Neomycin, spermine and hexaamminecobalt(III) share common structural motifs in
converting B- to A-DNA
Neomycin, spermine and hexaamminecobalt(III) share common structural motifs in converting B- to A-DNA
Howard
Robinson
and
Andrew H.-J.
Wang
*
Biophysics Division and Department of Cell and Structural Biology, University of
Illinois at Urbana-Champaign,
Urbana
, IL 61801,
USA
Received October 18, 1995;
Revised and Accepted December 11, 1995
ABSTRACT
The (dG)
n
[middot](dC)
n
-containing 34mer DNA duplex [d(A
2
G
15
C
15
T
2
)]
2
can be effectively converted from the B-DNA to the A-DNA conformation by neomycin, spermine and Co(NH
3
)
6
3+
. Conversion is demonstrated by a characteristic red shift in the circular
dichroism spectra and dramatic NMR spectral changes in chemical shifts.
Additional support comes from the substantially stronger CH6/GH8-H3
'
NOE intensities of the ligand-DNA complexes than those from the native DNA duplex. Such changes are
consistent with a deoxyribose pucker transition from the predominate C2
'
-
endo
(S-type) to the C3
'
-
endo
(N-type). The changes for all three ligand-DNA complexes are identical, suggesting that those three complex
cations share common structural motifs for the B- to A-DNA conversion. The A-DNA structure of the 4:1 complex of Co(NH
3
)
6
3+
/d(ACCCGCGGGT) has been analyzed by NOE-restrained refinement. The structural basis of the transition may be
related to the closeness of the two negatively charged sugar-phosphate backbones along the major groove in A-DNA, which can be effectively neutralized by the multivalent
positively charged amine functions of these ligands. In addition, ligands like
spermine or Co(NH
3
)
6
3+
can adhere to guanine bases in the deep major groove of the double helix, as is
evident from the significant direct NOE cross-peaks from the protons of Co(NH
3
)
6
3+
to GH8, GH1 (imino) and CH4 (amino) protons. Our results point to future
directions in preparing more potent derivatives of Co(NH
3
)
6
3+
for RNA binding or the induction of A-DNA.
INTRODUCTION
A number of aminoglycoside antibiotics are potent inhibitors of the interactions
of proteins and RNA. For example, neomycin (Fig.
1
) at 1 [mu]M concentration inhibits the binding of HIV Rev protein to the Rev-responsive element (RRE) RNA hairpin-bulge sequence (
1
). Similar inhibitions are found in the splicing of group I introns (
2
), ribozymes (
3
) and the RNA ligand of the 30S ribosome subunit (
4
). In contrast, streptomycin is not effective. The molecular basis for such
binding specificity is not yet known.
Neomycin has six primary amine groups distributed over three of its four sugars,
creating a network of positively charged amino hydrogen bond donors under
physiological conditions. Such a molecular architecture is reminiscent of the
hexaamminecobalt(III) ion [Co(NH
3
)
6
3+
], with six ammonia molecules surrounding the Co(III) ion. Co(NH
3
)
6
3+
is extremely potent in promoting, at sub-micromolar concentrations, the transition of poly[d(m
5
C-G)] from right-handed B-DNA to left-handed Z-DNA (
5
-
7
). It is equally effective in promoting formation of DNA four-way junctions (Holliday junctions) (
8
). In many ways Co(NH
3
)
6
3+
behaves similarly to the biologically relevant polyamines spermine and
spermidine, which are also capable of promoting the two above-mentioned DNA conformational transitions (
5
-
9
). Thus these three amine-containing ligands, neomycin, spermine and hexaamminecobalt(III) ion, may
share certain similar DNA/RNA interaction properties.
MATERIALS AND METHODS
The 34mer DNA d(A
2
G
15
C
15
T
2
) was chosen to contain a long stretch of consecutive (dG)
n
[middot](dC)
n
sequence which is capped with two AT base pairs to prevent concatenation of
duplexes. In addition, the DNA decanucleotide d(ACCCGCGGGT) was prepared for
high definition structural analysis. DNA oligonucleotides were synthesized at
the Genetic Facility of UIUC. They were purified and desalted on a Sepharose S-100 column. Solutions of the DNA oligomers for NMR studies were prepared
as described earlier (
1 8
,
19
). For the Na-form of the 34mer DNA sample 3.25 mg 34mer lyophilized powder was
dissolved in 0.55 ml H
2
O containing 20 mM phosphate buffer, pH 7.0, resulting in a 0.26 mM duplex
solution. For the ligand-DNA sample an ~0.4 mM DNA duplex solution was similarly prepared. Aliquots of the
stock solution of ligands [neomycin, spermine, Co(NH
3
)
6
3+
, streptomycin and Pt(NH
3
)
4
2+
were purchased from Sigma Chemical Co., St Louis, MO] were individually added
to the DNA solution to produce different ratios of ligand to duplex.
