Some useful CD applications aiming to investigate the B-Z
conformational change in nucleic acids as well as their inter actions
with a ruthenium complex of potential terapeutic interest are described in this paper.
The results presented here regard:
a) the energetics of the conformational transition from the B
right-handed to the Z left-handed helix in synthetic oligodeoxynucleotides with a cytosine-guanine alternating sequence;
b) The B to Z transition for sequences containing all four
canonical bases and the role of nickel ions and sodium perchlorate
in promoting this tranformation;
c) the inter action of Ru(II)(DMSO)4CI2a. compound exhibiting
a good antitumor activity in animals, with both mononucleosides
and polynucleotides

Franco Quadrifolio

Giorgio Manzini

Luigi Emilio Xodo

Sabina Cauci

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CROATICA CREMICA ACTA CCACAA 62 (2B) 293-300 (1989)CCA-1859YU ISSN 0011-1643Original Scientific PaperUDC 541.653Circular Dichroism as a Tool of Investigation in the B-Z Transitionof DNAFranco QuadrifogHoInstitute of Biology, Faculty of Medicine, University of Udine, 1-33100 Udine, ItalySabina Cauci, Luigi Emilio Xodo and Giorgio ManziniDepartment of Biochemistry, Biophysics and Macromolecular Chemistry,University of Trieste, 1-34127, Trieste, ItalyReceived September 2. 1988Some useful CD applications aiming to investigate the B-Zconformational change in nucleic acids as well as their inter actionswith a ruthenium complex of potential terapeutic interest are des-cribed in this paper.The results presented here regard:a) the energetics of the conformational transition from the Bright-handed to the Z left-handed helix in synthetic oligodeoxy-nucleotides with a cytosine-guanine alternating sequence;b) The B to Z transition for sequences containing all fourcanonical bases and the role of nickel ions and sodium perchloratein promoting this tranformation;c) the inter action of Ru(II)(DMSO)4CI2.a compound exhibitinga good antitumor activity in animals, with both mononucleosidesand polynucleotides.INTRODUCTIONNucleic acids share with other classes of biopolymers, as well as withmany smaller biomolecules, the property of being chiral and of frequentlyinducing optical activity in symmetrical molecules upon inter action withthem. Rence the important role that circular dichroism (CD) has assumed ina wide spectrum of biophysical investigations on nucleic acids, ranging fromthe study of the various chain conformations in solution and of their inter-conversion to the interactions of the chains with drugs, metal ions and theircomplexes, leading to non-covalent as well as to covalent adducts.In this paper we wish to present a brief survey of the results recentlyobtained in our laboratory by using CD and regarding few different topics,namely: 1) the energetic of the B-Z interconversion in (dCdG)n oligodeoxy-nucleotidesv": ii) the stabilization of the left-handed Z double helix in DNAoligomers of different sequence and composition by means on Ni2+; iii) thecovalent interaction of some promising antineoplastic ruthenium complexeswith DNA chains and with nucleotides",294 F. QUADRIFOGLIO ET AL.i) The B-Z Interconversion in OIigodeoxynucleotidesAfter the discovery of the novel left-handed helical structure (the ZDNA) in 19797,8, a wide spectrum of biophysical and biochemical studies wasdeveloped with the aim of clarifying its structural details, the effects of thebase sequence and of the solvent conditions on its thermodynamic stabilityas well as the energetics of the interconversion of this helix with the morefamiliar B one, and moreover to assess its presence in natural DNA, at leastin vitro if not yet definitely in viva, in search of an appealing biological roleof this conformation in the mechanisms of gene activity regulation (seereviews 9-11).In the investigations of the ability of oligo- and poly-deoxynucleotidesof different sequences to assume this conformation in solution, CD soon gainedwide popularity due to the relevant and typical changes observed in the CDspectrum of. B DNA .as a c0I?