Volume 12 Number 20 1984
Nucleic Acids Research
Synthesis and conformations! studies of ribooUgonudeotides which contain an alternating C-G
sequence and show unnsual drcnlar dichroism spectra
Seiichi Uesugi, Mitsuru Ohkubo, Eiko Ohtsuka, Mono Ikehara, Yuji Kobayashi* and Yoshimasa
Kyogoku*
Faculty of Pharmaceutical Sciences, and 'Institute for Protein Research, Osaka University, Suita,
Osaka 565, Japan
Received 17 August 1984; Revised and Accepted 2 October 1984
ABSTRACT
The poly pr(C-G)] duplex shows an unusually large negative
CD band in the long wavelength region. In order to elucidate
this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were
synthesized by a phosphotriestjer methoji and their properties
were examined by UV, CD,
H and
P NMR spectroscopy.
These ribooligomers form
self-duplexes at low temperature,
the CD spectra of which show negative bands at around 290 nm
and
positive bands at around 265 nm. The^esults of H
nuclear Overhauser effect experiments,
H chemical
shift-temperature
profiles of base protons, and the sharp
singlet observed for all HI 1 protons are consistent with a
normal A-RNA structure but not with a Z-DNA like structure.
The CD-temperature profiles and
P NMR spectra support this
conclusion. These results indicate that RNA duplexes with an
alternating C-G sequence can give an unusually large negative
CD band in the long wavelength region despite their
right-handed helical structure.
INTRODUCTION
Since the discovery of a left-handed DNA duplex structure
(Z-DNA) in d(C-G-C-G-C-G) crystals , much effort has been
focussed on studies of oligo- and polydeoxyribonucleotides
containing alternating pyrimidine-purine base sequences. In
Z-DNA, the dG residues adopt an unusual syn conformation about
the glycosidic bond and a C3'-endo furanose puckering while
the dC residues are characterized by the anti and C2' -endo
conformations that are commonly observed in B-DNA. It has
been shown that oligo [d(C-G)] s
also take the Z-form in
2-4
solutions of high salt concentrations.
CD spectra of the
Z-form DNAs, poly[d(G-C)] and oligo [d(C-G) ] in 4M NaCl, show a
characteristic negative band in the 280-300 nm region. '
It is also known that poly[r(G-C)] shows an unusually
© IRL Pren Limited, Oxford, England.
7793
Nucleic Acids Research
large negative CD band in the 280-300 nm region. '
It might
be assumed that the unusual band is due to a Z-forra structure
of the ribopolynucleotide. Recently, the possible existence
of Z-form structure in RNA duplexes has been suggested by
conforraational studies on a C-G analogue containing
fl ft ft
8-bromoguanosine, r(C-br G) . .
The dimer, in which the br G
residue is assumed to adopt a syn glycosidic conformation,
forms a stable duplex and the CD spectrum is very similar to
that of poly [r (C-G)].
In order to elucidate these phenomena, we have studied
the conformation of oligo[r(C-G)] using
UV, CD,
H and
P
Q
NMR spectroscopy.
The results strongly suggest that these
oligomers do not form a left-handed duplex of a Z-form type
but a right-handed duplex of an A-form type. These studies
thus establish that
A-forrn RNA with an alternating C-G
sequence can give an unusually large negative CD band in the
280-300 run region.
MATERIALS AND METHOD
General Procedures
Reverse-phase thin layer chroraatography (RTLC) was
performed on a sheet of silica gel 60 F 2 5 4 silanized (Merck)
with acetone-water mixtures. For column chromatography of the
protected oligomers, silica gel 60H (Merck) and alkylated
silica gel (C-18, 35-105 p, Waters) were used under moderate
air pressure. High pressure liquid chromatography (HPLC) was
performed on a column of alkylated silica gel (TSK gel ODS-120
T, Toyo Soda) using an Altex 332 MP apparatus.
UV spectra were recorded on a JASCO UVIDEC 610C spectrophotometer. CD spectra were measured on a JASCO J-500 A
spectrometer. The molar absorption coefficient, [c] , and
molar ellipticity, [8], are presented in terms of per base
residue value. NMR spectra were recorded on a Bruker WM-360
wb spectrometer (360 MHz for 1 H and 145.8 MHz for
P) . The
H chemical shifts were determined relative to internal
2-methyl-2-propanol, which had in turn been referenced to DSS
(sodium
l-trimethylsilyl-propyl-3-sulfonate).