NMR spectra were collected on a Varian VXR500 500 MHz spectrometer and the data
were processed with FELIX version 1.1 (Hare Research, Woodinville, WA). The non-exchangeable two-dimensional NOE spectra for the 34mer were collected at 25oC at a mixing time of 100 ms and a total recycle delay of 4.4
s, where the average
T
1
relaxation was 2.0 s. The data were collected by the States/TPPI technique (
2
0) over a sweep width of 5000 Hz with 512
t
1
increments and 2048
t
2
complex points, each the average of 24 transients. Apodization of the data in
the
t
2
and
t
1
dimensions consisted of 6 Hz exponential multiplication with a sine-bell squared function extending over the last quarter of the data to
reduce truncation effects. The data set was zero-filled in
t
1
to 2048 points prior to transformation. TOCSY spectra were used together with
the NOE spectra to derive the assignment. The chemical shifts (in p.p.m.) are
referenced to the HDO peak, which was calibrated relative to 2,2-dimethyl-2-silapentane-5-sulfonate at different temperatures.
Two-dimensional NOESY spectra at 25oC in 90% H
2
O/10% D
2
O were collected with the 1-not-1 pulse sequence as the read pulse of the NOESY. Twenty four
transients were averaged with a recycle delay of 2.9 s and a mixing time of 100
ms. The excitation offset was set to one quarter of the spectral bandwidth,
which was set to 12 000 Hz.
Refinements of both the Na-form and Co-form of the decamer d(ACCCGCGGGT) have been carried out. Starting
models for the decamer in the B- and A-DNA conformations were built using QUANTA, version 4.0 (MSI,
Massachusetts, MA). Structural refinement was carried out by the procedure
SPEDREF (
2 1
). The mixing time used in the refinement was 100 ms. The inversion recovery
experiment determined the
T
1
relaxation time for every spin. The recycle time was 4.41 s. The correlation
time [tau]
c
was determined to be 7 ns using the SPEDREF procedure described before (
2 1
). The starting models were refined within the program X-PLOR (
2 2
) in conjunction with the SPEDREF procedure. During the first 40 refinement
cycles the molecules were given a random set of velocities equivalent to 300 K.
NOE-restrained molecular dynamics were then run for 0.1 ps, the temperature
coupling bath lowered by 25 K and the process repeated until the molecules had
been cooled to 50 K. After each annealing the molecular dyad symmetry was
slowly imposed while running an additional 100 cycles of NOE-restrained conjugate gradient minimization. During the last 20 cycles the
molecules were refined with only NOE-restrained conjugate gradient minimization. The simulated NOE relaxation
rates and NOE intensities for the refined models were calculated by the program
MORASS (
23
). Simulated two-dimensional NOESY spectra were produced by the program CSL (in the SPEDREF
package) using the NOE intensities from the simulation and the line shapes and
chemical shifts previously obtained from MYLOR. The final structure of the Co-form was derived from NOE-restrained conjugate gradient minimization only, since the starting
model was very close to the observed data and simulated annealing could not
further improve on the refinement. The NMR
R
factor was defined as
R
= [Sigma][brvbar]
N
o
-
N
c
[brvbar]/[Sigma]
N
o
, where
N
o
and
N
c
are the experimental and calculated NOE integrals respectively.
Circular dichroism (CD) spectra were recorded on a Jasco J720 spectrometer. The
concentration of the 34mer DNA solutions was kept at 10 [mu]M duplex in 20 mM phosphate buffer, pH 7.0.
RESULTS AND DISCUSSION
Evidence of B- to A-DNA transition
Circular dichroism and one-dimensional NMR spectra were taken to monitor titration of the 34mer DNA.
Figure
2
A shows the CD spectra of the titration of neomycin. The characteristic spectral
shift toward the longer wavelength associated with a B- to A-DNA transition is evident. It appeared that the conformational
transition was nearly complete at a 4:1 ratio of neomycin. Comparisons of the
difference spectra from the titration of five multivalent cations [neomycin,
spermine, Co(NH
3
)
6
3+
, streptomycin and Pt(NH
3
)
4
2+
] were made to more easily see the changes (Fig.