-sequenceof its transition to Z (in fact an appro-ximate inversion of the bands ranging from about 300 down to less than180 nm takes place). As to the Z inducing agents in aqueous solution, themost widely used were initially concentrated NaCI, up to saturation, andethanol, up to 60%; soon after the effects of multivalent cations as Mg?",Mn2+, Co(NH3)63+,spermine, etc., and of different anions, as in the case ofNaCI04, ard organic solvents were explcred., providing the evidence thatcations of higher charge w.ere generally more effective and that at high eon-centration the nature of the anion was not irrelevant. (NaCI04 'is a better Zinducer than NaCI).One of the investigations undertaken in our' laboratory concerns the ener-getics of the B-Z interconversion in alternating cytosine-guanine oligodeoxy-nucleotides induced by conceritrated NaCl.· In this case CDhas proved to bewell suited for deterrnining the equilibrium amounts of Bvdouble helix,Z-double helix and coil conformations of the oligomers in' aqueous solutionas a function of temperature and, NaCI content. Figure ..1 shows a sample casefor d (CGCGCG) in 2.5 MNael:' the CD spectra recorded at different tempe-ratures show that the effect of increasing temperature is a shifting of theequilibrium from Z to B and then to coil. A knowledge of the CD spectraof the pure B form (low temperature, low NaCI. content), pur.e Z form (lowtemperature, saturated N~CI) andcoil (high temperature) allows one todetermine, from a CD spectrum recorded at equilibrrum in each given eon-dition, the amounts of'the three conformations satisfying the following systemof equations: . l . ./1,,0295= ~eB 295'f B + Mc:Z 295 f Z + ~ec295 f e~E2SS = ~eB2SS fE + ~ez2SS fz + ~ec2SS fel = fE + fz + feThe enthalpy cha,ngys relative to the B-Z transition have been obtainedby plotting In K = In (fE/fz) versus l/T. It was, therefore, possible to estimatethe therrnodynamic parameters of the B-Z inter~onversion which show thatfor: short d (CG)n oligomers the Z double helix has a lower enthalpic content,wliereas the B form has a higher entropy. Increasing NaCI promotes theB to Z transition since it affects more the' enthalpy difference between theCD OF DNA 2951= 81.8 ·C2- 72.83:63.84=54.85= 45.86- 3!i.87= 27.8 ·C8=18.89· 9.810: .0.811 - -5.5iJe /-.-." / 295O -, -, /-2 -1 ~/ ~./-2 / \. // \..--3,/255O 40 80 -eFigure 1. CD spectra for d(CGCGCG) in 0.1 M Tris. HCl (pH= 7.5), 2.5 MNaCl,as a function of temperature. The temperature values from 1 to 8 are as follows:72.8, 54.8,45.8,36.8,29.8, 18.8,9.8, 0.8°C.two forms, favouring the Zone, than the entropy. However, the balance bet-ween enthalpy and entropy changes is rather subtle and thus easily affectedby both the nature of Z promoting agent and the length of the oligomers.ii) Z Stabilizing Effect of Nickel Ions in High Salt Souitum.Regarding the effect of base composition and sequence on the stabilityof the Z structure, the general view is that: a) pyrimidine-purine alternationis a compelling, if not absolute, requirement to get this left-handed helix,b) the higher the percentage of G/C base pairs, the easier is the formationof Z DNA. However, the correlation between Z stability and base sequenceis not yet known in sufficient detail. In linear DNA oligomers indeed theusual high salt conditions, generated with either NaCl or MgCb, are capableof inducing the Z conformation only in a very limited number of sequences,i. e. almost only in d (C-G)n and some others chemically derived from these,as d (G-Br5C)n and d (G-m5C), precluding the possibility of observing thisconformation in linear fragments for a wider spectrum of sequences, includingthose capable of assuming the Z double helix under torsional stress 'in cova-lently closed circular DNAs.12Taking advantage of the fact that the combined action of concentratedNaCl04 and a few mM Ni2+ is highly effective in promoting the ze confor-mation-", we have started a CD study of the relative Zpropensity of a num-ber of oligomers of different sequence and composition, tinding that most of2965-. 