DNA structure studies by resonance Raman spectroscopy

Journal of Molecular Structure, 214 (1989) 43-70 Elsevier Science Publishers B.V., Amsterdam - Printed

DNA STRUCTURE SPECTROSCOPY

P.-Y. TURPIN*,

43 in The Netherlands

STUDIES BY RESONANCE

L. CHINSKY,

RAMAN

A. LAIGLE and B. JOLLES

Laboratoire de Spectroscopic Biomokkulaire (CNRS UA 198), Universitk Paris VI and Znstitut Curie, 75231 Paris Cddex 05 (France) (Received 4 January

1989 )

ABSTRACT The application of ultraviolet resonance Raman spectroscopy (RRS ) in DNA structure studies is reviewed. In exciting the purine and pyrimidine bases of DNA in their allowed electronic transitions at wavelengths shorter than 300 nm, the resonance Raman effect offers a specific source of information at each level of complexity. For the nucleic bases and mononucleotides, it gives information on the excited state geometries of the molecules through their intensity excitation profiles. For oligo- and poly-nucleotides, the Raman hypo/hyperchromism observed under the resonance conditions is very sensitive to base-base stacking interactions: thus it can monitor slight changes in these interactions, due to changes in the polymer geometries. Lastly the resonance Raman effect provides structural information through vibrational couplings between different components of the macromolecular buildings. Thus resonance Raman spectroscopy is shown to be a powerful technique for studying DNA conformational changes and gives very promising results in the selective investigation of chromophores in such elaborate molecular complexes as nucleic bases within DNA duplexes.

INTRODUCTION

When a light beam impinges on any molecular system, most of it passes through unaffected if the system is transparent, a small fraction is scattered at the same wavelength as the incident beam (Rayleigh scattering) and a much smaller fraction is scattered at wavelengths slightly different from the incident: this latter phenomenon constitutes the Raman effect, after the name of its discoverer [l]. These wavelength- or frequency shifts correspond to the vibrational modes of the atoms constituting the molecules being examined. A whole frequency shift pattern of the scattered light constitutes a Raman spectrum, which is very useful in determining vibrational properties from which structural information may be deduced. When the wavelength of the incident light beam falls in the vicinity or within *To whom correspondence

0022-2860/89/$03.50

should be addressed.

0 1989 Elsevier Science Publishers

B.V.

44

an electronic absorption band of the molecule, the Raman cross section is largely enhanced (by several orders of magnitude in favorable cases) for those vibrational modes which mimic the molecular distortion in the resonant excited state. Hence the resonance Raman effect (RRE) provides the advantage of the enhanced intensities of the lines from chromophores having electronic transitions in resonance with the excitation source. A spectrum obtained in resonance Raman spectroscopy (RRS) usually yields less structures than the corresponding ordinary Raman spectrum of the same molecule, since the enhanced modes are restricted to the atoms comprising the chromophoric group. However, this may constitute an advantage in the study of very large molecules (such as DNA) for which the Raman spectrum becomes crowded with many bands that have close or similar frequencies. This holds also in the investigation of molecular complexes (such as drug-DNA complexes) via vibrational spectroscopy: a correct choice of the excitation wavelength allows one to obtain selectively the RRE from a determined chromophore of the complex without interference of the other constituent(s). An additional bonus to the selectivity effect of the RRS is the sensitivity: because of the large increase in the intensity of scattering due to resonance, sample concentrations as low as 10P5 M may be used. Such concentrations are comparable to those needed in other spectroscopic techniques, such as fluorescence or circular/linear dichroism, thus they permit the running of same sample experiments with other techniques. They sometimes allow problems of material quantities, which may be encountered for specific applications, to be overcome. The eventual drawback of the exposure to exciting radiation, which the chromophore absorbs, is that it may lead to either fluorescent reemission or thermal decomposition of the sample. Numerous biological molecules possess chromophores of low fluorescence quantum yield: this is true for the DNA compounds. Besides this, there exist numerous experimental tricks to get rid of the fluorescence (excitation in higher orders of the electronic transitions of the molecules, for instance) and the photochemical or thermal damage (stirring of the sample, free-flowing stream systems, low temperature measurements in cryostats). Hence RRS offers an efficient, nondestructive means of obtaining valuable structural information. There are several recent, excellent review articles [ 2-91 and books [ 10, lla] that discuss various applications of RRS on biomolecules or molecules of biological interest. The purine andpyrimidine bases of the nucleic acids and DNA have strongly allowed electronic transitions at wavelengths shorter than 300 nm. Excitation in or near the first strong nucleotide absorption bands has been made possible by using doubled flashlamp-pumped dye lasers [ 12-151 or via frequency doubling of the 514.5 nm line of the Ar+ laser [ 16-181. These first approaches yielded the advantages of the UV-excited spectra over the visible spectra, par-