The
P
chemical shifts were determined relative to external trimethyl
7794
Nucleic Acids Research
phosphate.
Nuclease PI was obtained from Yamasa Shoyu Co. (Choshi,
Japan). Oligomers (ca. 2 A,,, units) were digested with the
enzyme (0.25 mg/ml) in 0.05 M ammonium acetate (pH 5) (total
volume, 12 yl) at 25°C for 3 h. ZnBr- was supplied by Aldrich
Chemical Co.
HO[C-G1X (4)
(MeO)2TrO[C-GJX (2_) (431 mg, 0.329 mmol) was treated with
1 M ZnBr 2 in isopropanol-CH2Cl2 (15:85, 16 ml) at room
temperature for 20 min with stirring. 1 M ammonium acetate
(30 ml) was added and the mixture was shaken. The mixture was
extracted with CH 2 C1 2 (40 ml x 3) . The organic fraction was
evaporated to dryness. The residue was dissolved in a small
volume of CHC1, and applied on a column of silica gel 60H (10
g) . Elution with methanol-CHCl, mixtures (0-8 %) and
precipitation with n-hexane gave the desired product (4) . The
recovered 2_ was again deprotected as described above. The
total yield of 4_ was 222 mg (0.22 mmol, 67 % ) .
(MeO)2TrO[C-G-C-G]x (5)
A mixture of (MeO) _TrO[C-G]£ {3_, 234 mg, 0.15 mmol) and
HO [C-G] x (4_, 138 mg, 0.13 mmol) was dried by repeated
evaporation with anhydrous pyridine. The residue was treated
with mesitylenesulfonyltetrazolide (MSTe, 75 mg, 0.30 mmol) in
pyridine (1 ml) at 30°C for 50 min with shaking. 50% aqueous
pyridine (6 ml) was added to the reaction mixture cooled in an
ice-bath. The mixture was extracted with CHC1,-pyridine (3:1,
80 ml). The organic fraction was washed with 0. 1M
triethylaramonium bicarbonate (TEAB) buffer (pH 7.5) and
evaporated to dryness. The residue was dissolved in a small
volume of acetone and water was added until the solution
turned slightly turbid. The solution was applied on a column
(3 x 4.5 cm) of C-18 silica gel. Elution was carried out with
acetone-0.2 % aqueous pyridine mixtures (6:4, 6.5:3.5, 7:3 and
7.5:2.5, 100 ml each).
The fractions containing 5_ were
collected and concentrated under reduced pressure. 5_ was
precipitated with n-hexane.
The starting materials were
similarly recovered and condensed again as described above.
The total yield of 5 was 174 mg (0.07 mmol, 54%).
7795
Nucleic Acids Research
r(C-G-C-G)
5_ (111 rag, 0.044 nnnol) was treated with 1 M
tetramethylguanidium syn-pyridine-2-carboxaldoximate (TMG-PAO)
in 50% aqueous dioxane (12 ml) at 30°C for 3 days with
shaking. The solvent was removed. The residue was dissolved
in pyridine (2 ml) and treated with cone. NH 4 OH (15 ml) at
55°C for 6 hr. The volatile materials were removed and the
residue was dissolved in 30% aqueous pyridine (20 m l ) . The
solution was passed through a column of Dowex 50 (pyridiniura
form) resin (20 m l ) . The column was washed with 30% aqueous
pyridine (200 ml) . The combined effluents were evaporated to
dryness. The residue was evaporated twice with toluene.
0.01N HC1 (50 ml) was added and the pH of the solution was
adjusted to 2 by using a pH meter. The mixture was shaken at
room temperature for 3 hr, neutralized with 0.5 N NH 4 OH and
washed with ethyl acetate.
The aqueous fraction was
evaporated.
The residue was dissolved in 20 raM tris-HCl
buffer (pH 7.5)- 7M urea (100 ml) and applied on a column (2.5
x 30 cm) of DEAE-cellulose (DE-52, chloride form). Elution
was carried out with a linear gradient of NaCl (0.05-0.20 M,
total volume 6 1) in the same buffer-7 M urea system. The
fractions containing the desired tetramer were combined,
diluted with water ( 3 fold) and applied on a column (2.5 x 8
cm) of DEAE-cellulose (chloride form). The column was washed
with 0.05 M TEAB buffer (1 1 ) . The tetramer was eluted with 1
M TEAB buffer (180 m l ) . The appropriate fractions were
concentrated and the residue was desalted by repeated
evaporation with water. The residue was dissolved in water
(10 ml) and passed through columns of Dowex 50 (pyridinium
form, 0.7 x 1 c m ) , Dowex 50 (sodium form, 0.7 x 1 c m ) , and
finally Chelex 100 (0.7 x 1 cm) resins. A powder of the
sodium salt of r(C-G-C-G) was obtained by lyophilization. The
yield was 1000 A 2 6 0 units (0.029 ramol, 66%).
