONDED COMPLEXES OF OLIGONUCLEOTIDES IN AQUEOUS SOLUTION. I. pdG-dC AND pdG-dT Thomas
R. Krugh* Department University Rochester,
Received
January
and Michael
A.
YOUng
of Chemistry of Rochester New York 14627
3,1975 Summary
The self-interaction of two deoxydinucleotides, pdG-dC and pdG-dT, was studied (in aqueous solution) by 100 MHz FT NMR spectroscopy. Concentration studies show that the self-complementary pdG-dC forms hydrogen bonded complexes. An analysis based on the concentration dependence of the chemical shift of the guanine amino protons strongly suggests that hydrogen bonded dimer formation occurs with a K for the dimerization equilibrium of 7.8 2 0.7 M-l. On the other hand, the non-self-complementary pdG-dT does not give evidence of similar complex formation in the same concentration range which thus illustrates the importance of complementarity in the formation of hydrogen bonded complexes of deoxydinucleotides. Further impetus is therefore provided for the use of dinucleotides in modeling interactions common to nucleic acids. Nuclear opportunity tons
of
in
proteins used
double
previous
complex other
the
Raszka protons
hydrogen
bonded
Cross the
association
(6) have
of mononucleotides
with
In
this
in
of the
Crothers
communication
while,
(4) observed protons. aqueous
for
solution
shifts the
we will
this
On the
(e.g.
downfield
is evidence
et al.
NH protons
d(T-T-G-T-T),
bases
the
NH and NH2 pro-
ring
and Crothers
observed
which
the
example,
nonexchangeable
stacking
(1) provide
exchangeable
of mononucleotides
vertical
Copyright Q 19 75 by Academic Press, Inc. All rights of reproduction in any form reserved.
the
of
For
of d(A-A-C-A-A)
by monitoring
base pairs.
(2).
observe
in D20 solution,
and Kaplan
H20 solutions
resonances
acids
to
complex
in the
in
important
approach
helical
results
although amino
this
work
hand,
the
studies
(1) and nucleic
formation
marily
resonance
of observing
(3) have of the
magnetic
pri-
see ref. of
5),
the
formation
of
show that
the
Vol. 62, No. 4, 1975
BIOCHEMICAL
G-m
C-H6
I
AND BIOPHYSJCAL RESEARCH COMMUNICATIONS
G-NH2
3
2 I wm Figure 1. The 100 MHz proton NMR spectra of pdG-dC in H20 at several concentrations; temperature 2.2+0.3OC, pH 7.15kO.15. Notes: Spectra were accumulated using a 180°-T-90' pulse sequence (WEFT method) with a supplemental homogeneity spoiling pulse applied to further reduce the H20 peak, as previously described (10). The sample, purchased from Collaborative Research Corp., was purified by passage through a Chelex-100 column and recovered by lyophilization. The dilutions were performed by adding weighed portions of glass distilled and filtered water. The molarity of the most concentrated sample was determined spectrophotometrically. Lower scale: ppm downfield from the H20 peak used as reference. concentration group
of
dependence the
of a hydrogen
nucleic
bonded
This
field
data
are as the
concentration
H5 and C-H6 protons
two of
of
the
guanine
2-amino
straightforward these
provides
as model
interactions
shown in
thus
shift provides
between
and ribo-dinucleotides acid
chemical pdG-dC
complex
The 100 MHz proton trations
the
deoxydinucleotide
deoxydinucleotides. deoxy
of
self-complementary
support
compounds
evidence
for
for
the
studying
use of the drug-
(7-9). nmr spectra Fig.
1. of
move upfield
of pdG-dC
The guanine the
2-amino
nucleotide with
1026
in
increasing
is
H20 at several protons increased, concentration.
concen-
move downwhile
the The
C-
Vol. 62, No. 4, 1975
G-H8 proton tion
chemical
range.
tainly
results
the
from
Pate1
pdG-dC
spectrum
performed
The cytosine titration.