2
B). Neomycin, spermine and Co(NH
3
)
6
3+
all caused a transition at 8:1, but streptomycin induces only a partial
transition and no change was detected for Pt(NH
3
)
4
2+
at 8:1.
Structural refinement of A-DNA
CONCLUSIONS
Our results here demonstrate that neomycin, spermine and Co(NH
3
)
6
3+
are capable of inducing the A-DNA conformation in a (dG)
n
[middot](dC)
n
sequence. Many DNA oligonucleotides having a (dG)
n
[middot](dC)
n
sequence have been found to crystallize in the A-DNA conformation. Thus the GpG sequence seems to have a high propensity to
adopt the A conformation, possibly due to its unique intrastrand base-base stacking interactions (
1 1
). It is likely that some factors may influence the B- to A-DNA transition. Here we have shown that three complex ions rich in
hydrogen bond donors (amine functions), which are distributed over a rigid
molecular frame and are highly positively charged, are potent in inducing the
formation of A-DNA. Note that streptomycin is less effective in promoting the transition.
This characteristic may mimic the arginine-rich RNA binding [alpha]-helical peptides (
2 7
). This hypothesis may point out future directions for preparing more potent
derivatives of simple ligands for RNA binding or the induction of A-DNA. For example, a bis-platinum(II)(triamine) complex with a butanediamine tether has been
found to be very potent in promoting the B- to Z-DNA transition for the (dC[middot]dG)
n
sequence (29). It is conceivable that a similar bis-cobalt(III)(pentaamine) ion may be equally or more potent. Such bis-metal ion complexes may be very effective in promoting the B- to A-DNA transition for (dG)
n
[middot](dC)
n
sequences or in binding to certain RNA sequences. This can be tested and
experiments are in progress.
.
Chemical shifts (p.p.m.) for the Na-form (top) and Co-form (bottom) of d(ACCCGCGGGT)
2
at 2oC
H2/5/Me
H8/6
H1'
H2'
H2''
H3'
H4'
H5'
H5''
Ade 1
8.10
8.31
6.29
2.76
2.89
4.88
4.30
3.78
3.78
Cyt 2
5.39
7.50
5.88
2.21
2.44
4.84
4.25
4.13
4.20
Cyt 3
5.57
7.53
6.00
2.15
2.48
4.86
4.21
4.12
4.12
Cyt 4
5.63
7.49
5.52
2.13
2.41
4.86
4.13
4.09
4.09
Gua 5
7.93
5.90
2.66
2.74
5.00
4.39
4.04
4.12
Cyt 6
5.35
7.26
5.65
1.85
2.30
4.82
4.13
4.21
4.12
Gua 7
7.85
5.50
2.66
2.71
4.96
4.31
3.97
3.97
Gua 8
7.71
5.74
2.62
2.73
4.98
4.39
4.11
4.17
Gua 9
7.75
6.02
2.57
2.74
4.92
4.39
4.20
4.20
Thy 10
1.51
7.39
6.25
2.22
2.22
4.55
4.07
4.08
4.26
Ade 1
8.11
8.46
6.34
2.95
3.02
4.89
4.37
4.02
4.02
Cyt 2
5.50
7.74
5.97
2.49
2.68
4.88
4.40
4.41
4.28
Cyt 3
5.65
7.83
6.05
2.49
2.68
4.90
4.36
4.27
4.39
Cyt 4
5.68
7.74
5.89
2.41
2.63
4.97
4.33
4.35
4.26
Gua 5
7.99
6.14
2.81
2.81
5.04
4.48
4.29
4.29
Cyt 6
5.38
7.55
5.82
2.39
2.55
4.87
4.31
4.40
4.27
Gua 7
7.91
5.95
2.82
2.82
4.97
4.40
4.41
4.28
Gua 8
7.66
6.04
2.82
2.82
4.89
4.41
4.39
4.27
Gua 9
7.53
6.12
2.62
2.82
4.80
4.43
4.37
4.24
Thy 10
1.44
7.69
6.39
2.37
2.46
4.67
4.24
4.36
4.25
ACKNOWLEDGEMENTS
This work was supported by NIH (GM-41612) and American Cancer Society (DHP-114) grants to AH-JW. The Varian VXR500 NMR spectrometer was supported in part
from NIH shared instrumentation grant 1S10RR06243.
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