ABSTRACT
The solution conformations of the dinucleotide d(TT) and the modified duplex
d(CGCGAATTCGCG)
2
with N3
' ->
P5
'
phosphoramidate internucleoside linkages have been studied using circular
dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex
conformation is similar to that of isosequential phosphodiester RNA, an A-type helix, and is different from that of DNA, a B-type helix. NMR studies of model dimers d(TpT) and N3
' ->
P5
'
phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2
'
-endo to C3
'
-endo when the 3
'
-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that
all furanose rings of 3
'
-aminonucleotides adopt predominantly N-type sugar puckering.
Antisense and antigene therapeutic strategies are attractive because they potentially offer highly specific targeting of the nucleic acids of
interest (
1
,
2
). Such selectivity in action can significantly reduce the toxic side effects
observed with other nucleic acid-addressed therapeutic agents. Antisense and antigene compounds developed to this time use Watson-Crick and Hoogsteen nucleobase recognition rules for specificity of
interaction with targets and contain nucleosides connected through various
linkers (
1
,
2
). These linking groups are generally based on structural similarity to the
original phosphodiester. Phosphodiester-linked oligonucleotides are too easily degraded by cellular nucleases to
be used as
in vivo
therapeutic agents. To date phosphorothioate backbone-modified oligonucleotides have been shown to be one of the best antisense
agents and several oligonucleotide phosphorothioates are in clinical trials
against a variety of targets (
1
,
3
). Typically, however, duplexes and, especially, triplexes formed by
phosphorothioate strands are less stable than those derived from corresponding
phosphodiesters (
4
,
5
). Consequently, it should be possible to improve efficiency in antisense
therapeutics by development of modifications that retain the favorable
properties of phosphorothioates, such as increased resistance to nuclease
degradation, but that have enhanced duplex and triplex stability.
In antisense applications of oligonucleotides their targets are single-stranded RNA regions with the nucleoside C3'-endo sugar conformation and preferences for A-type duplexes. This duplex structure preference could
be important in the development of improved antisense agents. There is also
evidence that the base stacking in triple helical DNA is closer to an A-type conformation than is observed in DNA duplexes, although many of the
sugars retain the C2'-endo conformation (
6
). These data suggest that oligonucleotides which prefer an A-type conformation in the single-stranded state should provide pronounced enhancements in stability
of duplexes with RNA and of triple helixes with duplex DNA.
It was recently reported that oligonucleotides with an internucleoside N3' -> P5' phosphoramidate linkage (Fig.
1
) form very stable duplexes and triplexes with complementary nucleic
phosphodiester compounds (
7
,
8
). The N3' -> P5' phosphoramidate monoester internucleoside linkage is not chiral, is
nuclease resistant and has the same formal charge as phosphodiesters. Initial studies with the dinucleoside monophosphate d(TnpA) suggested that the
sugar with a 3'-phosphoramidate linkage has a greater percentage of the N
conformation (C3'-endo) than sugars with a phosphodiester linkage (
7
). We have initiated a combined circular dichroism (CD) and NMR study on the
duplex d(CGCGAATTCGCG)
2
formed by uniformly modified oligonucleotide N3' -> P5' phosphoramidate strands to determine whether the sugar
preference for the N conformation will remain in a double-stranded complex and to determine whether the duplex conformation corresponds to an A-helical family. The oligonucleotide sequence d(CGCGAATTCGCG)
2
was chosen because its structure has been investigated in detail as DNA and RNA
phosphodiesters by X-ray crystallography and NMR methods (
9
,
10
). We find that the 3'-amino-substituted nucleosides in the duplex have sugar rings with a
high percentage of the N-type conformation and that the duplex adopts an A-type conformation, even though it consists of 2'-deoxyribonucleosides. To our knowledge this is the
first report of a mixed sequence duplex formed by 2'-deoxynucleosides with a phosphate-based backbone that adopts the A conformation in solution under
physiologically relevant conditions.
Thymidine (dT
OH
) and the dimer d(TpT) were purchased from Sigma (St Louis, MO). The
phosphoramidate and phosphodiester oligonucleotides used in this research were
synthesized, purified and characterized as previously described (
7
,
8
).
Thermal denaturation experiments were conducted on a Cary 4 spectrophotometer,
as previously described (
11
), in 0.01 M PIPES buffer, pH 7.0, containing 1 mM EDTA, 0.1 M NaCl and 6 * 10
-6
M nucleic acid strand concentration.
T
m
values were determined by fitting the data with a non-linear least squares computer program that includes sloping base lines in
the duplex and single-strand regions.
CD spectra were obtained with a JASCO J-710 spectrophotometer interfaced to an IBM computer as previously described (
12
). CD experiments were conducted at temperatures from 15 to 90oC in 1 cm path length cuvettes with a buffer adjusted to pH 7.0 containing 3.75 mM NaH
2
PO
4
, 1 mM EDTA, 0.1 M NaCl and 2.1 * 10
-6
M strand concentration. Phosphate buffer was substituted for PIPES buffer in
order to obtain CD spectra at lower wavelengths.
