Contribution of the resonance Raman spectroscopy to the identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Resonance Raman spectroscopy of polynucleotides

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.     

Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA.

Raman spectra of poly(dG-dC) . poly(dG-dC) in D2O solutions of high (4.0M NaCl) and low-salt (0.1M NaCl) exhibit differences due to different nucleotide conformations and secondary structures of Z and B-DNA. Characteristic carbonyl modes in the 1600-1700 cm-1 region also reflect differences in base pair hydrogen bonding of the respective GC complexes. Comparison with A-DNA confirms the uniqueness of C = O stretching frequencies in each of the three DNA secondary structures. Most useful for qualitative identification of B, Z and A-DNA structures are the intense Raman lines of the phosphodiester backbone in the 750-850 cm-1 region. A conformation-sensitive guanine mode, which yields Raman lines near 682, 668, or 625 cm-1 in B (C2'-endo, anti), A (C3'-endo, anti) or Z (C3'-endo, syn) structures, respectively, is the most useful for quantitative analysis. In D2O, the guanine line of Z-DNA is shifted to 615 cm-1, permitting its detection even in the presence of proteins.     

Contribution of the Resonance Raman Spectroscopy to the Identification of Z DNA

Abstract

Poly(dG-dC)?poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition.

With 257 nm excitation wavelength the 1580 cm^?1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm^?1 and at 1193 cm^?1 and the bands of the cytosines at 780 cm^?1, 1242 cm^?1 and 1268 cm^?1.

By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Identification of a new electronic transition in the Z form of poly(dG-dC)·poly(dG-dc) by infrared absorption and resonance Raman spectroscopy

Infrared absorption and resonance Raman spectroscopy (RRS) are used to study poly(dG-dC)·poly(dG-dC) in two different forms: the right-handed B form at low ionic strength and the left-handed Z form at high ionic strength. The existence of a new electronic absorption band in the 290–300-nm region is evidenced by uv RRS studies of the Z form at different wavelengths of excitation. Infrared absorption spectra prove that this new electronic band is polarized perpendicularly to the cytosine plane. The possibility of a nπ* character of this transition moment is discussed.     

Kinetics of exchangeable protons in Z DNA: a UV resonance Raman study.

We have studied the hydrogen-deuterium exchange kinetics of the exchangeable protons of the poly(dG-dC).poly(dG-dC) in the Z form of the polymer, using resonance Raman spectroscopy with 257 nm and 284 nm excitation wavelengths. In our experimental conditions (4.5 M NaCl, phosphate buffer pH7, 2 degrees C) the two amino protons and the imino proton of guanine are exchanged with the same exchange half-time of 13 min, whereas the two amino protons of cytosine are exchanged with the same exchange half-time of 51 min.     

Salt induced transitions between multiple conformations of poly (rG-m5dC).poly (rG-m5dC).

Salt induced transitions between four conformations of the methylated ribo-deoxyribo co-polymer poly (rG-m5dC).poly (rG-m5dC) have been studied using phosphorous-NMR, Raman spectroscopy, and circular dichroism. A high salt A-Z transition is observed for the polymer. However, the methylated polymer does not enter the high salt Z form more readily than the analogous unmethylated polymer, unlike the effect of methylation on the fully deoxy polymer poly (dG-dC).poly (dG-dC). The methylated polymer fails to undergo a low salt A-Z transition in 5 mM Tris buffer, unlike the unmethylated poly (rG-dC).poly (rG-dC). However, if the counterion is changed to triethanolamine buffer, an A-Z transition does take place. In 5 mM Tris buffer the phosphorous-NMR spectrum of poly (rG-m5dC).poly (rG-m5dC) shows one resonance in the absence of NaCl that splits into two closely spaced resonances as the NaCl level is increased to 30 mM. The Raman spectrum of poly (rG-m5dC).poly (rG-m5dC) shows that it is in the A conformation at intermediate salt concentrations. From this we conclude that poly (rG-m5dC).poly (rG-m5dC) is in a regular A conformation in Tris buffer at low Na+ levels, shifting to an alternating A conformation with a dinucleotide repeat at intermediate salt concentrations.     

Raman spectroscopy study of the B-Z transition in (dG-dC)n.(dG-dC)n and a DNA restriction fragment

The B to Z conformational transition of (dG-dC)n.(dG-dC)n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy. The 157 bp DNA has a 95 bp segment from the E. coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences. Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition. A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands. Profiles of relative intensity vs. sodium chloride concentration are shown for bands at 626, 682, 831-833 and 1093 cm-1. Both (dG-dC)n.(dG-dC)n and the 157 bp DNA show changes in the guanine vibration at 682 cm-1 and backbone band at 831-3 cm-1 preceding a highly cooperative change in the 1093 cm-1 PO2- vibration. This result indicates that there are at least two conformational steps in the B to Z conformational pathway. We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment. Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)n.(dG-dC)n indicate that in 4.5 M NaCl the (dC-dG) segments are in a Z-conformation. Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation. However, a substantial portion of this region no longer has a B-type backbone vibration.     

Raman spectroscopic study of left-handed Z-RNA

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.     

Intensity Changes of Base and Backbone Raman Modes in the B to Z Transition of poly(dG-dm5C)

Abstract

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm-⁵C). The transition midpoint was about 37°C for a solvent containing 20 mM Mg²⁺. A 10-fold change in Mg²⁺ concentration altered the transition midpoint by at least 60°C. Raman spectra of the B and Z forms of poly(dG-dm⁵C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm^?1 guanine mode and 835 cm^?1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm^?1, 805 and 819 cm¹. Several bands unique to poly(dG-dm⁵C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Interactions of water-soluble metalloporphyrins with nucleic acids studied by resonance Raman spectroscopy.

