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.     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

Toroidal condensation of Z DNA and identification of an intermediate in the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC)

Using a combination of spectroscopic techniques, quasi-elastic laser light scattering (QLS), and electron microscopy (EM), we have been able to show that the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC) is accompanied by extensive condensation of the DNA in both low and high ionic strength buffers. At low concentrations of NaCl (2 mM Na+), an intermediate rodlike form, which exhibits a circular dichroism (CD) spectrum characteristic of an equimolar mixture of B and Z forms, is observed. This is produced by the orderly self-association of about four molecules of the polymer after prolonged incubation of a concentrated solution at 4 degrees C. On addition of 5 microM Co(NH3)63+, the CD spectrum of the intermediate changes to that of the Z form, which is visualized as a dense population of discrete toroids on an EM grid stained with uranyl acetate. On the other hand, addition of NaCl to a solution of poly(dG-m5dC) X poly(dG-m5dC) in the absence of any multivalent ion condenses the polymer to toroidal structures at the midpoint (0.75 M NaCl) of the B to Z transition. Further addition of NaCl unfolds these toroids to rodlike structures, which show characteristic Z-form CD spectra. These results show that Z DNA can take up a variety of tertiary structural forms and indicate that its inverted CD spectrum is due to its left-handed helical sense rather than to differential scattering artifacts.     

Covalent attachment of an alkylamine prevents the B to Z transition in poly(dG-dC)

Covalent complexes of n-butylamine and double-stranded poly(dG-dC) were prepared by coupling the amine to exocyclic amino groups of guanine bases with CH2O. Neither the absorption spectrum above 230 nm nor the s020,w of the complexes in low to moderate ionic strength solvents, freed of excess unreacted reagents, differs significantly from that of unreacted poly(dG-dC) or a control which had been exposed only to CH2O. In contrast, the CD spectra are profoundly altered. The minimum at 252 nm becomes more negative, and the rotational strength of the positive band above 260 nm is reduced as a linear function of the extent of amine attachment. At 0.22 mol of amine per mole of nucleotide, the transformation is similar to that observed by others in poly(dG-dC) when complexed to core histones in reconstituted core particles or in concentrated LiCl solvents at temperatures below the B----Z transition. Sedimentation studies reveal that these changes in the circular dichroism (CD) spectra reflect secondary structural effects rather than the formation of aggregates or psi type structures. Raman spectra reveal, however, that these secondary structural changes must occur within the B family as the amine complex retains B backbone geometry. The conformation produced by the attachment of the amine is probably a higher winding angle (overwound) B variant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental and calculated study of the vibrational modes of poly(dG-dC).poly(dG-dC) in B and Z conformations

Infrared spectra of the B and Z forms of poly(dG-dC).poly(dG-dC) are presented. Experimental assignments relative to certain vibration modes have been confirmed by calculation based on the GF-Wilson method. The calculated results show that only the geometry change between B and Z forms, is responsible for the observed modifications in the vibrational spectra.     

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.     

Comparison of the conformation of poly(dI-dC) with poly(dI-dbr5C) and the B and Z forms of poly(dG-dC). One- and two-dimensional NMR studies

One- and two-dimensional nuclear Overhauser effects (2D NOE) have been used to compare the conformational properties of 60-80 base pair long duplexes of the synthetic DNA polymer poly(dI-dC) with those of poly(dI-dbr5C) and poly(dG-dC) in the B and Z conformations. Cross peaks in the 2D NOE spectra arising from proton-proton dipolar interactions which are more or less independent of the DNA conformation are used to assign the spectra of these molecules. Other cross peaks are sensitive to the conformational details, and these are used to make deductions about the average conformation in solution. The proton-proton interactions that give rise to the cross peaks in the 2D NOE spectrum of poly(dI-dC) are indicative of a B family conformation and rule out the possibility of some alternative conformations, including A, Z, alternating B, and left-handed B-DNA. The spectra are similar to those obtained from B-form poly(dI-dbr5C) and poly(dG-dC) but different from Z-form poly(dG-dC). Taken together, these results indicate that the solution conformation of poly(dI-dC) is not unusual but more closely resembles that of other B-form DNAs.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies

The photoaffinity analogue ethidium monoazide was used to prepare samples of poly(dGdC).poly(dGdC) containing covalently attached ethidium. The effects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly(dGdC).poly(dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC)4, (dGdC)5, and (dGdC)6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2 +/- 1.1 kcal mol-1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumably by locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies

The effects of covalent modification of poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) by ethidium monoazide (a photoreactive analogue of ethidium) on the salt-induced B to Z transition are examined. Earlier studies have shown ethidium monoazide to bind DNA (in the absence of light) in a manner identical to that of the parent ethidium bromide. Photolysis of the ethidium monoazide-DNA complex with visible light results in the covalent attachment of the photoreactive analogue to the DNA. This ability to form a covalent adduct was utilized to probe the effects of an intercalating irreversibly bound adduct on the salt-induced B to Z transition of the poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) polynucleotides. In the absence of drug, the salt-induced transition from the B to Z structure occurs in a highly cooperative manner. In contrast, this cooperativity is diminished as the concentration of covalently attached drug is increased. The degree of inhibition of the B to Z transition is quantitated as a function of the concentration of covalently attached drug. At a concentration of one drug bound per four base pairs for poly(dGdC).poly(dGdC) and seven base pairs for poly(dGm5dC).poly(dGm5dC), total inhibition of this transition is achieved. Lower concentrations of bound drug were effective in the partial inhibition of this transition. The effects of the covalently bound intercalator on the energetics of the B to Z transition were determined and demonstrated that the adduct is effective in locking the alternating copolymer in a right-handed conformation under high salt conditions.     

The effect of intercalating drugs on the kinetics of the B to Z transition of poly(dG-dC)

We have measured the ability of the intercalating drugs proflavine, ethidium bromide, actinomycin D, and bismethidiumspermine to inhibit the salt induced transition of poly(dG-dC) from the B to the Z form. While all of the drugs studied slowed the B to Z transition, the effectiveness of the drugs correlates much better with their DNA binding kinetics than their DNA binding constants. In studies where the binding densities of ethidium and actinomycin were varied we have found that high levels of ethidium, more than 1 per 20 base pairs, were required to inhibit the B to Z transition while low levels of actinomycin, less than 1 per 450 base pairs, reduced the transition rate. Studies of the B to Z transition in the presence of both actinomycin and ethidium suggest that the drugs inhibit the transition by different mechanisms. The results are interpreted in terms of a modification of the kinetic model proposed by Pohl and Jovin in which, depending on the DNA binding kinetics of the drug, the drug may inhibit nucleation and/or propagation of the B to Z transition.     

A temperature-dependent Z to B to single-strand transition in d(CGCG)

A thermally induced left- to right-handed helical interconversion for the self-complementary tetradeoxynucleotide d(CGCG) has been observed in the presence of NaCl at concentrations from 1.5 to 2.25M. Analysis of the Raman spectrum of d(CGCG) in aqueous solution as a function of temperature indicates that at low temperature a left-handed Z-helical form is the predominant species. An increase in temperature results in a decrease in the population of the left-handed form and an increase in the population of the right-handed form. Further elevation in temperature results in extensive disruption of base stacking and a loss of secondary structure. This unstacking presumably represents dissociation to a single-stranded structure. The data suggest that a temperature-dependent Z to B to single-strand transition occurs under the conditions employed. A direct Z to single-strand conversion was not identified. In contrast to previous examples, the thermally induced left- to right-handed helical conversion for d(CGCG) can occur in the absence of chemically modified nucleic acid bases, alcohol solutions, or divalent ions.     

A circular dichroism study of the binding of CC-1065 to B and Z form poly(dl-5BrdC).poly(dl-5BrdC)

CC-1065, Benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1,6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8,8a-tetrahydro-7-methyl-4- oxocyclopropa[c]pyrrolo[3,2-e]indol-2(1H)-yl)carbonyl]benzo [1,2-b:4,3-b']dipyrrol-3(2H)-yl]carbonyl]-1,6-dihydro-4-hydroxy- 5-methoxy-, (7bR,8aS), binds to the B form of poly(dl-5BrdC).poly(dl-5BrdC) to yield a reversibly bound species whose stability with respect to an irreversibly bound species (presumably the inosine N-3 adduct) is much greater than it is for other DNA polymers. Competitive binding experiments with netropsin, show that this reversibly bound species of CC-1065 contains CC-1065 in the minor groove of the double helix. A review of the CC-1065 binding data obtained on other synthetic DNA polymers suggests that the widely different rates of species conversion shown by these polymers may result from small differences in DNA secondary structure rather than from different alkylating abilities of the adenine or inosine N-3 active site. CC-1065 converts the Z-form of poly(dl-5BrdC).poly(dl-5BrdC) in 3.5 M sodium chloride to the B form and does not bind to the Z form in this solvent system. CC-1065 bound to the B form polymer inhibits the formation of the Z form if the helix is saturated with CC-1065. Regions of the polymer without bound CC-1065 can convert to the Z form with added salt, producing a situation where the polymer contains both the B and Z conformations. In 4.0 M sodium chloride, where the Z conformation is also predominate, the addition of CC-1065 causes chiral aggregates to form, and CC-1065 binds to the aggregates. The addition of dimethylformamide in the absence of CC-1065 or a simple dilution of the 4.0 M sodium chloride polymer solution with water also causes aggregation, indicating that the Z form of this polymer in 4.0 M sodium chloride is unstable with respect to an aggregated form.     

