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.     

A study of the thermal stability of the Z form of (m5dC-dG)3 by resonance Raman spectroscopy.

The thermal stability of the hexanucleoside pentaphosphate m5dCpdGpm5dCpdGpm5 dCpdG has been studied by resonance Raman spectroscopy with 257 nm excitation wavelength. At low temperature and in 3M NaClO4, the Raman spectrum resembles that of poly(dG-dC).poly(dG-dC) in the Z conformation. As the temperature is increased, the position and the intensity of several bands (1312 cm-1, 1482 cm-1, 1584 cm-1 and 1632 cm-1) are modified. The variation of intensity versus temperature is biphasic. Analysis of the results suggests that the increase of temperature induces first a transition from the Z form to an intermediate stable form which then melts. These results and those previously obtained by circular dichroism and 31P nuclear magnetic resonance suggest that the intermediate form belongs to the left family but with changes in the stacking of the bases and the geometry of the phosphate groups as compared to the canonical Z form.     

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.     

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.     

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.     

Salt-induced conformational transition of poly(d2NH2A-dT) studied by ultraviolet resonance Raman spectroscopy.

The conformational changes of poly(d2NH2A-dT) in aqueous solution, induced by increasing the NaCl concentration from 0.1M to 4M, have been monitored by ultraviolet resonance Raman spectroscopy, in using the 222-, 257- and 281 nm excitation wavelengths. These changes have been interpreted in comparing the polymer spectra to those of the mononucleotide compounds on one hand, and to those of other alternating purine-pyrimidine polymers on the other hand, i.e. poly(dG-dC) and poly(dA-dT) which showed a B to Z transition in going from low- to high salt concentrations. The high salt poly(d2NH2A-dT) spectra do not show any Raman marker line of the Z conformation. The spectroscopic results indicate that most of the ribose puckering goes from C2'-endo/anti to C3'-endo/anti in increasing the salt concentration. In addition the base stacking interactions, to which the resonance Raman effect is very sensitive, are not drastically changed upon salt variations. Thus the high salt structure of poly(d2NH2A-dT) remains a right-handed helix, likely under a dominant A conformation.     

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.     

Conformational Transitions of Poly(dl-dC) in Aqueous Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries, which strongly depend on salt nature and concentrations. These equilibrium structures have been monitored by resonance Raman spectroscopy (RRS) measurements in the ultraviolet region, i. e. by using 257 and 281 nm laser excitation wavelengths which favor the resonance enhancement of the Raman contributions from inosine and cytosine residues of poly(dI-dC), respectively. Spectral changes depending on the NaCl concentration and on the presence of Ni²⁺ ions have been observed and interpreted in comparison with RRS results previously obtained for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA- dT) and poly(dA-dC). poly(dG-dT), which also showed B to Z conformational transitions in varying the salt concentrations. It is shown here that: i) the base stacking geometries are nearly the same in the high-salt form (5 M NaCl) of poly(dl-dC) as in the low-salt form (0.1M NaCl) of the polymer, ii) however, the high-salt structure yields important differences from a B-helix (obtained in low-salt solution) as regards the nucleoside conformations (sugar puckering and base-sugar orientation), and: iii) the addition of 9 mM NiCl₂ in the high-salt (5 M NaCl) solution of poly(dI-dC) induces the Z-conformation of the polymer.     

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.     

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.     

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.     

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.     

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.     

The B goes to Z transition of poly(dG-dC) . poly(dG-dC) modified by some platinum derivatives.

Poly(dG-dC) . poly(dG-dC) was modified by chlorodiethylenetriamino platinum (II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine platinum (II), respectively. The conformation of these modified poly(dG-dC) . poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular dichroism spectra of poly(dG-dC)dien-Pt (0 less than or equal to rb less than or equal to 0.2) are similar (rb is the amount of bound platinum per base). It is concluded that the conformation of these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z goes to B transition of poly(dG-dC)dien-Pt(0.12) is at 0.2 M NaCl. Moreover another B goes to Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small amounts of EDTA. Poly(dG-dC) . poly(dG-dC) modified by cis-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.     

