Volume 15 Number 14 1987
Nucleic Acids Research
Z form of poly d(A-C).poly d(G-T) in solution studied by Raman spectroscopy
J.-P.Ridoux, J.Liquier and E.Taillandier
Laboratoire de Spectroscopie Biomoleculaire, 74 rue Marcel Cachin, F93 012 Bobigny Cedex, France
Received April 13, 1987; Revised and Accepted June 22, 1987
ABSTRACT
Poly d(A-C).poly d<6-T) structures have been studied In solution by
Raman spectroscopy, in presence of Na*, Mn 2 * and Ni 2 * a counterions.
Increase of the Na* concentration or addition of Mn * ions up to 1M
MnClz does not modify the B geometry of the polynucleotide. On the
contrary, in conditions of low water activity <4M NaCl), the presence of
small amounts of nickel ions (65 mM) induces a left-handed1 geometry of the
DNA. The shift of the guanine line located at 682 cm" in B form to 622
cm"1 reflects unambiguously the C2/-endo/anti->C3/-endo/svn reorientation
of the deoxyribose-purine entities. Moreover modifications in the phosphate
backbone lines indicate that the polymer is in a Z conformation. New or
displaced lines corresponding to adenosine vibrations
are correlated with the
left-handed structure. An interaction of the Ni 2 * ions specifically with
the N7 site of purines, combined with a low water activity is necessary to
promote the B->Z transition.
INTRODUCTION
The existence of left-handed structures of DNA has been proposed by
Pohl and Jovin (1) to explain the inversion of the CD spectrum of poly d(G-C)
in solution at high salt concentrations. The X-ray diffraction study of a
d(C-G) 3 crystal has shown that this oligonucleotide is in a left-handed
geometry called Z DNA (2). Since then Z DNA has been extensively studied by a
variety
of
physico-chemical
and biological
techniques. Various
ollgonucleotldlc sequences incorporating increasing amounts of A-T base pairs
In G-C tracts have been shown to be able to adopt this left-handed geometry
(3-6) as well as high molecular weight polynucleotides with regularly
alternating purine-pyrimidine bases, poly d(A-C).poly d(G-T> (7-11) and poly
d(A-T) (12,13). In solution, Raman spectroscopy has been widely used as a
sensitive method to characterize the secondary structures of DNAs. In
particular lines can be assigned to the Z conformation allowing to detect
left-handed structures in oligonucleotides (6,14,15), polynucleotides (16-18)
or more complex systems such as form V DNA (association of complementary
single stranded circles (19)). We present here results concerning the Raman
study of poly d(A-C).poly d(G-T). The spectra of right-handed helixes of this
polymer have been published by many groups (20,21), however the left-handed
structure Raman spectra have been breafly discussed only in the case of poly
d(A-C).poly d(G-T) modified by methylatlon of cytosines (8). Our results
IR L Press Limited, Oxford, England.
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present the left-handed structure of unmodified poly d(A-C).poly d(G-T> and
allow to point out several characteristfcs of A-T base pairs in left-handed
configuration.
MATERIALS AND METHODS
Poly d(A-C).poly d(G-T) was purchased from P.L. Biochemicals (lot
517940). Raman samples were prepared in the following manner: 10 0D of
lyophyllzed material were dissolved in 20ul of either ,1M NaCl or 4M NaCl.
Progressive amounts of 200 mM NiClz, 4M NaCl or 200 mM MnCl 2 , 4M NaCl
were gently added. The final concentration was 60 mM in nucleotide base, 4M
NaCl, 65 mM N1CI2. The samples were placed in a microcell and exposed to
the 514.5 nm line from a Spectra-Physics model 2025 argon laser. The output
power used was 400rawat the source. The Raman spectra were recorded between
2000 and 400 cm"1 using a DILOR OMARS 89 multichannel spectrophotometer.
Integration time varied between 4 and 11 seconds. Each spectrum is an average
of 2S0 integrations. The spectral slit width was 5 cm"1. Spectra were
treated using an IBM AT3 computer coupled to the spectrophotometer. Smoothing
was performed following the Savitsky and Golay proceduce using generally 17
points. Solvent background correction was obtained when necessary by
substract ing the solvent spectrum recorded in the same conditions. A simple
base line adjustment procedure assuming a straight but sloped base line was
used.
RESULTS AND DISCUSSION
The Raman data will be discussed using expanded spectra in three
different regions : 600-700 cm-', 700-900 cm"1 and 1000-1400 cm" 1 . The
relative intensities of the lines in these regions being extremely different,
spectra plotted with various expansions are used to visualize the
experimental results.