45

titularly by the selective enhancements which allow one to alleviate the overlap of Raman bands associated with different nucleic acid constituents. The advent of pulsed Nd-YAG lasers has opened a much wider region of the UV spectrum for resonance Raman studies: the high peak powers of very short laser pulses allow very efficient wavelength shiftings through non-linear optical processes. The Nd-YAG 1.06fim fundamental wavelength may be doubled or tripled, these overtones being used in pumping a dye-laser which delivers continuously tunable wavelengths over the fluorescence domain of a variety of dyes. The dye-laser lines may be in turn doubled and/or mixed with the 1.06 ,um fundamental, and these combinations allow one to cover continuously the 215-750 nm excitation domain. Other authors use higher overtones of the NdYAG laser [ 19-211 and H, Raman shift cells to generate shorter wavelengths, by multiples of the 4155 cm-’ Hz stretching frequency [ 22,231. However it is to be noted here that, owing to the very high peak powers one can reach with these pulsed sources, it is sometimes possible to obtain Raman spectra of the molecules in their excited states by a pump-probe process: although this may be useful for some applications, it may sometimes constitute a drawback for other applications. It is necessary to be aware of this contingency in interpreting the spectra. Nevertheless it is now possible to probe continuously the first allowed transitions of the purine and pyrimidine bases, and higher lying transitions: the spectra show dramatic changes in enhancement patterns and reveal vibrational features which have not been previously resolved. In incorporating the bases into nucleic acid duplex structures, perturbations of the spectra are expected from the electronic effects of base pairing and stacking and from alterations in vibrational coupling to modes of the ribose-phosphate backbone (Fig. 1) . These perturbations in the purine and pyridimidine RR spectra, observed on duplex formation, are important for RR studies of DNA structures. A number of the RR bands are highly sensitive to the DNA conformation in solution: one can observe very important hypo- or hyperchromic effects of some of them, much larger than those observed in classical Raman or in electronic absorption. These effects are mainly due to changes in the base stacking interactions, and can probe order-disorder or conformational transitions. A number of vibrational frequency changes may be also seen with UV excitation, correlated to DNA structure transitions such as the B-+2 conformational transition. ULTRAVIOLET RRS OF NUCLEIC ACID BASES: RR EXCITATION PROFILES AND EXCITED STATE GEOMETRIES

The first Raman intensity versus excitation wavelength profiles of the four nucleic acid bases in the 647.1-351.1 nm range (Kr+ and Ar+ laser lines) have been obtained by Tsuboi and coworkers [ 18, 241: they showed that some vibrations of the base chromophores derive their intensities from various al-

46 BASES

NH2

Adenlne

Rib&e

Guanine

kibose

Cytosine

I

Ribose

Thymlne 0

H

0

H#

H

Ns 2 L?

O

4

5

Uracil

1 6 i

H

Ribose

Fig. 1. Covalent structure of the nucleic acid deoxyribopolymer chain (left-hand side) and the structures of the purine (A, G) and pyrimidine (C, T, U) bases (right-hand side).

47

lowed electronic transitions in the UV range. They drew their assignments from theoretical prediction involving a comparison of the normal modes of vibration of the molecules with the corresponding bond order changes obtained from molecular orbital calculations on the excited states. The general rule stated by Tsuboi and coworkers [ l&24] is the following: if the ground state deformation of the molecule in a normal mode of vibration resembles the distortion of the molecular geometry in going from the ground- to an excited state of the molecule, then the Raman active line corresponding to this vibrational mode tends to get its intensity from that electronic state through a resonance effect, i.e. it is strongly enhanced for an excitation lying in this electronic state. Conversely, the examination of the intensity enhancement factors of the normal modes of vibration as a function of the excitation wavelength in the region of the allowed electronic transitions, i.e.genuine resonance Raman conditions, should give valuable information about the molecular geometry in its excited states. This constituted one of the main goals in seeking detailed UV RR excitation profiles of the nucleic bases. The gap between 351.1 nm and 257.3 nm (the frequency doubled 514.5nm line from an Ar+ laser) was filled by the use of a pulsed dye laser containing a KDP frequency doubler. This allowed Blazej and Peticolas [ 121to present the RR excitation profile of AMP from 514.5-268.0 nm, and to fit the experimental profile with a calculation using a simple form of the vibronic theory of Raman scattering. In the same period, Chinsky et al. [ 14,251 obtained RR spectra of nucleotide derivatives in solution as dilute as 10e4M, with the 300 nm excitation wavelength, the pyrimidines and the purines being brominated in the 5and &position, respectively; these substitutions induce an enlargement of the low energy limb of their electronic absorption profiles, enabling a genuine resonance effect at 300 nm to occur. Blazej and Peticolas developed a semi-empirical theoretical method, based on a Kramers-Kriinig transformation, for calculating the RR excitation profile from the measured absorption profile [ 131. They obtained the A&; values for a number of normal coordinates of UMP and CMP for their lowest excited states, AQ being the shift of the potential energy minimum in the electronic excited state from that in the ground electronic state (Fig. 2): it is given as a vector in the normal coordinate space, and has dimensions of A amu112. Thus the estimation of a good AQ vector can lead to the excited state geometry. The Kramers-Kriinig transform technique has been modified to permit the calculation of overtone and combination band intensities in a resonance Raman spectrum relative to the fundamentals [ 161. This was applied to the UV (300, 257 and 248 nm) RR spectra of UMP, which yield five fundamental Raman lines (700-1700 cm-l ) and very rich overtone and combination band contributions in the 2000-5000 cm-l region (Fig. 3). For matching the theoretical and experimental results, no internal standard for the experimental determination of the Raman intensities is required as in ref. 13, nor is it nec-

,.-------__ Tp

48

m’c(O

____.

-

,)

-___

m'=(O,O)

,__-----__

EMD

w

___--.. ___ ,-,___- - - - - - ___- . --_ _--. _--

i

m=(m,,m2)

II

m=(l,l) m=(O.Z.)

LM

A%

;,-,b, 4

:

(:_I

Fig. 2. Representation of the potential energy surfaces of the ground- (G) and excited (M & M’ ) states, showing the AQi, shifts of the minimum of the mth electronic state from that of the ground state, in the normal coordinate space.

essary to assume any value of the vibronic linewidth r, which appears in the Kramers-Heisenberg expression for the Raman scattering tensor. Only the shape of the absorption band and relative intensities of the harmonics to the fundamentals are sufficient for obtaining the value of AQej, the shift of the eth excited state potential minimum along the jth normal mode for each of the five RR active vibrations (Table 1) . After obtaining these AQejvalues, it is possible to translate them into a change in the bond lengths and/or bond angles, through the use of the transformation matrix L from the normal coordinate vector Q to the more familiar intramolecular coordinates: R = L Q, in using (see Tsuboi et al. [lib]) AR=L

AQ

Since then, a few groups of spectroscopists have made a number of investigations in the 280-200 nm range. Ziegler et al. [ZO] used the 266 and 213 nm excitations (i.e. the fourth and fifth harmonics of the Nd-YAG laser) for obtaining RR spectra of the nucleoside 5’-monophosphates UMP, CMP, AMP and GMP: with 213 nm they obtained several new features relative to the 266

49

her-tones and combination bonds

b

Fundamental:s

300 nm

n

?48 nm

5000

a+JJJyJc, 4500

4000

3500

3000

1

I

2500

2000

1.