(MeO)2TrO C-G-C-G p (7)
A mixture of 2 (408 mg, 0.274 mmol) and HO [C-G]£an
(<5) (269 mg, 0.205 mmol) was dried by repeated
evaporation
with anhydrous pyridine, dissolved in pyridine (1 ml) and
treated with mesitylenesulfonyl-3-nitrotriazolide (MSNT) (148
7796
Nucleic Acids Research
mg, 0.50 mntol) at around 30°C for 40 rain with shaking. The
condensation reaction was followed by RPTLC (acetone-water,
7:3).
50% aqueous pyridine (4 ml) was added to the mixture
cooled in an ice bath. The mixture was extracted with CHC1,
(80 m l ) . After washing with 0.1 M TEAB buffer, the organic
fraction was evaporated. The fully protected tetramer was
purified by reverse-phase column chromatography as described
for 5^ and precipitated with n-hexane. The yield was 366 mg
(0.13 mmol, 63%). The fully protected tetraraer was treated
with isoamyl nitrite (0.85 ml, 6.5 mmol) in pyridine-acetic
acid (5:4, 4.5 ml) at 30°C for 3.5 hr with shaking. The
deprotection reaction was followed by RPTLC (acetone-water,
7.5:2.5).
The reaction mixture was added dropwise to
n-pentane-ether (1:1, 100 m l ) . The gummy solid obtained was
dissolved in CHC1,-pyridine (3:1, 80 ml). The solution was
washed with 0.2 M TEAB buffer (50 ml x 3) and evaporated to
dryness. 7_ was precipitated with n-pentane from its solution
in CHC1 3 . The yield was 355 mg (0.13 mmol, 100%).
(MeO)2TrO C-G-C-G-C-G X (8)
A mixture of 7_ (355 mg, 0.13 mmol) and £ (150 rag, 0.15
mmol) was dried by repeated evaporation with pyridine and
treated with MSNT (95 mg, 0.32 mmol) in pyridine (1 ml) at
30°C for 50 min with shaking. The reaction mixture was worked
up as described for 1_. 8^ was purified by chromatography on a
column (2 x 7.5 cm) of C-18 silica gel. Elution was carried
out stepwise with acetone-0.2% aqueous pyridine mixtures
(6:4-8:2) as described for j>. Precipitation with n-pentane
gave 316 mg of J3 (0.086 ramol, 66%).
r(C-G-C-G-C-G)
£ (147 mg, 0.04 mmol, 2060 AjgQ units) was deprotected as
described for r(C-G-C-G).
The deprotected hexamer was
purified by chromatography on a DEAE-cellulose column
(chloride form, 2.5 x 30 cm) with 7M urea system. Elution was
carried out with a linear gradient of NaCl (0.1-0.3 M, total
volume 6 1) . Desalting and salt form exchange were performed
as described for r(C-G-C-G). The yield was 1270 A 2 60 un:"-t3
(0.025 mmol, 63%).
7797
Nucleic Acids Research
(Mt0) 2 Tr0CC-0Jpon (««0) 2 TrOtC-OK
1
I
?
3
I
y-yOH-0
H0IC-O3X
3
4
I
(M«O)jTrO(C-G-C-G3p'
7
MSNT
(MtO)jTrOtC-G-C-0]X
IMSNT
(M»0)jTrO[C-G-C-G-C-G3X
5
I
I U 0 5MTMG-PAO
2)cone NH3
|3)0.01NHCI
I D05MTMG-PA0
2)cone NH3
| 3 ) 0 01NHa
C-G-C-G
C
§
jilMSNT 2)
I ZnBr2
(MtO)2TrOCC-G]p"
KOCC-Gllpcin
j
C-G-C-G-C-G
]
prottcttd txctpt for ttrmlnl
p
o-chloroph*nyt pho«phoryl
pan o-chlorophtnyl photphoro -p-anUktat*
Figure 1. Synthetic schemes for r(C-G-C-G) and r(C-G-C-G-C-G).