3Eo-..~ 1(Al<J -1-3-5-7It)Oo)C\I(Al -1<JF. QUADRIFOGLIO ET AL.3252235 265A,nm295o o10C\I-2 (Al<J1 2 3 4NaGI04,MFigure 2. CD spectra of d(ATACGCGCGTAT) relative to the NaCI04 titration in10 mM Tris . HCI (pH = 7.4), 100 mM NaCI04 and in the presenee of 5 mM Ni(C104)2.Solid lines represent the initial and final spectra, while broken lines are the inter-mediate one. The insert shows the ellipticity values at 295 nm as a function ofNaCI04.1-2-3-4CD OF DNA 297the sequences considered do assume the left-handed helix in these conditions(as shown for a sample case in Figure 2). Table I summarizes the first resultsobtained, and shows that even without the support of torsional stresses:a) sequences with AT content up to 500/0 can afford the B-Z transition inthe presence of a central (C-G)n tract; b) a perfect pyrimidine-purine alter-nation is not a very stringent requirement for the left-handed helix. However,amore extended and quantitative investigation is in progress in our laboratoryand will further contribute to a better characterization of this phenomenon.TABLE ISummary of Titration Data for the B-Z Interconversion. The Numbers Indicate theAdded Salt Concentrations at the Semitransition Points, when Measured. B-ZIndicates the Presenee of not Negligible Amounts of Both Conformations Near SaltSaturation. No Z Indicates the Absence of Appreciable Amounts of Z Even NearSalt Saturation - not TestedOligomer NaCI NaCI04 NaCI04*(M) (M) (M)d(CGCGCGCGCGCGCG) 2.7 1.8d(CGCACGCGCGTGCG) 3.3 2.9 2.1d(ATACGCGCGTAT) no Z B-Z 2.9d(CATACGCGCGTATG)O no Z B-Z 2.0d(CGACGCGCGTCG) no Z B-Z 4d(GGTCGCGCGACC) no Z 2.5d(ACACACGCGTGTGT) 2.7d(ACACACATGTGTGT) no Z* titrations carried out in the presenee of 5 mM Ni2+° titration carried out in the presenee of 10 mM NP+iii) Reaction of Ru(II)(DMSO)4Cl2 with DNA Chains and WithNucleotuiesPrevious studies have shown that Ru(II)(DMSO).!Clz exhibits a good anti-tumor activity in experimental animals'", Both cis and trans isomers comparewell with the properties of cisplatin, a well known and widely used anti-blastic, but show an apparently lower toxicity. Their mutagenic propertiesstrongly suggest that the target in vivo for these drugs is DNA14.We have performed a study of the reaction between the drugs andnatural and synthetic DNA and have demonstrated that both complexes bindirreversibly to the DNA chain. CD spectra obtained on DNA-complex adductsshowed that whereas the cis isomer does not change the B conformation ofthe chain, even at a high drug/DNA ratio, the trans isomer does change theCD bands of the polymer beyond a certain ratio, thus suggesting at least alocal modification of the conformation, similar to that already found followingthe reaction between cisplatin and DNA.15When the reaction with the drug is performed with a synthetic poly-nucleotide, poly(dGdC), at very low ionic strength and is followed by CDmeasurements as a function of time, the circular dichroism spectrum of theoriginal B conformation is gradually changed to that typical of the left-handedZ form. The rate at which this conversion is brought about is higher for thetrans isomer than for the cis form (Figure 3). These measurements allowed298 F, QUADRIFOGLIO ET AL,4---_ ...heat denatured""" ,250 300 350 400 ). (nml 450Figure 3. CD spectra of poly(dGdC): poly(dGdC), incubated with an equimolaramount of Ru(II)DMSO)4C12 at 37=c in 0.2 mM NaCI04, taken at different reactiontimes, The transition is completed within 10 hours.,., ..": ,2! \,\ ,,,,,Or---~'~------~~-~---'---~-~~~------~~----~-1250 350 450AJnmFigure 4. CD spectra of the two products (exhibiting the sameU'v spectrum) obtainedfrom the reaction of trans Ru(II)(DMSO)4C12 with equimolar amounts of 5'dGMP inaqueous solution.CD OF DNA 299us to conclude that the main target of reaction of both isomers on the DNAchain is the N7 of guanine residue. The bulky complexation at this site, infact, pushes