The samples were dissolved in 0.6 ml 99.96% D
2
O or 90% H
2
O solution containing 7.5 mM phosphate, 0.01 mM EDTA, 100 mM NaCl, pH 7.0. All
NMR spectra were acquired with a Varian Unity plus 600 MHz spectrometer at 25oC, except for the temperature study of the phosphoramidate duplex, which
was obtained with a Varian Unity plus 500 MHz spectrometer. The one-dimensional (1D) NMR data were processed by Vnmr 4.3 software from Varian.
The two-dimensional (2D) NMR data were transferred to a Silicon Graphics work
station and processed with Felix 2.3 software from Biosym. 1D spectra were
collected with a spectral width of 5000 Hz and 32K data points. A 2D NOESY
spectrum at 300 ms mixing time was acquired using 90% H
2
O as solvent with spectral widths of 14 000 Hz in D1 and 7198 Hz in D2 and data
sizes of 4096 points in
t
2 and 512 in
t
1. Data were zero-filled to 4K points in both dimensions. A 2D NOESY spectrum at 100 ms
mixing time was recorded with a spectral width of 5000 Hz in both dimensions
and 4096 data points in
t
2, 512 in
t
1. After zero-filling a data set of 4K * 4K was obtained. The double-quantum filtered COSY (DQF-COSY) spectrum was collected with a spectral width of
5000 Hz in both dimensions and 4096 data points in
t
2, 512 in
t
1. The
t
2 dimension was then zero-filled to 4K. The TOCSY spectrum was obtained using a spin-lock time of 40 ms with 5000 Hz spectral width in both dimensions
and 2048 data points in
t
2, 512 in
t
1, zero-filled to 2K * 2K. All 2D NMR data were acquired in the phase-sensitive mode using the States-Haberkorn method of phase cycling and were apodized
with a sine-bell function shifted by 90o.
Melting curves for the sequence CGCGAATTCGCG as oligonucleotide RNA, DNA and N3' -> P5' phosphoramidate are shown in Figure
2
A and illustrate the remarkable stability of the phosphoramidate duplex. The
melting studies were repeated at several salt concentrations and the
T
m
values are plotted as a function of log[Na
+
] in Figure
2
B. The lines have similar slopes of 16 +- 2. It is clear from these results that substitution of the N3' -> P5' phosphoramidate linkages for phosphodiesters
significantly stabilizes the nucleic acid duplex at all salt concentrations used and the effect of increasing salt concentration for the phosphoramidate
duplex is very similar to that for DNA and RNA. The
T
m
curve for the phosphoramidate duplex is steeper than for the phosphodiester
duplexes and this suggests that a greater enthalpy component is, at least
partially, responsible for the increased stabilization of the modified duplex.
NMR studies have been conducted with the phosphoramidate-substituted dinucleoside monophosphate d(TnpT), where np indicates the N3' -> P5' phosphoramidate linkage, with the d(TpT)
phosphodiester as a reference. The percentage of N conformer was evaluated from
the standard two-state N-S deoxyribose equilibrium assumption using the Karplus equation as
modified by Altona and co-workers (
13
). In d(TnpT) the nucleoside linking group is the N3' -> P5' phosphoramidate, while the 3'-terminal nucleoside has a 3'-OH group. Significant differences
in vicinal hydrogen coupling constants between the two 2'-deoxyriboses in the phosphoramidate dimer are apparent in the H1' spectral region of the NMR spectrum (Fig.
3
). In the 3'-amino nucleoside no significant coupling between H1' and H2' protons was observed, indicating that the dihedral
angle between H1' and H2' is close to 90o, as expected for the C3'-endo conformation (
10
,
13
). In contrast, significant coupling for the same protons was observed in the 3'-terminal nucleoside (Table
1
), indicating a significantly higher percentage of the C2'-endo conformation. The increase in percentage of the N conformation
in the dinucleoside for the 3'-amino- versus 3'-hydroxyl-substituted sugar correlates with the
trend observed for 3'-NH
2
versus 3'-OH mononucleosides, where replacement of a 3'-hydroxyl by an amino group increases the amount of C3'-endo conformation (Table
1
) (
14
). These results indicate that in the dimer the nucleoside sugar puckers are
relatively independent and the sugar conformation is strongly affected by
changes in the 3' substituent (
14
).
1D NMR spectra of the N3' -> P5' phosphoramidate duplex d(CGCGAATTCGCG)
2
as a function of temperature are shown in Figure
4
. As can be seen from the melting curve in Figure
2
, the phosphoramidate sample is in the duplex state below 70oC and is completely melted above 90oC at 6 * 10
-6
M strand concentration. The signals in 1D NMR spectra from 25 to 55oC sharpen as expected, due to increased thermal motion in the duplex, and
exhibit some small spectral shifts, due to changes in base stacking and end
fraying of the duplex, with increasing temperature. At 65oC the signals begin to broaden again, due to intermediate exchange between
the duplex and single strands. At 85oC the signals are very broad, due to the exchange process between
populations of native and denatured strands (
15
).