RESULTS AND DISCUSSION
Synthesis of oligo [r(C-G)] s
r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a
modified phosphotriester method with a tetrahydrofuranyl group
for protection of the 2'-OH groups and a p_-methoxyanilido
group for temporary protection of the 3'-terminal phosphate
groups. '
The dimer block condensation method was employed
as shown in Fig. 1. The fully protected oligomers were
purified by reversed phase chromatography on alkylated silica
gel columns. After deprotection, the tetramer and hexamer
were purified by DEAE-cellulose column chromatography in 7M
urea system.
Reversed phase HPLC analysis of each oligomer
showed a sharp single peak. The sequences of the oligomers
were proved by mobility shift analysis '
and identification
of the 5 *-terminal nucleotide residue. The molar absorption
coefficients and hypochromicities were determined by complete
digestion experiments (Table 1) . The UV absorption spectra
before and after digestion and the difference spectra are
shown in Fig. 2. The UV spectrum of r(C-G-C-G-C-G) shows no
apparent shoulder around 270 run which is present in that of
r (C-G-C-G) . The same phenomenon is also observed in the
corresponding deoxyoligonucleotides,
d(C-G-C-G-C-G) and
3
d(C-G-C-G).
7798
Nucleic Acids Research
Table 1.
UV absorption properties of r(C-G)
X
max
''max1
X
min
E
260
hypochromicity
at
r (CGCG)
255
(9000)
260 run
224 (5000)
8700
9.5 %
227 (4800)
8400
12%
271(sh) (7700)
r (CGCGCG)
258
(8500)
a) The E per residue and hypochromicity were calculated from
the results of digestion experiments with nuclease PI. The UV
spectra were measured in 0.1 M NaCl, 10 mM phosphate buffer
(pH 7.5) at 20°C.
CD and UV Spectral Properties
CD spectra of the ribooligomers are shown in Fig. 3.
Both hexa- and tetramers (1 A_, n unit/ml) show similar spectra
zou
at 10°C that contain a negative band at around 290 ran and a
positive band at around 265 nra. These spectra are also
similar to that of poly [r(G-C)].
The CD spectral patterns of
these RNAs in the 250-300 run region are very similar to that
of Z-DNA 5,3 (the spectrum of a d(C-G-C-G-C-G) in 4M NaCl is
included in Fig. 3A). At higher oligomer concentration and
320
WwritngtMnm)
Figure 2. UV absorption spectra before (
) and after
(
) digestion of r(C-G-C-G) (A) and r(C-G-C-GC-G) (B) with nuclease PI and the difference spectra
(
) . The spectra were measured in 0.1 M NaCl, 10
mM phosphate buffer (pH 7.5) at 20°C.
7799
Nucleic Acids Research
' IS
"A
fp
v y'
\ / "
Wav»ltnQth(nm)
Woo«1tnQth(nm)
Figure 3. A: CD spectra of the oligomers (1 A n unit/ml) in
0.1M NaCl, 10 mM phosphate buffer ((pB 7.5);
7.5);
,
r(C-G-C-G-C-G);
(
( — . — , r(C-G-C-G); and in 4 M NaCl;
10 mM phosphate buffer (pH 7.5);
,
d(C-G-C-G-C-G) at 1°C; B: CD spectra of
r(C-G-C-G)(100 A 2 , 0 units ml) in 0.1 M NaCl, 10 mM
phosphate buffer IpH 7.5)(
) and in 4M NaCl, 10
mM phosphate buffer (pH 7.5)( — • — ) at 1°C.
low temperature, the oligomers show negative bands as large as
the positive band (see the spectrum of r(C-G-C-G) in 0.1M NaCl
in Fig. 3B).
In contrast to the case of d(C-G) n , the CD spectral
patterns of r(C-G) in 4M NaCl are essentially the same as
those in 0.1 M NaCl (Fig. 3B) though the intensity of the
negative band significantly decreases. With increasing
temperature, the intensity of the negative band decreases and
9
finally disappears.
The CD-temperature profile for the
negative band shows a sigmoidal curve with a relatively sharp
transition (Fig. 4) . The CD spectrum at high temperature is
quite different from that at low temperature and is similar to
that of r(C-G) which is in a single-stranded form under
o
similar conditions.