The resonance Raman spectra of water-soluble porphyrins, M(TMpy-P4) (M = Cu(II), Ni(II) and Co(III] and their mixtures with poly(dG-dC)2, poly(dA-dT)2 and calf thymus and salmon DNAs were measured using a divided rotating cell to determine the magnitudes of frequency shift and intensity variation resulting from M(TMpy-P4)-nucleic acid interactions. Bands II(C beta-H bending, approximately 1100 cm-1) and VIII(C beta-C beta stretch, approximately 1570 cm-1) show a large and small upward shift, respectively, when Cu(TMpy-P4) and Ni(TMpy-P4) are intercalated at the G-C sites. In contrast, these bands show a small upward and downward shift, respectively, when Co(TMpy-P4) is groove-bound at the A-T sites of nucleic acids. Both Bands V (approximately 1260 cm-1) and IX (approximately 1646 cm-1) which originate in the N-methylpyridyl group always show small downward shifts due to coulombic interaction between the N-CH3+ group of TMpy-P4 and the PO2 group of the nucleic acid.     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Reactivity and binding of benzo(a)pyrene diol epoxide to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms

The physical and covalent binding of the carcinogen benzo(a)pyrene-7,8-diol-9,10-oxide (BaPDE) to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms were studied utilizing absorbance, fluorescence and linear dichroism techniques. In the case of poly(dG-dC).(dG-dC) the decrease in the covalent binding of BaPDE with increasing NaCl concentration (0.1-4 M) as the B form is transformed to the Z form is attributed to the effects of high ionic strengths on the reactivity and physical binding of BaPDE to the polynucleotides; these effects tend to obscure differences in reactivities with the B and Z forms of the nucleic acids. In the case of poly(dG-m5dC).(dG-m5dC) the B-to-Z transition is induced at low ionic strength (2 mM NaCl + 10 microM Co(NH3)6Cl3) and the covalent binding is found to be 2-3-times lower to the Z form than to the B form. Physical binding of BaPDE by intercalation, which precedes the covalent binding reaction, is significantly lower in the Z form than in the B form, thus accounting, in part, for the lower covalent binding. The linear dichroism characteristics of BaPDE covalently bound to the Z and B forms of poly(dG-m5dC).(dG-m5dC) are consistent with nonintercalative, probably external conformations of the aromatic pyrenyl residues.     

Ultraviolet resonance Raman marker bands of the right to left helix structure transitions in DNA and polynucleotide model compounds.

The right to left helix structural transition in purine-pyrimidine alternating copolymers has been extensively studied by vibrational spectroscopies, amongst many other experimental approaches. Here, the use of resonance Raman spectroscopy in the ultraviolet region (223-, 257- and 281 nm excitation wavelengths) to monitor such structural changes is reviewed in the light of new results obtained on poly(dA-dC).poly(dG-dT) on one hand, and the previous results obtained on poly(dG-dC)2, poly(dA-dT)2 and natural DNA (Chicken erythrocytes) on the other. It is now possible to define B----Z transition marker bands involving the proper bases, which show a similar behaviour on structural transition whatever the composition of alternating purine-pyrimidine sequences: the 1580- and 1487 cm-1 lines of the purines, the 1486- and 1294 cm-1 lines of the pyrimidines are good markers in the vibrational spectra recorded at various UV excitation wavelengths.     

Comparison between poly(dG-dC).poly(dG-dC) and DNA modified by cis-diamminedichloroplatinum (II): immunological and spectroscopic studies

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG).poly(dC) is larger than to poly (dG-dC).poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT). In the competition between poly(dG-dC).poly (dG-dC) (B conformation) and poly(dG-br5dC).poly(dG-br5dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)n.(GC)n sequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diamminedichloroplatinum (II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m5dC) in the Z conformation. When they bind to poly(dG-m5dC) in the B conformation, the conformations of poly(dG-m5dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

The Z-conformation of poly(dA-dT).poly(dA-dT) in solution as studied by ultraviolet resonance Raman spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

The concentration dependence of the right to left conformational transition in natural DNA identified by Raman spectroscopy

The classical and resonance Raman spectra of DNA from Chicken Erythrocytes have been obtained for different DNA concentrations in solution with low and high ionic strengths. The classical Raman spectra of 30 mg/ml DNA solutions were measured in varying the sodium chloride concentration from 0.1 to 4.5 M NaCl. An increase in the salt content of the solution leads to spectral changes in the 600-700 cm-1 region, indicating a C2' endo/anti to C3' endo/syn conformational transition of the purine residues. Other changes around 840 cm-1, due to the antisymmetrical stretching vibration of the PO2 group, are also detected: they were characteristic for the B----Z transition in model systems such as poly(dG-dC).poly(dG-dC). The resonance Raman spectra of low (1 mg/ml) and high (30 mg/ml) concentrated DNA solutions were obtained with low (0.1 M) and high (4.5 M) NaCl contents, in using a 284 nm excitation wavelength. No change was observed in the intensities and band positions in the low and high salt solutions of low concentrated DNA. Thus it is assumed that the DNA structure remains unchanged whatever the salt concentration for low concentrated DNA. In contrast, great modifications of the intensities and positions of some lines were found in the spectra of high DNA concentration solution when the NaCl content is increased up to 4.5 M: these changes resemble to some extent those observed in the study of B----Z transition of several polynucleotide model compounds. It is assumed that the right-handed to left-handed conformational transition may occur in certain sections of natural DNA, likely containing alternating purine-pyrimidine sequences, when the DNA concentration is sufficiently important.     

Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.