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.     

Resonance Raman studies of the flavin and iron-sulfur centers of milk xanthine oxidase

Resonance Raman spectroscopy has been used to study milk xanthine oxidase, an enzyme containing molybdenum, binuclear iron-sulfur clusters, and FAD as cofactors. The contribution of FAD dominates the resonance Raman spectrum at frequencies above 500 cm-1. As expected, no bands assignable to FAD are observed in deflavo xanthine oxidase. The resonance Raman spectrum below 500 cm-1 reveals the contribution of the Fe2S2(Cys)4 groups with frequencies similar to those of adrenodoxin and putidaredoxin. Resonance enhancement profiles of the Fe2S2(Cys)4 clusters indicate intensity variations among the Fe2S2(Cys)4 peaks that are attributed to different excitation wavelength maxima of their bridging and terminal iron-sulfur vibrations. No evidence for Mo-ligand vibrations could be obtained by using excitation wavelengths between 363.8 and 514.5 nm.     

Chain flexibility and hydrodynamics of the B and Z forms of poly(dG-dC).poly(dG-dC).

The solution properties of the B and Z forms of poly(dG-dC).poly(dG-dC) have been measured by static and dynamic laser light scattering. The radius of gyration, persistence length, translational and segmental diffusion coefficients, and the Rouse-Zimm parameters have been evaluated. The persistence length of the Z form determined at 3 M NaCl is about 200 nm compared to 84 and 61 nm respectively for the B forms of poly(dG-dC).poly(dG-dC), and calf thymus DNA, both determined at 0.1 M NaCl. The data on persistence length, diffusion coefficients and the Rouse-Zimm parameters indicate a large increase in the chain stiffness of Z DNA compared to the B form. These results are opposite to the ionic strength effects on random sequence native DNAs, for which the flexibility increases with ionic strength and levels off at about 1 M NaCl.     

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.     

DNA Methylation Detection Using Resonance and Nanobowtie-Antenna-Enhanced Raman Spectroscopy

We show that DNA carrying 5-methylcytosine modifications or methylated DNA (m-DNA) can be distinguished from DNA with unmodified cytosine by Raman spectroscopy enhanced by both a bowtie nanoantenna and excitation resonance. In particular, m-DNA can be identified by a peak near 1000 cm^?1 and changes in the Raman peaks in the 1200–1700 cm^?1 band that are enhanced by the ring-absorption resonance. The identification is robust to the use of resonance Raman and nanoantenna excitation used to obtain significant signal improvement. The primary differences are three additional Raman peaks with methylation at 1014, 1239, and 1639 cm^?1 and spectral intensity inversion at 1324 (C₅=C₆) and 1473 cm^?1 (C₄=N₃) in m-DNA compared to that of DNA with unmodified cytosine. We attribute this to the proximity of the methyl group to the antenna, which brings the (C₅=C₆) mode closer to experiencing a stronger near-field enhancement. We also show distinct Raman spectral features attributed to the transition of DNA from a hydrated state, when dissolved, to a dried/denatured state. We observe a general broadening of the larger lines and a transfer of spectral weight from the ～1470 cm^?1 vibration to the two higher-energy lines of the dried m-DNA solution. We attribute the new spectral characteristics to DNA softening under high salt conditions and find that the m-DNA is still distinguishable via the ～1000 cm^?1 peak and distribution of the signal in the 1200–1700 cm^?1 band. The nanoantenna gain exceeds 20,000, whereas the real signal ratio is much less because of a low average enhanced region occupancy even with these relatively high DNA concentrations. It is improved when fixed DNA in a salt crystal lies near the nanoantenna. The Raman resonance gain profile is consistent with A-term expectations, and the resonance is found at ～259 nm excitation wavelength.     

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.