These results clearly demonstrate that
oligo [r(C-G)]s form duplexes at low temperature. It should be
noted that oligo [r(C-G)] duplexes give the same melting
temperature (Tm) whether it is obtained by the UV method or by
the CD method (Fig. 4 ) . This result is in contrast to that
7800
Nucleic Acids Research
CD
-,0 7
-ffi
0.35
2 0.30
§0.25
<
0.20
0
10
20
30 «
50 60
T»mp»ratur»(#C)
70
Figure 4. CD and UV-temperature profiles for r(C-G-C-G)
(strand concentration, 3mM) in 0.1 M NaCl, 10 mM
phosphate buffer (pH 7.5).
for oligo[d(C-G)]s in 4M NaCl where the CD-temperature profile
are not sigmoidal .
Tra's for oligofr(C-G)]s as well as for oligo[d(C-G)]s are
presented in Table 2. The effect of NaCl concentration on T
Table 2.
Melting temperatures of r(C-G)
Compound
Concentration of
oligomer (A_,n/ml)
d(C-G)3b
l
l
r(C-G) 3
l
l
l
l
l
d(C-G)2b
r(C-G) 2
100
113
Concentration of
NaCl (M)
0.1
0.0
0.1
T
m( °
C )
41.5
43
0.5
50
54
1.0
54
2.0
3.0
0.1
54
0.1
40
51
38
a) Measured in 0.1 M NaCl, 0.01 M phosphate buffer (pH 7.5).
b) Measured in 0.1 M NaCl, 0.01 M cacodylate buffer (pH 7.0)
The data were taken from ref. 3.
7801
Nucleic Acids Research
C'HS
50
40
Owmiuil ShiH(pp<n)
Figure 5.
H NMR spectra of r(C-G-C-G)(strand concentration,
14 mM) in D_O containing 0.1 M NaCl, 10 mM phosphate
buffer (pD 7.5) at 30°C.
was examined with r (C-G-C-G-C-G) . The T increases with
increasing NaCl concentration up to 0.5 M NaCl and begin to
decrease from 3.0 M NaCl. This result again appears to be
inconsistent with a Z-DNA structure.
It is noted that
r(C-G-C-G-C-G) duplex is more stable than the d(C-G-C-G-C-G)
duplex in 0.1 M NaCl at least at low strand concentration.
1
H NMR Spectra
In order to obtain more detailed information on the
oligomer conformations, H NMR spectra (360 MHz) were measured. The spectrum of r(C-G-C-G) in 0.1 M NaCl is shown in
Fig. 5. Assignments of these proton resonances were made by
decoupling and nuclear Overhauser effect (NOE) experiments.
The H6 signal (signal 18 in Fig. 5) of the first C residue,
C H6, was assigned by the observed mutual NOEs with the H5'
signal (signal 1 in Fig. 5) of the free 5'-terminal residue,
1
9
1
C H 5 , which appears in the highest field.
Irradiation of
the C H5" signal also gives a small NOE on the signal 17 and ,
therefore, was assigned to G H8. This leaves signals 16 and
4
3
15 to be assigned to G H8 and C H6, respectively. From decoupling experiments, signals 14 and 9 were assigned to C H5
and C H5, respectively. The sugar-1' proton resonances were
7802
Nucleic Acids Research
o>
CMS I
t.0
TO
Figure 6. NOE difference spectra for J: (C-G-C-G)(14 mM) at
22°C: a) irradiation at C M6; b) irradiation at
C H6; c) irradiation at G H8. Irradiation was
applied for 0.3s.
The resonance numbers are
indicated in Figure 5.
assigned as indicated in Fig. 5 by irradiation of base proton
1
9
resonances (Fig. 6 ) . The C HI 1 also shows a NOE peak , which
is apparently due to spin diffusion, on irradiation of the
C 1 H5 1 .
We can obtain some qualitative information about the
glycosidic conformation from the NOE results. When irradiated
at CH6, the largest NOE is observed on the CH5 of the same
o
residue (the distance is ca. 2.4 A) and NOEs observed in the
H2' , H3' region are larger than that of the HI' .
These
results are consistent with an anti conformation of the C
15 4
residue but not with a syn conformation ' . When irradiated
at GH8, larger NOEs are also observed in the H2',H3' region
with respect to the HI 1 suggesting that the G residue also
adopts an anti conformation. The relatively large NOEs in the
H2', H3' region can be partly explained by the close proximity
o
(about 2A) between the base proton of a residue and the H2' of
its 3'-adjacent residue in the A-RNA conformation.