the nucleotide residue to assume the syn conformation at theN-glycosidic bond which, in turn, stabilizes the left-handed form of the alter-nating chain.When trans-Ru(II)(DMSO)4Cb is incubated with 5'dGMP, only two pro-ducts are formed and can be separated with gel filtration HPLC. The relativeamount of the two species is dependent on the temperature, thus showingthat the two compounds are interconvertible. The UV spectra are nearlyidentical whereas the CD spectra taken in aqueous solutions of the twospecies are almost mirror images of one another at high wavelengths (Fi-gure 4), thus suggesting that the compounds are enantiomers with respectto the metal center. (Actually, they are diastereoisomers as deoxyribose isa chiral compound and for this reason the CD spectra are not exact mirrorimages at low wavelengths where the transitions of the nucleotide are loca-ted). Subsequent high resolution proton and phosphorus NMR measurementsshowed that 5'dGMP reacts with the metal complex with al: 1 stoichiornetryat neutral pH's and chelates the octahedral ruthenium atom via' N7 andphosphate group in two different steric arrangements'", At the moment it isnot clear which coordination sites are occupied by the chelating ligand inthe two isomers.REFERENCE S1. F. Qua d r i f o g 1i o, G. Man z i n i, N. Ya t h i n d r a, and R. C r e a, in:16'" JerusaLem Symposium »Nucleu: Acids: the Vectors of Life«, B. P u Il manand J. J ort ner (Ed.), Dordrecht, Holland, 1983, p. 61.2. F. Qua d r i f o g 1i o, G. Man z i n i, and N. Y a t h i n d r a, J. MoL BioL175 (1984) 419.3. L. E. X o d o, F. Qua d r i f o g 1i o, G. Man z i n i, G. A. van der Mar e 1,and J. H. van B o o m, Nucieic Acids Res. 14 (1986) 5289.4. G. Manzini, L. E. Xodo, F. Quadrifoglio, G. A. van del' Mare],and J. H. van B o o m, J. BiomoL Struci, Dyn. 4 (1987) 651.5. L. E. X o d o, G. Man z i n i, F. Qua d r i f o g li o, G. A. van der Mar e 1,and J. H. van B o o m, Biochemistry 27 (1988) 6327.6. S. C a U c i, E. Ale s s i o, G. Me str o n i, and F. Qua d r i f og 1i o, Inorg.Chim. Acta 137 (1987) 19.7. A. H.-J. Wang, G. J. Quigley, F. J. Kolpak, .J. L. Crawford, J.H. van B o o m, G. A. van der Mar e 1, and A. Ric h, Nature 282 (1979)680.8. H. R. D r e w, T. Tak a n o, S. Ta n a k a, K. Ita k ura, and R. E. D i c-k e r s o n, Nature 286 (1980) 567.9. R. E. D i c k e r s o n, H. R. D r e w, B. N. C o n ner, R. M. W i n g, A. V.Fra t i n i, and M. L. K o p k a, Science 216 (1982) 475.10. A. Ric h, A. N o r d he i m, and A. H.-J. Wa n g, Ann. Rev. Biochem. 53(1984) 791.11. T. M. J o v i n, D. M. S o u m p a s i s, and L. P. Mc I n t o s h, Ann. Rev. Phys.Chem. 38 (1987) 521.12. E. Ta i Il and i e r, J. A. Ta b o u r y, S. Adam, and J. Li q u i e r, Bioche-mistry 23 (1984) 5703.13. G. Sava, T. G ira 1d i, G. Me str o n i, and G. Za s s i n o vic h, Chem.BioL Interact. 45 (1983) 1.14. C. M o n t i - Bra gad i n, M. Ta mar o, and E. B a n f i, Chem. BioL Intemct.11 (1975) 469.15. R. C. S r iva s t a v a, J. F r o eli c h, and J. L. E i c hho r n, Biochimie 60(1978) 879.16. Submitted to J. Amer. Chem. Soc.SOO F. QUADRIFOGLIO ET AL.SAZETAKCirkularni dikroizam kao oruđe u istraživanju B-Z prijelaza DNAFranco QuadrifogLio, Sabina Cauci, Luigi Emilio Xodo i Giorgio ManziniOpisane su neke korisne primjene CD u istraživanju konformacijske izmjeneB-Z u nukleinskih kiselina, kao i njihova interakcija s rutenijevim kompleksi ma odterapeutskog interesa.a. energija konformacijskih prijelaza iz B-desnoruke u Z-lijevoruku uzvojnicusintetskih oligodeoksi-nukleotida s alternirajućom sekvencijom citozin-guanin.b. prijelaza iz B- u Z-konformaciju za sekvencije koje sadrže sve četiri kanon-ske baze, kao i utjecaj nikal-iona i natrij-perklorata na poticanje ovih pri-jelaza.c. interakcije Ru(II)(DMSO)4C12 spoja koji pokazuje dobru antitumorsku aktiv-nost na životinjama, s mono- i polinukleotidima.

Circular Dichroism as a Tool of Investigation in the B-Z Transition of DNA

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