600-700 cm"1 region
This spectral region is extremely sensitive to changes in DNA
secondary structure and contains in particular one of the most important
Raman marker lines of the Z conformation. Thus in the case of poly d(G-C) an
intense line found at 662 cur1 in 0.1M NaCl characteristic of a guanosine
vibration (anti geometry, B form) is shifted to 625 cm"1 in 4 M NaCl (svn
geometry, Z form) (fig. la,b) (14-18). A simple increase of the ionic
strength is sufficient to induce the B->Z transition and can be followed by
the shift of this Raman line in the case of poly d(G-C). In the case of poly
d(A-C). poly d(G-T), high salt concentration (4M NaCl) does not affect the
spectrum in any way as can be seen fig. lc. This spectrum is quite similar to
that recorded at 0.1M NaCl (not shown) and that obtained in presence of CsF
(20). Divalent and trivalent ions are known to be much more efficient in
inducing the left-handed structure of poly d(G-C> (22). If Mn a * long are
added to the initial 4M NaCl poly d(A-C).poly d(G-T) solution, and up to 1M
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a
<D
_
*
•
Figure 1 Raman Spectra in the 600-700 cm"1 region of: a) Poly d(G-C) 0.1M
NaCl B form, b> Poly d(G-C) 4M NaCl Z form, c> Poly d(A-C).poly d(G-T> 4M
NaCI B form, d) Poly d(A-C).poly d(G-T> 4M NaCl, 40mM N i C U B + Z forms, e)
Poly dCA-O.poly d(G-T> 4M NaCl, 65mM NiCl 2 Z form, f> Poly d(A-T) 4M NaCl
B form, g) difference spectrum obtained by substraction (Z-B) of poly
d(A-C).poly :: d(G-T) after normalization; guanosine \v.v\ thymidine /////
adenosine r-••:•••
MnCla, no important modification of the spectrum is detected in this
region. On the contrary low amounts of divalent transition metal ions induce
a progressive modification of the DNA structure which can be followed by the
gradual evolution of the Raman spectrum (Fig. lc,d,e). A doublet at 682-669
cor' in B form has been previously assigned respectieIy to guanosine and
thymidine modes in the case of native DNAs (23-25) and poly d(A-C).poly
d(G-T) (20). When Ni a * ions are added, the guanosIne component is
progressively shifted from 682 cnr* to 622 cm"1, while the thynldlne
contribution remains unaffected at 669 cm~* (Fig. ld,e). The shift of the
guanosine component, characteristic of the B->Z transition in oligo and
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Figure 2
Raman Spectra of the phosphodiester backbone of poly d(A-C).poly
d(G-T): a) 0.1M NaCl, (B form); b) 4M NaCl, (B form); c> 4M NaCl, 1M MnCU,
(B form); d) 4M NaCl, 40mfi NiCU, <B + Z forma); e) 4M NaCl, 65mM NiCla,
<Z form); f) difference spectrum obtained by substraction (B-Z) after
normalization; antisymmetric 0P0 stretch •*.•;.?}
symmetric 0P0 stretch /////
polydeoxynucleotides, reflects the C2/-endo/anti->C3/-endo/svn reorientation
of the guanosines.This experimental result is in excellent agreement with
calculations based on the GF-Wilson method, showing that only the geometry
change
between the B and Z forms is responsible for this observed
modification in the vlbratlonal spectrum (26,27). Similarly a contribution of
the adenosines in svn conformation may be expected around 622 cm~* as
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>••>
_ t-
Raman Spectra in the 1000-1400 era"1 region of poly d(A-C).poly
G-T): a) 4M NaCl, <B form); b> 4M NaCl, 65mM NiCl a , (Z form); c)
Difference spectrum obtained by substractioni of (B-Z) after normalization.
Symmetric P0 a ~ stretch —.
adenosine \-h y:-A guanosine
predicted by force field calculations (28). Spectra presented fig If and lg
will be discussed further.
700-900 cm~' region
In this spectral region are found Raman lines characteristic of the
phosphodiester backbone geometry, which are also sensitive to the secondary
structure of the DNAs. Fig. 2 presents the spectra of poly d(A-C>.poly d(G-T)
recorded in 0.1H NaCl (Pig. 2a), 4M NaCl (Fig. 2b) and in presence of Mn a *
ion3 (Fig. 2c) and Nl 2 * ions (Fig. 2d,e). The strong line located around
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790 cm"1 in the B form spectrum (Fig. 2a) involves a cytosine vibration
superimposed on the phosphodiester symmetric stretching vibration (20). The
antisymmetric stretching vibration of the phosphates i3 found at 836 cm"*.