I

1 so0

1

1000

c M-l

Fig. 3. Observed resonance F-&man spectra of uracil in UMP at 248,257 and 300 nm excitation wavelengths, showing (in the 2000-5000 cm-’ region) the overtones and combination bands of the fundamentals.

TABLE I The excited state displacements

dQ=j for resonance

Normal vibration j (cm-‘)

AQej

1680 1630 1396 1232 783

0.8kO.l 0.5 k 0.1 0.7kO.l 1.3 + 0.2 1.OkO.l

Baman active modes of UMP [ 161”

(relative value)

nm spectra, particularly some modes of the pyrimidines with a character similar to the e‘&& mode of benzene, and a strong enhancement of the 1670 cm- ’ C=O stretching mode of GMP. Shortly after, Fodor et al. [ 221 investigated the deoxyribonucleotides of uracil, thymine, cytosine, adenine and guanine (dUMP, dTMP, dCMP, dAMP and dGMP) by the use of a quadrupled Nd-YAG laser (266 nm) and an H2 Raman shifter. For dUMP and dTMP the enhancements were interpreted in terms of resonance with electronic transitions based on the C6=C5-C4=0 and C2=0 fragments (see Fig. 1) at long and short wavelengths,

50

respectively. For dGMP and dAMP, the intensities are related to an N7=C8 localized transition at long wavelengths and a series of triene-based transitions throughout the UV region. Kubasek et al. [ 231 reported UV Raman spectra of the four ribonucleotides AMP, GMP, UMP and CMP, obtained with eight laser excitations between 299 and 200 nm. They constructed low-resolution excitation profiles for the strongest bands, using the phosphate band at 994 cm- ’ as an internal reference supposed to have no resonance enhancement in this region (Fig. 4). It was clear from their data that a full description of the RR profiles of the nucleic

1

t

CMP

I

UMP

c: 4) 3.4

2w

209

2.18

229

240

253

266

299

Excltatton frequency, nm Fig. 4. Resonance Raman excitation profiles for the four ribonucleotides AMP, GMP, CMP and UMP. All panels are on the same intensity scale, and the curves are labeled by the vibrational band in question (in cm-‘) (from ref. 23).

51

acids would have to include several electronic excited states, and that the fitting of resonance data to so called “Albrecht’s A-type terms” may fail in some cases and no longer be sufficient. In addition, Kubasek et al. [ 231 show cases where ionic species of mononucleotides can be distinguished by using far-UV excitation, these species being indistinguishable with 266 nm excitation: this demonstrates the utility of far-UV RRS for obtaining structural information. Finally, there is a large difference in the maximum intensity of the bands for each nucleotide, depending on the excitation wavelength: this means that the base-specific differential excitation permits selective enhancement of particular bases within large molecular complexes such as DNA or RNA. In the same way interesting secondary structure changes may have significant effects on the Raman excitation profiles because of shifts in electronic excitation energies. This will be demonstrated below. HYPO-HYPERCHROMISM

IN PRERESONANCE

TRA OF POLYNUCLEOTIDES:

AND RESONANCE RAMAN SPEC-

MOLECULAR SIGNIFICANCE

The classical Raman spectra of polynucleotides and DNA give some information for both sugar-phosphate backbone and base moieties, related to the various structures of the polymers (see Fig. 1) . The phosphodiester backbone is a fully saturated type molecule whose electronic transitions involve a-type bonding electrons: the absorption bands arising from electronic excitation of the backbone chain lie in the vacuum UV. Hence the information linked to the backbone is lost in the RR spectra of nucleic base containing polymers in solutions as dilute as 10e4 M in base, for excitations at ca. 250 nm. However, Peticolas and coworkers reported in a series of papers [26-281 that, in classical Raman, the intensity of the lines from the base vibrations show variations upon base stacking, due to a preresonance Raman effect of these lines coupled with hypochromic absorption bands, corresponding to lowlying excited states of the bases. When the bases are juxtaposed, either longitudinally by means of stacking interactions, or laterally by means of WatsonCrick hydrogen bonding interactions, the electronic transition moments may interact for changing the shape and intensity of the absorption profiles, and consequently the Raman line intensities deriving from them. From Peticolas we adopt the term “Raman hypochromism” to signify the decrease in Raman intensity which occurs upon formation of a stacked or ordered nucleic base structure. Actually, the effect can be measured as an increase in the Raman intensities upon melting of the ordered structure. In classical Raman, the relative intensities of the hypochromic bands are measured relative to the backbone chain mode at 994 cm-‘, due to the O-P-O symmetric stretching vibration supposed to be independent of the structure variations. Since in principle one can monitor the Raman hypochromism of each line of each base separately, the Raman effect provides a very sensitive