CD spectra for the oligomer CGCGAATTCGCG as DNA, RNA and N3' -> P5' phosphoramidate are very similar above the
T
m
. At temperatures below the duplex
T
m
values the CD intensities increase and the spectrum for RNA corresponds to a
classic A-type helical pattern, while the spectrum for the DNA duplex shows a B-form helical pattern. The CD spectrum for the phosphoramidate at low
temperature is very similar to the RNA A-type pattern and is quite different from the B-helical spectrum (Fig.
9
). These experiments indicate that at high temperatures the oligonucleotides are in a similar conformational state as denatured single strands, but both RNA and phosphoramidate assume an A-type of helix in the duplex form while the DNA exists as a B-type duplex below the
T
m
. The results for RNA and DNA are expected (
9
,
10
), but the result for the phosphoramidate is somewhat surprising for a 2'-deoxyribose-containing structure. Since the DNA and phosphoramidate
duplexes have the same sequence and 2'-deoxyribose sugars, their conformational differences must be caused
by substitution of nucleoside 3'-amino for 3'-hydroxy groups.
NMR and X-ray studies on the DNA duplex have demonstrated that it has a B-type conformation, while experiments with the isosequential RNA
counterpart have indicated that it has an A-type duplex structure (
9
,
10
,
20
, and references therein). The NMR data presented here for the phosphoramidate
duplex strongly support the conclusions from CD experiments that this nucleic
acid adopts an A-type helical structure. Analysis of coupling constants indicates that all
of the nucleoside 2'-deoxyriboses with a 3'-amino substituent have predominantly N sugar
conformations (Table
1
). The fact that no H1'-H2' coupling can be detected (Fig.
8
) suggests that all of the 3'-amino sugars are close to a pure N-type conformation. NOESY connectivities also strongly support
an A-type conformation. In a duplex with the C3'-endo sugar ring conformation base H6/H8 aromatic protons are
close to the
n
- 1 sugar H2'/H2'' protons, while in a duplex with nucleoside C2'-endo conformations the aromatic protons
are close to the
n
sugar H2' and the
n
- 1 sugar H2'' protons. The strong H8/H6 cross-peaks observed with the phosphoramidate duplex are
all to the
n
- 1 H2'/H2'' protons, while much weaker cross-peaks can be seen to the
n
H2'/H2'', in support of an A-form helical structure (Fig.
7
). A particularly important cross-peak for defining the A-form geometry is observed from AH2 to the interstrand
m
+ 1 residue H1' (where
m
refers to the complementary strand). This distance is long in B-form helical conformations (
10
,
16
) and no or a weak cross-peak is usually observed. Strong interstrand cross-peaks are observed for A5H2-C9H1' and A6H2-T8H1' in the phosphoramidate duplex and
confirm that the grooves in the duplex have a characteristic A-form helical topology, as observed in the r(CGCGAAUUCGCG)
2
sequence (
10
). From these NMR and CD spectral data it may be concluded that in the N3' -> P5' phosphoramidate duplex both base stacking and sugar
conformation are characteristic of the A-type helical family.
Duplexes with electronegative substituents at the 2' position, such as RNA, are generally found in an A-form conformation. DNA, with a 2'-deoxysugar can adopt either the A- or B-helical conformation, but in solution the
B-form conformation is observed under a wide range of conditions that
bracket those observed in biological systems (
21
). Analysis of substituent effects at the 3' position of 2',3'-dideoxyribose derivatives by Chattopadhyaya and co-workers (
14
, and references therein) has indicated that as a consequence of the gauche
effect there is a trend in the sugar conformation from the S conformational
state with very electronegative substituents (for example -OH, -F, -NO
2
) to an N conformational preference as the electronegativity is decreased (for
example -H and -NH
2
3'-substituents). We have conducted studies with the dinucleotide
monophosphate d(TnpT), which has a N3' -> P5' phosphoramidate linkage and a terminal nucleoside with a 3'-hydroxyl group. The results for the two
nucleosides in d(TnpT) are different: the 2'-deoxyribose substituted with a 3'-NH is strongly biased to the N conformational state
while that with the 3'-OH has a much higher percentage of the S conformation, as do both
nucleosides in the phosphodiester dimer d(TpT) (Table
1
). These results indicate that substitution of the normal DNA phosphodiester by
the N3' -> P5' phosphoramidate linkage strongly biases the nucleoside
sugar puckering to the N state, as expected from the gauche effect. This sugar
conformation preference must account for a large part of the observed A-type conformational preference of the phosphoramidate duplex. Our
analysis, however, suggests that the percentage N sugar puckering is higher in
the phosphoramidate duplex than in the TnpT dimer and it is possible that there are
additional structural or functional (e.g. hydration) constraints for the
phosphoramidate group in the duplex that act in a cooperative fashion to shift
the conformation even more strongly to an A-form helix.
This research was supported by NIH grants AI-27196 and AI-33363 and Lynx Therapeutics Inc. The NMR, UV-visible and CD instruments were purchased through funds from
the NSF and the Georgia Research Alliance. We appreciate some early spectra in
this project that were provided by Dr George Gray of Varian Associates.
REFERENCES
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