It is
7803
Nucleic Acids Research
noted that upon irradiation of H6 or H8 of C or G residue, a
significant NOE is observed on HI 1 of its 5'-adjacent residue
(Fig. 6b and 6c). This phenomenon is also consistent with
both rG and rC residues in the A-RNA conformation (the internucleotide H8- or H6-H1' distance £ 4.7 A ) 1 6 but not with a dG
residue in the Z-DNA conformation (the internucleotide H8-H1'
distance = 6.5 A) . Similar results are obtained in the
two-dimensional NOE study of r(C-G-C-G-C-G). 9 ' 18
The H NMR spectral data for r(C-G-C-G) are summarized in
Table 3. The coupling constants between HI' and H2' (J,,-i)
are very small for all residues. Only the G4 residue shows a
measurable J-,,,. o f 2 - 2 H z a t 30°C in 0.1 M NaCl. The result
suggests that all the residues adopt predominantly a C3'-endo
19
sugar puckering form
which is characteristic of the A-RNA
structure. In the case of Z-DNA, the dC residues adopt a
C2'-endo form while the dG residues adopt a C3'-endo form.
The chemical shift-temperature profiles for base and
sugar-1' protons of r (C-G-C-G) in 0.1 M NaCl and in 4M NaCl
are shown in Fig. 7 and 8. The profiles in 0.1 M NaCl and 4M
NaCl are essentially the same suggesting again that no drastic
conformational change is induced by high salt concentration.
The chemical shift-temperature profiles of the CH6 and,
especially, CH5 resonances are similar to those for d(C-G-C-G)
in 0.1 M NaCl (B-form) '
but are different from those for
d(C-G-C-G) in 4 M NaCl (Z-form). 3
In the case of r(C-G-C-G)
and d (C-G-C-G) in 0.1 M NaCl, the C 1 H5 signals do not show a
Table 3.
1
H NMR data for r(C-G-C-G) a
Chemical shift
Residue
H8(H6)
Cp-
7.91
-pGp-
7.78
-pCp-
7.63
-pG
7.59
H5
5.90
(ppra)
HI'
5.42
5.77
5.34
5.55
5.85
Coupling
constant
J112,(Hz)
<1
<1
<1
2.2
a) The tetramer (strand concentration, 15 mM) was measured in
D 2 O containing 0.1 M NaCl, 0.01 M phosphate buffer (pD 7.5) at
30°C.
7804
Nucleic Acids Research
10 10 »
g
'
«
»
(0 »
10 20 30 W
SO GO
H Chemical shift-temperature profiles for base
proton resonances of r(C-G-C-G) (strand
concentration, 2.8 mM) in D_0 containing 0.1 M NaCl
(A) or 4 M NaCl (B) and 10fflMphosphate buffer (pD
7.5).
temperature dependence while the C H5 signals show a marked
downfield shift upon duplex melting.
The C H6 and C H6
signals also show quite different behaviours.
The C H6
3
signals show an upfield shift while the C H6 signals show a
downfield shift with increasing temperature.
These phenomena
are consistent with a right-handed
structure
helical
A
B
6.0
G*
o
°
0
o
G 4 H1'
o-
^°c HI*
• 5.5-
Figure 8.
^—o-o
G 2 Hl'
C 3 H1'
10 20
30
A0
of
50
60
PS*
/C'HI'
70
10 20 30
Temperature ("C)
40
50
60
H Chemical shift-temperature profiles for HI
resonances of r(C-G-C-G)(strand concentration, 2.8
mM) in D-0 containing 0.1 M NaCl (A) or 4 M NaCl (B)
and 10 mB phosphate buffer (pD 7.5).
7805
Nucleic Acids Research
a)
-10
-15
v-vV-*-10
-15
-*J)
Chnrtul fWtt (PHOT)
Figure 9. Totally decoupled 3 1 P NMR spectra of r(C-G-C-G)
(15 mM) (a) and r(C-G-C-G-C-G) (15 mM) (b) in 0.1
M NaCl, 10. mM sodium phosphate buffer (pD 7.5) at
27°C and
P chemical shift-temperature profiles
(c) for the tetramer.
1
2
C-G-C-G where the C and G residues have little stacking
interaction and, moreover, C H5 and C H6 are located distant
2
2
3
from the adjacent G guanine while the G and C residues
stack well and the cytosine protons are close to the adjacent
guanine ring . In the case of d (C-G-C-G) in 4 M NaCl, both
H6 and both H5 signals show a downfield shift upon duplex
melting . The characteristic upfield shift as observed for
C H6 of r(C-G-C-G) is usually observed for ribooligonucleotide duplexes containing a 5'-terminal cytidine residue
(r(C-A-U-G) , r(C-C-G-Gp) ) . Similar trends are also
observed in deoxyribooligonucleotide duplexes (d(C-G-C-G), '
d(C-C-G-G) 2 4 ) .