52

tool for studying specific base-to-base interactions. Because it is much more sensitive, it may become, under resonance conditions, far more important than the UV electronic absorption hypochromism, as we shall see below. However, the Raman hypochromism of each nucleic base component is highly dependent on the polynucleotide structure in which it participates, e.g. ribo- or deoxyribonucleotide, A, B, 2 or heteronomous form, and obviously the degrees of hypochromicity of the normal modes differ from each other [ 291. In RRS in the 250 nm region, although the information pertaining to the backbone is generally lost because of low sample concentration and out-ofresonance conditions for the sugar-phosphate chain, one can observe very important hypochromic effects on the nucleic base bands upon structure transition, often much more important than in classical Raman. This was observed for the first time by Chinsky et al. [25] in the RR spectrum of a bromodeoxyuridine containing DNA, i.e. poly (5BrdU-dA)a, with an excitation at 300 nm. For this excitation the adenine contribution to the spectrum remains very weak (the base concentration was 1.3x 10m4M), while the 5BrdU contribution is largely enhanced owing to the bromo-substitution. Upon thermal melting of the polymer, the intensities of the 5BrdU lines at 1220 cm-’ (“Kekulk-type” mode), 1352 cm-’ (ring mode) and 1627 cm-’ (C-O stretching) were increased by a factor of 2.2, 2 and 1.8, respectively. In contrast the measured absorption hypochromism is only 35% on melting of the same sample. A similar observation has been reported in the case of conformational changes of poly (dA-dT), from random coil to ordered structure with stacked bases [ 151: some of the thymine lines show a hypochromism of about 200% (instead of 40% in absorption) when the polymer is excited at 300 nm in preresonance conditions (Fig. 5). This intense effect was monitored to study the poly (dAdT),-RNase and poly(dA-dT),-histone Hl interactions, as models for mechanisms of destabilization and stabilization of the DNA secondary structure by proteins, respectively. However, the preceding observations show that the hypochromism in preresonance or resonance Raman does not merely follow the rule of proportionality to the square of the molar extinction coefficient, as could be stated in a first approximation from an Albrecht’s A-type term interpretation [30]. A more precise investigation of the Raman hypochromism as a function of the exciting wavelength showed that this effect can be inverted near resonance, i.e. hypochromism may become hyperchromism: this was observed in tracing the hypochromism excitation profile on melting of poly (rU ) between 363 and 290 nm [31] and of poly(dA-dT), between 320 and 257 nm [32]. These excitation profiles exhibit negative values in the 290 nm region, although the absorption profiles of the random (melted) and ordered structures do not show any crossing in this region. Chinsky and Turpin [ 311proposed a theoretical explanation for these ob-

53

Fig. 5. Preresonance Raman spectra of poly(dA-dT), 2 mg ml-‘, 3 X 10e2 M NaCl aqueous solution, excited withA= nm. Temperature: (a) 2O”C, (b) 55”C, 2’,=49”C.

servations, involving both the relative variations of the molar extinction coefficient, and the relative variations of rej, the damping factors of the excited vibronic levels on the order-disorder transition of the polymer. Figure 6 shows the results of a computation of the variations in Raman hypochromism AI/I versus the excitation frequency near and within the resonance conditions, the vibronic damping factor r being as a parameter, expressed relative to the vibrational frequency u Vof the mode under investigation. In particular, for large values of r (r= IJ,,, which is reasonable since the absorption curve of polynucleotides is broad and unstructured) it is possible to see a large hyperchromism when the excitation falls within the uoo-uol interval. These theoretical curves are found to fit adequately the experimentally measured hypochromism excitation profiles obtained for melting transitions of poly (rU) and poly (dA-dT),, over the experimental excitation range [31,32]. Hence it appears that knowledge of the complete hypochromism excitation profile is important in following order-disorder transitions of a polynucleotide in using RRS. This is also the case for the line intensity variations of a chromophore upon intercalation within an ordered structure. For instance, in investigating the binding of a copperporphyrin to a DNA duplex by RR in the Soret band, the trend of the hypochromism excitation profile of the chromophore lines is to follow the variations of the absorption profile. However, the hypochromism excitation profiles of the lines are different [ 331: this was in-

-- 2AE &

L V 00

V

01

vI

V”

Fig. 6. Calculated Raman hypochromism AI/I excitation profiles near and within the resonance conditions (see text). The abscissa unit is the vibrational quantum u”, uoois the energy gap between the minima of the ground- and electronic states. Al/I is compared to AC/E, the relative variation of the molar extinction coefficient upon structure changes.

terpreted in terms of core ring additional deformation (distortion on excitation) when intercalated. STRUCTURE AND DYNAMIC INFORMATION ON DNA IN UV RRS

The highly polymorphic nature of double helical DNA is now well established [ 341. The interconversions between alternative conformations (A, B, 2 and the so-called “heteronomous forms”) are processes of interest not only to the physical chemist but also to the molecular biologist interested in relating sequence features to functional consequences for gene expression. Among the various forms, the 2 conformation (left-handed helix) has been the subject of

55

intense investigation in the recent past (Fig. 7): Z-DNA has been detected in fibres and films of DNA oligomers and polymers, in supercoiled plasmids, in chromosomes and cells. Raman spectroscopy is widely recognized as a powerful tool in determining nucleic acid structures, and classical Raman spectroscopy has been extensively used for that purpose. A few years ago it became evident that DNA conformational information may also be obtained from the resonance enhanced vibrations of the chromophoric portions of the nucleic bases. These RR investigations are now available not only from excitations lying in the first allowed transitions of the bases, but also from higher energy transitions. G-C containing oligo- and polyndeotides Pohl and Jovin [ 351 showed by circular dichroism that the conformation of poly (dG-dC), is changed merely by raising the salt ( NaCl) concentration. The existence of a left-handed helix was proven by an X-ray diffraction study of a crystal oligonucleotide d ( CPG)~ [ 36-381. This left-handed structure was also observed in fibers of poly (dG-dC),. Thamann et al. [ 391 showed that the Raman spectra of the d (CpG), crystal and of the poly (dG-dC), solution at high salt concentration are similar. Hence they proved that the structures of the crystal and of the polymer in high salt solution are at least very close to each other. Thereafter a tremendous effort was made using Raman spectroscopy for

Z.DNA

S-DNA

Fig. 7. Schematic Van der Waals side views of the DNA molecule, in two canonical forms: the right-handedB double helix (right-hand side), and the left-handedZdouble helix (left.-hand side) showing the zig-zag phosphate chain (from ref. 81).