These
restricted
protons
have
rotation
addition,
the
can also
help
resonances
will
transform
of
exchange
in Fig.
of the for
broaden be discussed
spectroscopy
pdG-dT
been
cytosine
this
shift (Fig.
of
aqueous the
(2~4,6,
and
300 MHz proton in
connection dependence
(11)
assumed
to monitor
(6,12)
in
This
3) changes
Figure
with
of the
cytosine
may not
a separate
paper
the
of
2. solvent
enough
saturation
on proton
fourier
(13).
2-amino less
than
protons 5 Hz over
of
the the
deoxyconcentra-
W
3
2
I
wm
Figure 2. The 100 MHz proton NMR spectrum 87 mM, temperature O.O°C, pH 7.00. Lower H20 used as reference.
1027
In
amino
be fast
transfer
the
due to
protons
exchange
solutions
guanine
that
during
as shown in
4-amino
resonances.
in detail in
cer-
(50 mM, pH 5.8).
low intensity
though
almost
shown to be nonequivalent
group
the
the
Pate1
are difficult
amino
1, even
The chemical dinucleotide
the
to account
to substantially effect
protons
the
a concentration
discussion
concentra-
protons
H20 solution,
However,
4-amino
this
formation
reported in
as monomers
over
amino
bond
recently
D.
exist
the
hydrogen
and in his
molecules
of
of pdG-dC
RESEARCH COMMUNICATIONS
constant
shift
(11)
on actinomycin
was not
essentially
intermolecular
resonance
a study
is
AND BIOPHYSICAL
downfield
therein).
magnetic
study
shift
The large
references
with
BIOCHEMICAL
of pdC in H20; concentration scale: ppm downfield from
Vol. 62, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
T-l-6 G-H8
-
r
4
G-NH,
i\.;ui[
20
+\1-
Difference
3
2
mM D,O
Spectwn
I
PPm
Figure 3. The 100 MHz proton NMR spectra temperature 1.7t0.3'C; pH (meter reading) apply here also.
tion
range
pdG-dT this
of
are
5 mM to 80 mM.
not
involved
concentration
complementary mentary
not
expect
base protons this
concentration
5 mM to
80 mM.
pdG-dC
vertical
chemical
dinucleotide base
concentration
are
stacking
in
dependence
(Fig.
Although
the
1) must the
observed
less
result
the
from
chemical
(14)
shifts
of not
the
formation
of the the
the
non-comple-
et al.
(15)),
non-exchanging in
non-exchanging
concentration
range
non-exchanging
protons
of of
by intermolecular then
shifts
of
of the
hydrogen
are
self-
concentration
range,
changes
1028
the
on ribodinucleo-
the
influenced
concentration chemical
for
or TS'O
of
shifts
in
of complementarity
dinucleotide
4 Hz over
shift
for
of
formation
experiments
shifts
likewise
this of
than
spectra
effect
and Chan
increasing
protons
bond
spectra
of previous
The chemical
changed If
the
significant of
range.
of pdG-dT
tons
to observe
the
the
Bangerter
as a function
protons
the
(e.g.
of
with
basis
a-amino
hydrogen
illustrates
On the
monophosphates
we did
pdG-dC
pdG-dT
9).
guanine
A comparison
dinucleotide
see ref.
the
intermolecular
range.
dinucleotide
(e.g. side
in
Thus
of pdG-dT in H20 and D20; 7.0 The notes of Figure 1
in
the
the
observed
C-H5 and C-H6 pro-
accord
bonded with
complex. the
Vol. 62, No. 4, 1975
formation
BIOCHEMICAL
of a double
further
experiments
helical are
The concentration 2-amino
protons
may
hydrogen
bond
formation.
sults
from
the
AND BIOPHYSICAL
complex
RESEARCH COMMUNICATIONS
containing
Watson-Crick
required
before
a detailed
dependence
of
chemical
be used
to determine
Assuming
formation
the
of
the
that
a dimer
P 'dG
is warranted.
of
equilibrium
the
(i.e.
analysis shift
the
guanine
constant
hydrogen
bond
a double
pairs,
base
of
formation
re-
helix)
P
K\
2