An increase of the ionic strength up to 4M NaCl (fig. 2b) and the addition of
Mn a * ions (Fig. 2c) does not modify the spectrum in an important way. Only
a slight splitting of the 790 cm"1 line similar to that detected by
addition of high CsF amounts (20) may reflect a subtle change in the
phosphodiester conformation. On the contrary, addition of Ni 2 * ions
strongly modifies the backbone geometry, as reflected by the drastic decrease
of the relative intensities of both the symmetric and antisymmetric phosphate
stretching vibrations located at 832 cnr1 and 790 cnr1 and the emergence
of two new lines, detected around 810 cm"' and 742 cm"' (Fig.2e>. This
can be clearly seen on the computer generated difference spectrum, B form
minus Z form, presented in fig.2f. Peaks pointing in the upward direction
correspond to lines detected in the right-handed geometry while peaks
pointing in the downward direction correspond to lines of the left-handed
geometry. Two negative peaks around 810 and 742 cm"1 are characteristic of
the Z phosphodiester chain of poly d(A-C).poly d(G-T) in presence of nickel.
Moreover the complexity of the Raman line observed at 790 cm"1 in the B
form spectrum of poly d(A-C).poly d(G-T) (fig.la) is evidenced by the
existence of one negative (784 cm" 1 ) and two positive (790 and 771 cm" 1 )
peaks in the difference spectrum.
1000-1400 cm"1 region
Figure 3 presents the Raman spectra of poly d(A-C).poly d(G-T) in 4M
NaCl (fig. 3a, B form), in 4H NaCl, 65 mH N 1 C U (Fig. 3b, Z form) and the
difference B-Z spectrum (Fig. 3c). This region contains on one hand the
P0 2 " symmetric stretching vibration found at 1092 cm"1 for both
structures, and several lines due to base vibrations and which we shall now
discuss in detail. Three lines are detected in the B form spectrum, at 1374,
1342 and 1304 cm" 1 . The first one involves thymidine, adenosine and
guanosine motions while the two other ones are due to adenosine vibrations.
When nickel Is added the intense contributions of guanosine and adenosine in
the 1374cm" 1 line are shifted to 1354 cm" 1 , while the thymidine
contribution remains unaffected at 1374 cm"1 (shoulder). The line observed
at 1342 cm"1 in the B geometry involves a deformation motion of the adenine
imidazollc ring (27). It is displaced to 1328 cnr1 in the Z form spectrum.
A strong line is observed in the Z form spectrum at 1314 cm"1 corresponding
to a guanosine mode; a similar intense line is observed in the Z form
spectrum of poly d(G-C) at 1316 cm"1, (Fig. 4b) and only weakly in the B
form one (17).
These spectral modifications can be clearly evidenced on the
difference spectrum presented fig.3c. B and Z form spectra have been
normalized using the phosphate symmetric stretching vibration as a standard
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Figure 4
Raman Spectra in the 1000-1400 cm"1 region of: a) poly
d(A-C).poly d(G-T) 4M NaCl, 65mM N i C U (2 form); b) poly d<G-C) 4M NaCl (Z
form); c) Difference spectrum obtained by substraction of a) -.5b); d) poly
d(A-T) 4M NaCl (B form). Adenosine -p?..? guanosine \ \ \ \ cytoaine /////
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(1092 cm- 1 ). The <B-Z) computed spectrum is displayed. Three lines at 1354,
1328 and 1314 cm"' are characteristic of the Z form while a line at 1374
cm"1 reflects the B geometry. Moreover a broad line found at 1251 cm"' in
B form and Involving both adenosine and cytosine is split into two components
well separated in Z form at 1257 and 1245 cm"'. We propose to assign the
1245 cnr1 line to the adenosine component in the Z form spectrum of poly
d(A-C>.poly d(G-T) by comparison with the Z form spectrum of poly d(G-C)
(Fig. 4b).
All these results are In excellent agreement with I.R. data obtained
on hydrated polydeoxynucleotide films. Thus an absorption band located at
1374 cnr 1 In B form poly d(G-C), poly d(A-C).poly d(G-T), and poly d(A-T)
I.R. spectra, is shifted to 1354 cm"1 in the Z form spectra of all three
polydeoxynucleotides (9,12,29). This effect has been correlated with the
anti->gvn reorientatlon of the purines when the B->Z transition occurs.
Moreover the GF-Wilson calculations concerning adenosines and guanosines
involved in polydeoxynucleotides have predicted the evolution of these Raman
lines under the B->Z transition. In particular the 1316 cm"' mode of
guanosine in Z form poly d(G-C) involves mainly deoxyribose vibrations (27).
The Intense line observed at 1314 cnr1 in Z form poly d(A-C).poly d(G-T)
should thus be due to vibrations of deoxyribose bound to guanines in svn
geometry. Moreover a contribution of deoxyribose bound to adenines should be
expected at this position as predicted by the GF-Wilson calculations (28).