56

studying oligonucleotide crystals, polynucleotide fibers and solutions and for characterizing the right- and left-handed structures unambiguously [ 40-451. Jolles et al. [45] first obtained the poly (dG-dC), spectra in low salt (0.1 M NaCl) and high salt (4.5 M NaCl) solutions, corresponding to the B and 2 forms of the polymer, respectively, with an excitation at 257 and 295 nm, i.e. at the maximum absorption and at the maximum differential absorption of both forms (see Fig. 8). Since in a rough approximation the resonance effect enhances the Raman line intensities proportionally to the square of the molar extinction coefficient, it was not surprising that the line intensities were found to be about sevenfold enhanced, with the 295 nm excitation, upon the B--+2 transition. In addition, at this excitation, several lines provided by both G (620 and 1193 cm-‘) and C (780,1242 and 1268 cm-l) residues are sensitive to the conformation modifications of the polymer (Fig. 9). On the other hand, with the 257 nm excitation, the guanine contribution to the spectrum is largely predominant: the 1580 cm-’ G line (triene stretching motions) is greatly enhanced in the 2 form, but also an important enhancement of the cytosine 1630 cm-l line (carbonyl stretch) is observable in going from the B to the 2 form (Fig. 10). These results, related to the B-2 structure transition, may be essentially interpreted as follows: (i) The general intensity enhancements mentioned above are to be related & (M-l.cm-1) 7000

5000

3000

1000

225

250

275

3oo nm

Fig. 8. Electronic absorption profiles of the B (full line) and Z (dashed line) conformations of poly(dG-dC)2.

57

k

1750

1

1500

I

I

I

1250

1000

I

750

I 4-l

Fig. 9. Resonance Raman spectra of poly(dG-dC)z obtained with 295 nm excitation wavelength. A (x 7, see text): in 100 mM NaCl (B form), B: in 4.5 M NaCl (2 form), phosphate buffer pH 7.

,

1790

1500

I

1250

I

1000

Fig. 10. Same as Fig. 9, but A,,=257

1

750

I

I .I-1

nm.

to the changes in the stacking interactions between nucleic base cycles during the conformational transition. The changes in the helix geometry parameters (see Table 2) in going from B to 2 all trend towards weaker stacking interac-

58 TABLE 2 Some distinctive

parameters

of the B (right-handed)

and 2 (left-handed)

B helix Helix rotation

angle ( ’ )

‘Number of b.p. per turn Diameter (8) B.p. distance.(i) Helix pitch (A) Glycosidic x angle (’ ) Sugar pucker

36.0 (Monomer 10 20 3.4 34.0 260 anti C2’ endo

Z helix 60.0 (CpG unit) 12 18 3.1 45 65 syn C3’ endo

unit)

Syrl Anti OF

C2’

endo

Sugar

‘d-DNA

DNA helices

POSITION

POSITION

OF GUANINE

GUANINE

Pucker

C3’

endo

Sugar

Pucker

Z-DNA

Fig. 11. Perspective drawings of the anti and syn orientations about the glycosidic bond. The two major conformations of the sugar puckering are also represented (from ref. 77).

tions for the 2 helix: the B-t2 transition may mimic to some extent what happens on melting the B form (as far as the cycle stacking strength is concerned), i.e. the Raman lines are enhanced. (ii) The guanine line observed at 680 cm-l in the B form is assigned to a base residue ring breathing mode coupled with sugar-pucker vibrations: it shifts to 620 cm-’ in the 2 form of the polymer, owing to the C2’ e&o/anti to C3’ endo/syn reorientation of the sugar (Fig. 11). (iii) The cytosine 780 cm-’ and 1630 cm-l lines (Figs. 9 and 10, for the 295 and 257 nm excitation wavelengths, respectively), are assigned to cytosine ring vibrations coupled with C2=0 out-of-plane bending and in-plane stretching motions, respectively. As shown by Howard et al. [46], the C2=0 cytosine

59

frequencies of hydrogen bonded G-C pairs in polynucleotides may be influenced by interbase vibrational couplings. In the B form, the unit motif is one base pair, and all the base pairs are stacked along the axis of the helix. In the 2 form, the unit motif is the d (CpG ) dimer, and the base pairs are rejected at the periphery of the helix; however, the cytosine C2=0 groups are located inside the helix, pointing towards the axis and are almost superimposed along the 2 helix axis (see Fig. 12). Hence this geometry strongly favors C2=0 interbase vibrational couplings described by Howard [ 461. With this geometry in mind it is easy to understand that the Raman cross-section of the C2=0 carbonyl vibrational motions (and consequently the line intensities) may be

0

10;

B DNA

Z DNA Fig. 12. End views of the 2 (lower part) and B (upper part) of a complete turn of a DNA helix. In the 2 DNA, the guanine bases are found on the outer part of the helix, the center has an elliptical oxygen tunnel (cytosine C=O groups). In B DNA, the bases are clustered near the center of the molecule (from ref. 36).