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.     

Raman spectroscopic elucidation of DNA backbone conformations for poly(dG-dT).poly(dA-dC) and poly(dA-dT).poly(dA-dT) in CsF solution

Raman spectra have been recorded for poly(dG-dT) · poly(dA-dC) and poly(dA-dT) · poly(dA-dT) in low salt and at high concentrations of CsF. Poly(dG-dT) · poly(dA-dC) shows no change in the 682-cm^?1 guanine mode, demonstrating the absence of the Z-structure at high salt. The 790-cm^?1 phosphodiester symmetric stretch, however, shifts up 5 cm^?1 in 4.3M CsF, suggesting a slight conformational change, associated with ion binding or hydration changes. Poly(dA-dT) · poly(dA-dT) shows an additional broad band at 816 cm^?1, attributed to the phosphodiester modes associated with the C3′-endo deoxyribose units in the alternating B-structure. In this case, both the 841- and the 816-cm^?1 asymmetric phosphodiester stretches, associated with the C2′- and C3′-endo units, shift down on addition of CsF in a sequential manner. Correlation of this sequence with that previously observed for the two ³¹P-nmr resonances, establishes that the phosphodiester stretching frequencies depend on the conformation of the 5′-sugar, and not on the 3′-sugar.     

Binding of [(dien)PtCl] Cl to poly(dG-dC)-poly(dG-dC) facilitates the B goes to Z conformational transition.

Chlorodiethylenetriamineplatinum(II) chloride, [(dien)PtCl]Cl, bound to less than or equal to 10% of the nucleotide bases of poly(dG-dC) . poly(dG-dC) reduces the amount of ethanol necessary to bring about the B goes to Z conformational transition in proportion to the amount of platinum complex bound as monitored by CD spectroscopy. The transition may be effected by 25% ethanol with 9.3% of the bases modified polymer an ethanol with 5.4% of the bases modified. With an unmodified polymer an ethanol concentration of 55-60% is necessary to bring about the transition. The assignment of the Z conformation was supported by 31P NMR spectroscopy. This covalent modification of the DNA is reversed by treatment with cyanide ion after which the normal amount of ethanol is necessary to achieve the transition. The platinum complex shows no enhanced binding to DNA in the Z versus the B conformation. Between 20 and 33% (saturation binding) modification, [(dien)PtCl]Cl binds cooperatively to the heterocopolymer as judged by CD spectroscopy. At this high level of modification it is no longer possible to induce the Z DNA structure with ethanol. When [(dien)PtCl]Cl is bound to preformed (with ethanol) Z DNA at saturating levels the CD spectrum is altered but reverts to the spectrum of highly modified DNA upon removal of ethanol. The antitumor drug cis-diaminedichloroplatinum(II), cis-DDP, binds to poly(dG-dC) . poly(dG-dC) and alters the CD spectrum. It does not facilitate the B goes to Z conformational change, however, and actually prevents it from happening even at very high ethanol concentrations.     

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.     

Increase in temperature induces the Z to B transition of poly[d(G-C)] in water-ethanol solution

An increase in temperature from 20 to 50° C results in the complete transition from the Z to B form of poly(d(G-C)], dissolved in a 55% ethanol-water solution. The transition is fully reversible and displays a slow kinetics. The transition profiles for the free polynucleotide and for that in the presence of ethidium bromide, which is known to stabilize the B form, are obtained by circular dichroism. Based on these data the enthalpy value for the B-Z transition in our conditions is estimated to be ΔH_BZ = ?0.7 $\text{kcal}\text{mol}$.     

A one- and two-dimensional NMR study of the B to Z transition of (m5dC-dG)3 in methanolic solution

The deoxyribose hexanucleoside pentaphosphate (m5dC-dG)3 has been studied by 500 MHz 1H NMR in D2O (0.1 M NaCl) and in D2O/deuterated methanol mixtures. Two conformations, in slow equilibrium on the NMR time scale, were detected in methanolic solution. Two-dimensional nuclear Overhauser effect (NOE) experiments were used to assign the base and many of the sugar resonances as well as to determine structural features for both conformations. The results were consistent with the an equilibrium in solution between B-DNA and Z-DNA. The majority of the molecules have a B-DNA structure in low-salt D2O and a Z-DNA structure at high methanol concentrations. A cross-strand NOE between methyl groups on adjacent cytosines is observed for Z-DNA but not B-DNA. The B-DNA conformation predominates at low methanol concentrations and is stabilized by increasing temperature, while the Z-DNA conformation predominates at high methanol concentrations and low temperatures. 31P NMR spectra gave results consistent with those obtained by 1H NMR. Comparison of the 31P spectra with those obtained on poly(dG-m5dC) allow assignment of the lower field resonances to GpC in the Z conformation.