Raman spectrum of AT base pairs in Z conformation
The experimental
spectrum of poly d(A-T) in left-handed
configuration is not yet known. However some of its main features can be
predicted simply by comparison of the Z form spectra of poly d(G-C> and poly
d(A-C>.poly d(G-T). The substraction of the d(G-C) component from the poly
d(A-C).poly d(G-T> spectrum leaves us with an hypothetical computed Z form
spectrum of poly d(A-T). Spectra have been normalized using the phosphate
symmetric stretching line which is observed at 1092 cnr 1 . The difference
spectrum has been obtained by substracting from the poly d(A-C).poly d(G-T)
spectrum that of poly d(G-C) multiplied by a .5 factor so as to take into
account the base composition of the polymer. Thus we obtain both the
contributions of the A-T bases in Z form and of the corresponding
phophodiester chain and phosphate groups. Figure 4 presents the Raman spectra
in the 1000-1400 cnr1 region of Z form poly d(A-C).poly d(G-T) (4a), poly
d(G-C) (Fig. 4b), the difference spectrum (Fig. 4c) and for comparison the B
form spectrum of poly d(A-T) recorded in 4M NaCl (Fig. 4d). The three intense
lines of adenosine observed at 1374, 1342 and 1302 cm"1 in B form should be
deeply affected by the B->Z transition and should be expected as can be seen
on the difference spectrum (Fig. 3c) around 1354, 1332 and 1314 cm" 1 . In
particular the possible shift of the 1374 cm"1 line to 1354 cm"1 could be
satisfactorily compared with the corresponding displacement of the I.R.
absorption in poly d(A-T), found at 1374 cm-1 in B form and at 1357 cm"1
In Z form (12). Finally we must keep in mind that in the 600-800cnr' region
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Nucleic Acids Research
a line reflecting the a m geometry of adenosines should be expected around
622cnr'. This is shown in figure 1 which presents the Z form spectra of
poly d(A-C).poly d(G-T) (le), poly d(G-C) <lb>, the difference spectrum (lg)
obtained as in figure 4, by substraction of the G-C component from the poly
d(A-C).poly d(G-T) spectrum, and for comparison the B form poly d(A-T)
spectrum (If). As can be seen on the difference spectrum the thymidine line
around 669 cm"1 is unaffected while the intensity of the 622 cnr 1 line is
strongly decreased by the substraction of the guanosine contribution which
leaves present the proposed C3'-endo/sjm adenosine contribution.
The Raman spectra of poly d(A-C).poly d(G-T) obtained in high Na*
environment in presence of low amounts of divalent transition metal ions
<Ni 2 *) reflect a left-handed conformation of the polymer. We must notice
that the addition of either only high NaCl or only NiCla fails to induce
the Z conformation. If nickel chloride is added directly aggregation takes
place. It appears thus that a high sodium chloride content is necessary to
decrease the water activity and to allow interactions between nickel ions and
the N7 3ite of purines to take place. Such interactions have been shown to
exist in our I.R. study of poly d(A-C). poly d(G-T) selectively deuterated
on the N7 site of guanines, or of both purines (13). It may be proposed that
the Interaction of N K H a O ) * 2 * and the phosphate groups does favour an
aggregation of the B structure rather than a B->Z transition, and that the
left-handed geometry
is obtained only when N K H z O ) * 2 * binds
preferentially to the active N7 sites of the bases, possibly because of
electrostatic shielding of the phosphates by the excess sodium ions. Such a
competition between two types of interaction sites (phosphates and bases) has
been recently proposed to explain the different efficiencies of cobalt and
ruthenium hexaammines in stabilizing the Z form of d(CGCGCG) (30). Ruthenium
hexaammine has more potential binding sites available, however it is far less
effective at stabilizing Z DNA than the cobalt analogue. One possibility is
that its binding affinity for the groove phosphates is similar to that for
the convex surface. A dilution effect of the stabilizing effect occurs, the
non stabilizing groove binding modes competing for ruthenium binding with the
Z DNA stabilizing binding sites on the convex surface. Finally a recent
theoretical study calculating the salt dependent part of the free energy
determining DNA conformational stability in 1:1 electrolytes shows that
diffuse ionic cloud electrostatic effects can explain the B->Z high salt
transition for poly d(G-C), but not for other sequences for which specific
interactions may play an important role (31).
In summary, the Raman spectrum of Z form poly d(A-C). poly d(G-T)
has been characterized- in particular by the following marker lines : a line
at 622 cm"1 corresponding to purines in a SYJi conformation, phosphodiester
chain vibrations shifted from 790 and 832 cm"1 to 742 and 810 cm" 1 , a new
intense line of guanosine at 1314 cm" 1 , a shift of the adenosine lines from
1342 and 1374 cm"1 to 1328 and 1354 cm" 1 . All these features can be used
to characterize left-handed structures in sequences containing all of the
four ACG and T bases.
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