60

largely increased in the 2 geometry, relative to the B geometry, for the stretching as well as for the out-of-plane bending modes. These observations and interpretations were confirmed in two other papers reporting RRS results on poly (dG-dC), under the 2 form. In the first paper, Chinsky et al. [ 471 showed that the cytosine 780 cm-’ frequency does not take its intensity from the 260 nm electronic band, in either B or 2 forms of the polymer. Furthermore, IR spectra showed that this line is highly polarized along the axis of the 2 helix, i.e. perpendicular to the cytosine planes. For interpreting both the dramatic increase of the 780 cm-’ line in the Z form excited at 295 nm, and the polarized IR measurements, the authors assume that it may derive its intensity from an electronic transition which may be located in the 280-300 nm region in the 2 form of the polymer. This electronic transition could be perpendicular to the cytosine ring, thus having some kind of m* character, although it remained impossible to bring evidence of such a transition through UV electronic polarization measurements. The second paper reports RR spectra of poly (dG-dC), B and 2 conformations, taken with 224 nm excitation, i.e. in higher energy transitions [ 481. This excitation gives rise to predominant pyrimidine contributions: apart from the guanine 680-620 nm structure marker line which can be clearly seen, most of the other lines of the spectra are provided by the cytosine. The behaviour of the 780 cm-’ frequency (ring breathing coupled with C2=0 out of plane motions), reported for the 295 nm excitation, is now quite different since it is no longer in resonance with the same electronic state, and the 1640 cm-’ in-plane C=O stretching frequency is largely enhanced in the 2 form relative to the B form, owing to the geometry-induced stronger interbase carbonyl vibronic couplings of states which are still under resonance at 224 nm. These few examples demonstrate very well the variety as well as the complementarity of information that RRS can afford in using various wavelengths of excitation over the whole domain of electronic transitions of the nucleic bases, for gathering structural aspects concerning DNA in dilute solution. In addition, the whole range of vibrational frequencies can be explored in RRS until 2000 wavenumbers without disturbance from the bending motions of water, which usually forbid any valuable information in the very interesting 1550-1700 cm-l C=C and C=O region in classical Raman spectroscopy, even in highly concentrated samples. Instead of that, the high and broad stretching motion band of water around 3450 cm-’ may be used in RRS as a convenient internal standard of intensity when following the structure changes in the DNA samples. The pathways and mechanisms for the B-2 transition have been extensively explored with various methods, including Raman spectroscopy (see review [ 491). On the other hand, the canonical 2 form with a zig-zag phosphodiester chain (Fig. 7) is probably not unique in the wide family of the lefthanded helices. We now report the unexpected behaviour of the canonical 2

61

helix upon melting in solution, as clearly evidenced in UV RRS, which leads us to assume the existence of relaxed left-handed DNA structures. In dilute solution, a modification of the conformation of the 2 double helix as a function of the temperature was observed in UV absorption, circular dichroism and 31P NMR, for a temperature lower than the melting point of the helix [ 501. It was suggested that this new conformation was neither a B form nor a single strand polymer. Laigle et al. [51] made the study of the thermal stability of the hexanucleotide pentaphosphate (m5dC-dG)3, by RRS with a 257 nm excitation wavelength. The methylation in the 5-position of the cytosine is known to favor and stabilize the 2 conformation of d(G-C),. At low temperature, the spectrum of (m5dC-dG), in 0.2 M NaClO, resembles that of the B form of poly (dG-dC ) 2, while in 3 M NaClO, it looks like a 2 form spectrum. At first the authors showed that the melting at 75°C of both samples (low and high salt) leads to the same spectra of the denatured species. The first astonishing finding is, while the B form reveal hypochromic structure (actually the line intensities are weaker for the ordered than for the melted structure, see previous section), the 2 form reveals hyperchromic structure for this excitation wavelength (see Fig. 13). Remember that in going from B to 2 conformation, the absorption curve is lowered in the 260 nm region (Fig. 8)) and both ordered species are hypochromic relative to the melted species in the same region. Hence, both species would have been expected to show a hypoch-

Fig. 13. Normalized RR spectra of (m5dC-dG)3 2.7 X lop4 M, tris buffer 10 mM, pH 7.5, I,,= 257 nm. Dashed line: 0.2 M NaC104, O”C, I3 form. Alternate line; 3 M NaClO,, O”C, 2 form. Full line: 0.2 and 3 M NaC104, 75 ’ C, denatured form.

62

romic effect, and there is up to now no definite explanation for this surprising opposite behavior of the B and 2 structures observed in RRS. At least there is no unequivocal correspondence between geometry changes and electronic transition properties. Another very interesting observation made by Laigle et al. [51] is that the melting profile of several bands of the high salt 2 form versus temperature is biphasic: a first phase of the melting profile appears linear between 0°C and 30 ’ C, a second phase appears sigmoidal (cooperative) between 30” C and 75’ C (Fig. 14). A similar observation was made by circular dichroism experiments only for AeZg5,but not for Aen,7 nor for Ae255on the same sample. An analysis of the results suggests that the temperature increase induces at first a transition from the canonical 2 form to an intermediate stable form, which then melts at higher temperature. From a deconvolution technique, the authors characterized the RR features of this intermediate (Fig. 15) which, like the 2 form, is hyperchromic but to a lesser extent. Hence it probably belongs to the left-handed family, but with changes in the base stacking and in the geometry of the phosphate groups as compared to the canonical 2 form. Let us assume it is some relaxed left-handed form, and propose the thermal transition pathway Canonical 2 form-Relaxed

left-handed form-Denatured

species

As a last example of the possibilities of structure and dynamic investigation of alternating G-C containing polymers by RRS, we should like to quote an experiment dealing with the dynamics of hydrogenwdeuterium exchange in

Fig. 14. Three dimensional view of the RR spectra of the 2 form of ( m5dC-dG)3, temperatures. For conditions, see caption to Fig. 13.

taken at different

63

e-1

Fig. 15. Normalized RR spectra of the B form (dashed line), 2 form (alternate line), and intermediate left-handed form (full line) observed in the premelting of the 2 form. For conditions, see caption to Fig. 13.