Both GH8 signals of r (C-G-C-G) show
considerable downfield shifts upon duplex melting which are
absent in the case of d (C-G-C-G) in 0.1 M NaCl. This trend is
consistent with an A-RNA duplex structure in which a
pyrimidine-purine
sequence site contains
interstrand
purine-purine stacking . It should also be noted that C HI'
shows a profound upfield shift at low temperature. This could
be due to the change of glycosidic torsion angle suggested by
7806
Nucleic Acids Research
Bubienko et al. in studies on r(A-G-C-U) and
duplexes
31
r(A-C-G-U)
P NMR Spectra
Another characteristic property of Z-DNA is that it
shows two widely separated
P signals (66^1.5 ppm) in the
P NMR spectrum. '
This phenomenon is assumed to be due
to the different conformations about the P-0 bonds of GpC
(gauche - trans) and CpG (gauche - gauche ) fragments . The
31
P NMR spectra of r(C-G-C-G) and r(C-G-C-G-C-G) shown in Fig.
9 do not show such wide separation of the
P resonances,
r(C-G-C-G)(strand concentration, 15 mM) shows three peaks
(-3.51, -3.57 and -3.82 ppm in 1:1:1 ratio) at 27°C.
r(C-G-C-G-C-G) shows four peaks (-3.49, -3.58, -3.86 and -3.99
ppm in a ratio of 2:1:1:1) under the same conditions. In both
cases, the greatest chemical shift differences are less than
0.5 ppm. The
P NMR spectral patterns of r (C-G-C-G) and
r(C-G-C-G-C-G) are quite different from those 20 of d(C-G-C-G)
and d(C-G-C-G-C-G). The former appear to be more dispersed in
the chemical shifts. The
P chemical shift-temperature
profiles for r(C-G-C-G) are shown in Fig. 9C. The dispersed
signals at low temperature shift upfield with increasing
temperature and merge into a broad peak upon duplex melting.
CONCLUSION
The NMR data for r(C-G-C-G) and r(C-G-C-G-C-G) described
above are consistent with a normal RNA structure (A-form) but
not with a Z-DNA like structure. The NOE and J.,., data
strongly suggest that both C and G residues take an anti and
C3'-endo conformation. The observed NOE between GH8 and HI 1
of its 5'-adjacent residue and chemical shift-temperature
profiles for the base proton signals suggest that the
nucleoside residues are arranged in a right-handed helical
structure. It is also demonstrated that the conformation of
the ribooligomers in 4 M NaCl is almost the same as that in
0.1 M NaCl.
Moreover T ' s of the duplexes in 4M NaCl are
m
lower than those in 0.1 M NaCl.
The ribooligomers do not show
characteristic properties of Z-DNA in UV, CD,
H and
P NMR
spectroscopy.
7807
Nucleic Acids Research
The present results clearly reveal that oligo and poly[r(C-G)] duplexes can give an unusually large negative band at
around 290 run despite their right-handed helical structures.
Since oligo- and poly [d (C-G)] duplexes in the B-form do not
show such a negative band, the special geometry of the
cytosine and guanine arrangement in an A-RNA duplex may be
responsible for the negative band. Recently it has been
reported that calculation of the CD spectrum of the poly[r(G-C) ] duplex in the A-RNA structure using revised monomer
transition parameters gives a result in substantial agreement
27
with the measured spectrum
More recently, we have shown that r(C-G-C-G) analogues
containing 8-substituted guanosine residues form duplexes with
2 fl
Z-form structures . The Z-RNA duplexes show a positive CD
band at around 290 nm in contrast to the A-RNA duplex and also
in contrast to the Z-DNA duplex. The Z-RNA tetramer duplex
shows some of the chracteristic properties expected from the
case of Z-DNA; the same chemical shift-temperature profiles
for both CH6 residues; alternating sugar puckering and
glycosldic conformations; widely separated
P NMR signals;
and stabilization of the duplex with increasing salt
concentration.
CD spectra of all four possible forms of r(C-G-C-G) and
d(C-G-C-G) duplexes are now available. The validity of CD
theories may be tested by application to the B- and Z-forms of
oligo[d(C-G)] duplex and A- and Z-forras of the oligo [r(C-G)]
duplex.