relation to the flexibility of the polynucleotide. Indeed, the B and 2 forms of DNA differ not only in conformation but also in dynamic properties. In poly (dG-dC ) 2, five protons are spontaneously exchangeable in each base pair, i.e. two protons of the C2 amino-group and the proton of the Nl imino-group of the guanine, and two protons of the N4 amino-group of the cytosine residue. At first Leng and co-workers [52,53] showed that the exchange rates of the protons of the 2 form of poly (dG-dC), are lower than those of the B form by several orders of magnitude. In addition, it was established that in the 2 form itself, two protons are much more slowly exchangeable than the other three, but the precise assignments of the slow/fast protons were rather controversial [ 54-561. A definite answer has been given by Laigle et al. [ 571 in following the H+D and D-+H exchange rates in the poly(dG-dC), 2 form, in exciting at 257 nm (for which the guanine contribution is dominant) and at 284 nm (for which the cytosine mostly contributes to the spectrum), with a multiple fast scanning procedure of adequate exchange marker lines. They established that the three protons of the guanine are exchanging at the same rate, and faster than the two protons of the cytosine, which both exchange about tenfold more slowly (Fig. 16). It was then possible to confirm a model of “asymmetric opening process” of the polymer proposed by Ramstein et al. [ 551, in which the guanine protons are more easily in contact with the surrounding solvent than the cytosine protons.

64

Time

(hours)

Fig. 16. Hydrogen -+ deuterium exchange in the 2 form of poly (dG-dC)z, measured in following RR marker bands of guanine ( A ) and of cytosine (0 ). AZ(t) is the difference between the RR intensities measured at time t and after 24 h (complete exchange). The normalized amplitude of AZ(t) is plotted on a semi-logarithmic scale versus time.

A-T containing

polynucleotides

The polymorphism of A-T containing sequences is still far more diversified than that of G-C containing sequences: this is probably because AT bases engage only two hydrogen bonds to constitute a base pair, instead of three for the GC base pairs, which may result in a much higher flexibility of the AT duplexes. Thus it is not surprising that structural studies of A and T containing sequences give rise to a very abundant literature, most of the results being far from definitive and most of the time subject to severe controversy. For instance, Klug et al. [ 581 proposed an “alternating B structure” for the alternating copolymer poly (dA-dT),, but this failed to fit the fiber data [ 591. A good discussion was proposed by Gupta et al. [ 591 for elucidating the structure of this polymer, between Watson-Crick or Hoogsteen base-pairing, rightor left-handed helix, etc. The paired homopolymer poly (dA) *poly (dT) has also been extensively studied: two successive papers of Arnott et al. [60,61] proposed, from X-ray data on fibers, either a minor variant of B DNA structure or a heteronomous secondary structure for this polymer. Classical Raman data on poly (dA) *poly (dT) in low temperature solutions revealed the simultaneous existence of both C2’ -endo and C3’ -endo Raman marker bands [ 621, but it remains impossible to know what strand has C3’ -endo puckered ribose rings. Jolles et al. [ 631 obtained, with the 257 nm excitation wavelength, the spectra of the adenine residues participating in two single-stranded polynucleotides, poly (rA) and poly (dA) , and in three double-stranded polynucleotides, poly(dA)*poly(dT), poly(dA-dT), and poly(rA)*poly(rU). Poly(rA) and

65

poly (rA) -poly (rU) are known to adopt an A-family structure in solution. This is actually what Jolles et al. [63] found by RRS for poly (rA) and For low temperature aqueous solution of poly(rA)*poly(rU). poly (dA) -poly (dT) , they proved that the adenine strand adopts an A conformation with C3’ e&o-puckered furanose rings, while the other strand poly (dT) has the C2’ e&o-puckered ribose rings of the B family. Thus they confirm in solution the hypothesis of a heteronomous form proposed for fibers of the same compound by Arnott et al. [ 611, These results may be compared to those obtained in classical Raman spectroscopy by Benevides and Thomas [64] on a solution structure of the RNA-DNA hybrid poly (rA) l poly (dT) with C3’ -e&orA and C2’ -endo-dT nucleosides, indicating an unusual intrastrand hydrogen bond involving adenine donor (C8-H) and acceptor (05’ ) groups. For a long time, it had been impossible to find evidence for the existence of left-handed 2 helices in A-T alternating copolymers, such as had been observed some years ago in high-salt solutions of d (G-C),. Then, numerous sequences incorporating various amounts of A-T base pairs inserted in G-C tracks were investigated by a variety of physicochemical techniques [ 65-681 and were revealed as being able to adopt left-handed geometries, although the A-T pairs seemed less stable in the 2 conformation as compared to the G-C ones. Recently the B-t2 transition of poly (dA-dT), was observed for the first time in IR spectroscopy for hydrated films in the presence of divalent transition metal ions [ 691. In addition, for poly (dA-dT) 2 in 5 M NaCl aqueous solution the structural transition is induced by the addition of 95 mM of Ni2+ ions (UV absorption and CD measurements [ 70 ] ) . A classical Raman study of a similar sample yielded marker lines of the Z-form of the polymer, essentially characteristic for 2 DNA backbone vibrations and anti+syn reorientation of the purine geometry under the B+Z transition [71]. Unfortunately this last study was limited to the 600-850 and 1000-1400 wavenumber regions, and particularly dropped the information linked to the very interesting C=C and C=O 1500-1800 cm-’ region. The present authors, in collaboration with Miskovsky, made a UV RRS study of poly (dA-dT), solutions at high NaCl concentrations and in the presence of different amounts of Ni2+ ions, with the 223,257 and 281 nm excitation wavelengths, in order to profit from different resonance conditions and find specific information on the A and T residues [ 721. In lowering the water activity by the presence of 5 M NaCl (the strongly negatively charged phosphate groups of the polymer being screened by the high Na+ concentration), the effect of increasing the NiC12 concentration in the solution is a reorganization of the water distribution via local specific interactions of Ni2+ (H,O), charged complexes with the adenine N7 positions, and a stabilization of the syn geometry of the purine residues between 85 and 95 mM in Ni2+. It is shown that UV RRS provides good marker bands of the poly (dA-dT), left-handed helix in the whole 500-1800 cm-l domain (see for instance Fig. 17, taken with the 223 nm excitation wavelength), which are as follows: (i) The purine 627 cm-l ring-breathing mode coupled with the deoxyribose

66

1 1750

1500

1250

1000

750

CM-1

Fig. 17. RR spectra of poly(dA-dT)z in aqueous solution, 10 mM tris buffer, pH 7.8 (a) 5 M NaCl, B form, (b) 5 M NaC1+95 mM NiCl,, 2 form. I.,,=223 nm.

vibration in syn geometry (seen only on the 281 nm spectrum). (ii) A 1300-1340 cm-’ region characterizing the local chemical interactions of the Ni2+ ions with the adenine N7 position. (iii) Upshifts and intensity variations of important adenine marker lines at 1483 and 1582 cm-l (triene motions) correlated to the changes in the base stacking interactions (and to the anti+syn reorientation of the adenine residues) on B+Z structure transition. (iv) Marker bands of the couplings of the thymine carbonyl groups at 1680 and 1733 cm-l, due to the disposition of the pyrimidine residues in the 2 helix specific geometry. Alternating

purine-pyrimidine polymers and natural DNA

It has now been proven that the left-handed helices may be observed in all alternating purine-pyrimidine sequences, either in lowering the base hydration or under the effects of constraints (see ref. 49 for a review). The (dAdC ),, (dG-dT) n sequences can also adopt the 2 conformation [ 73 1, and vibrational marker bands of this left-handed polymer have been reported by IR [ 741 and by classical Raman [ 751. B-Z junctions have also been explored with a variety of physico-chemical techniques on model oligonucleotides, and their occurrence in inserts in recombinant plasmids or the corresponding restriction fragments has been proven [ 49 1. Z DNA tracts were identified in DNA from natural source ( [ 761 and refer-

ences cited therein). Although the repeating sequence (dG-dC ) n is not widely found in biological systems, the sequence (dA-dC),, (dG-dT), is widely distributed in eukaryotic genomes where it may be considered as a form of middle repetition DNA [ 771. These tracts are found in all gene systems that have been extensively sequenced [ 781. In most of the natural nucleic acids studied in vitro by conventional spectroscopic techniques, it was not possible to detect the occurrence of 2 forms unless the salt concentration in solution was increased. However Miskovsky et al. [ 791 recently showed by classical Raman spectroscopy that it is possible to find 2 form marker bands in natural DNA under specific conditions, i.e. high NaCl concentration and high DNA concentration (30 mg ml-‘). We obtained the RR spectra of chicken erythrocyte DNA in low and high salt solutions and for low- and high DNA concentrations [ 801. For that we used the 284 nm excitation wavelength, for which we showed in a previous paper [ 451 that it is possible to enhance selectively left-handed tracts’ contribution. It was found that a high salt concentration is not sufficient itself to give rise to the appearance of the left-handed geometry marker bands. However, in the case of high-salt-high-DNA concentrations, some of the previously mentioned marker lines are observed. (i) A 620 cm- ’ line, attributable to the syn orientation of the purines and a C3’ endo ribose puckering, specific for the left-handed structure. (ii) A large enhancement of a 1261 cm-’ frequency, attributable to a cytosine vibration and which was observed only in the 2 structure of G-C containing polynucleotides (see above ) . (iii) A large 1630 cm-’ line, due to the interbase couplings of pyrimidine carbonyl stretching modes, which are very efficient couplings in the left-handed structures. Up to now, no evidence has been obtained to show that classical or RR spectra are sensitive to mere condensation processes of the DNA. However, a mere decrease of the water activity was proposed as the leading factor accounting for the induction of the B+A or B-2 transitions in model polynucleotides, but was never sufficient alone for natural DNA. Here the high salt concentration, which modifies the water organization around the helix, together with the DNA high concentration, which modifies the hydrophobic environment of the chains, may play a part in inducing the B-+2 transition. Thus, the spectral features mentioned above led us to assume the existence, in DNA 30 mg ml-’ and 4.5 M NaCl, of sequences having a conformation quite different from the genus B form: our study suggests that these regions may be under 2 form, although it is not yet possible to give a reasonable estimate of the proportion of these regions. In the cell nucleus the DNA concentration is considerable: it can be estimated at 50 mg ml-‘, and is probably one order of magnitude larger in the highly condensed regions. It is obvious that the NaCl concentration we used is not encountered in the cell; however it was shown that cations of higher va-

68

lence, or polyanions at very low concentrations, may have the same influence upon the B-t2 transition as high NaCl concentrations [ 771. Hence we showed that under specific conditions DNA may locally adopt a structure quite different from the canonical B form, which is probably close to the 2 form. CONCLUSIONS

Vibrational spectroscopy offers a means to examine the structure of DNA. Base vibrations reflect base stacking and pairing interactions, sometimes coupled with sugar vibrations, and are thus sensitive to the backbone conformations. The UV RRS technique gives very promising results in the selective investigation of chromophores participating in highly elaborate molecular complexes, such as nucleic bases within DNA duplexes. Hence RRS is a very powerful tool for studying DNA conformational changes, and the flexibility of the laser sources now available in the UV region considerably widens this domain. The interpretation of the spectra obtained in RRS on large systems such as DNA is based on a very good knowledge of the spectroscopic properties of model compounds, starting from the nucleic bases and mononucleotides, then oligonucleotides and finally synthetic polynucleotides. At each level of complexity, RRS offers a specific source of information: excited state geometries of the molecules through the intensity excitation profiles, base-base stacking interactions through hypo/hyperchromism excitation profiles, and lastly structure information through vibrational couplings between different components of the macromolecular buildings.

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