ACKNOWLEDGMENT
We
thank Dr. Alexander F. Markham for reading
manuscript.
the
This research was supported in part by a grant-in-
aid for Scientific Research from the Ministry of Education,
Science and Culture of Japan.
REFERENCES
1. Wang, A. H. -J., Quigley, G.J., Kolpak, F.J., Crawford,
J.L., van Boom, J.H., van der Marel, G., and Rich, A.
(1979) Nature 282, 680-686.
2. Thamann, T.J., Lord, R.C., Wang, A.H. -J., and Rich, A.
(1981) Nucleic Acids Res., 9, 5443-5457.
7808
Nucleic Acids Research
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Uesugi, S., Shida, T., and Ikehara, M. (1981) Chem.
Pharm. Bull., 29, 3573-3585.
Patel, D.J., Kozlowski, S.A., Nordheira, A., and Rich, A.
(1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1413-1417.
Pohl, F.M., and Jovin, T.M. (1972) J. Mol. Biol. 67,
375-396.
Gray, D.M., Tinoco, I., Jr., and Chamberlin, M.J. (1972)
Biopolymers 11, 1235-1258.
Gray, D.M., Liu, J.-J., Ratliff, R.L., and Allen, F.S.
(1981) Biopolymers, 20, 1337-1382.
Uesugi, S. , Shida, T., and Ikehara, M. (1982)
Biochemistry, 21, 3400-3408.
A preliminary communication is presented in Uesugi, S.,
Ohkubo, M., Ohtsuka, E., Ikehara, M. , Kobayashi, Y. ,
Kyogoku, Y., Westerink, H.K., van der Marel, G.A., van
Boom, J.H., and Hansnoot, C.A. G. (1984) J. Biol. Chem.
259, 1390-1393.
Ohtsuka, E., Yamane, A., and Ikehara, M. (1983) Chem.
Pharm. Bull., 31, 1534-1543.
Ohtsuka, E., Yamane, A., and Ikehara, M. (1983) Nucleic
Acids Res., 11, 1325-1335.
Toralinson, R.U. and Tener, G.M. (1963) Biochemistry, 2,
697-702.
Sanger, F., Donelson, J.E., Coulson, A.R., KOssel, and
Fischer, D. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,
1209-1213.
Silberklang, M., Gillum, A.M., and RajBhandary, U. (1977)
Nucleic Acids Res., 4, 4091-4108.
Frechet, D., Cheng, D.M., Kan, L, -S., and Ts'o, P.O.P.
(1983) Biochemistry, 22, 5194-5200.
Arnott, S., Smith, P.J.C., and Chandrasekaran, R. (1976)
in CRC Handbook of Biochemistry and Molecular Biology,
3rd edn., Fasman, G.D. Ed., Nucleic Acids
Vol. II,
pp.411-422, CRC Press, Cleveland.
Wang, A. H. -J., Quigley, G.J., Kolpak, F.J., van der
Marel, G., van Boom, J.H., and Rich, A. (1981) Science
211, 171-176.
Westerink, H.P., van der Marel, G.A., van Boom, J.H., and
Haasnoot, C.A.G. (1984) Nucleic Acids Res, 12, 4323-4338.
Altona, C , and Sundaralingam, M. (1973) J. Am. Chem.
Soc. 95, 2333-2344.
Cheng, D.M., Kan, L.-S., Frechet, D. , Ts'o, P.O.P.,
Uesugi, S., Shida, T., and Ikehara, M. (1984) Biopolymers
23, 775-795.
Arnott, S. , Dover, S.D., and Wonacott, A.J. (1969) Acta
Crystallogr. B25, 2192-2206.
Romaniuk, P., Neilson, T. , Hughes, D.W., and Bell, R.A.
(1978) Canad. J. Chem. 56, 2249-2252.
Petersheim, M., and Turner, D.H. (1983) Biochemistry, 22,
269-277.
Patel, D.J. (1977) Biopolymers, 16, 1635-1656.
Bubienko, E., Uniack, M.A., and Borer, P.N. (1981)
Biochemistry, 20, 6987-6994.
Simpson, R.T., and Shindo, H. (1980) Nucleic Acids Res,
8, 2093-2103.
7809
Nucleic Acids Research
27.
28.
7810
Williams, A. Jr., and Moore, D.S. (1983) Biopolymers,
22, 755-786.
Uesugi, S., Ohkubo, M. , Urata, H., Ikehara, M.,
Kobayashi, Y., and Kyogoku, M. (1984) J. Am. Chera. Soc.,
106, 3675-3676.