Vibrational Circular Dichroism Spectroscopy and the Effects of Metal Ions on DNA Structure

Journal of Molecular Structure 661-662 (2003) 541–560 www.elsevier.com/locate/molstruc

Vibrational circular dichroism spectroscopy and the effects of metal ions on DNA structure

V. Andrushchenko, D. Tsankov, H. Wieser*

Department of Chemistry, University of Calgary, Calgary, AB, Canada T2N 1N4

Received 28 May 2003; accepted 6 August 2003 Dedicated to Professor Bernhard Schrader

Abstract The effects of manganese (II), nickel (II), chromium (III), and platinum (II) (cisplatin) on the double helical structure of selected oligonucleotides and DNA as detected by vibrational circular dichroism spectroscopy (VCD) are reviewed. For (dG–dC)20, the VCD spectra displayed unambiguously the transition from the normal right-handed B-form to the left-handed Z-from at the highest concentration of Mn2þ (8.5 [Mn]/[P]). For poly(rU)·poly(rA)·poly(rU), the double-triple- single helix transition induced by nickel (II) was clearly observable at increasing temperatures. For calf thymus DNA, chromium (III) at 0.6 [Cr]/[P] produced a 4-fold increase of the main VCD couplets, which are characterized as c-type spectra. These unusual features are due to condensed particles with dimensions comparable to the wavelength of the probing light and with regular arrangements of DNA double-helices inside the aggregates. c-Type spectra were also observed for DNA with manganese (II) between 55 and 60 8C at 2.4 [Mn]/[P]. Between 2.4 and 10 [Mn]/[P], intense and indistinct VCD features appeared above the DNA melting point indicating aggregation induced by Mn2þ ions at elevated temperature in a relatively wide range of experimental conditions, whereas DNA condensation with Mn2þ ions occurs only in a very narrow range of concentration and temperature. The complexes formed by coordination of cisplatin with d(CCTGGTCC)·d(GGACCAGG) and d(CCTCTGGTCTCC)·d(GGAGACCAGAGG) yielded very detailed VCD spectra, which distinctly conﬁrmed the Gp4pGp5 and Gp6pGp7 lesion sites, respectively. The octamer slowly isomerized to form other adducts, which was not the case for the dodecamer. q 2003 Published by Elsevier B.V.

Keywords: Vibrational circular dichroism; Metal ions; Cisplatin; Oligonucleotides; DNA

1. Introduction cesses. They can stabilize and destabilize biological structures. They are found in intimate association Metal ions are present in practically all biological with nucleic acids (NAs) in their natural environ- systems and participate in many biological pro- ment. Together with water molecules in the hydration shell they determine NA conformation [1,2]. Metal ion interaction with NAs plays an * Corresponding author. Tel.: þ1-403-220-4934; fax: þ1-403- important role in different biological processes such 289-7635. E-mail address: [email protected] (H. Wieser). as genetic expression, metallo-enzymatic processes,

0022-2860/$ - see front matter q 2003 Published by Elsevier B.V. doi:10.1016/j.molstruc.2003.08.037 542 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 mutagenesis, carcinogenesis, and DNA packing in can induce a transition from the natural right-handed living cells [3–6]. conformation to the left-handed Z-form [20,27–30]. Among varied other techniques, infrared The left-handed Z-form sequences of DNA are absorption spectroscopy (IR) has been used to believed to play a significant role in a number of probe the effects of metal ions on NA structures important life processes, such as gene regulation and [7–9]. In the present publication, we summarize our DNA replication [31–33]. A protein has been isolated recent applications of vibrational circular dichroism very recently in vitro and from several mammalian spectroscopy (VCD), which we expected to reveal tissues, which specifically recognizes Z-DNA important aspects of metal ion interaction with NAs [34–38]. In our study we used Mn2þ ions to induce that are not evident from IR. VCD is based on the the B–Z transition of (dG–dC)20 [20]. differential absorption of left and right circularly Another important NA conformational change polarized infrared light. It offers several advantages induced by metal ions is double- to triple-strand over conventional electronic circular dichroism transition and formation of triple helical structures (ECD) and conventional IR. The structural details of NAs [17,39,40]. Triple helical NAs are said to in ECD are usually limited by broad and less play a biological role as regulators of eukariotic resolved bands, which arise from electronic tran- gene expression [41]. Homo(purine)·homo(pyrimi- sitions of a small number of chromophores and give dine) sequences are frequently located at the 50-end limited detailed information about molecule struc- of many eukariotic genes and at sites involved in ture. In contrast, VCD intensities arise from normal genetic recombination [41,42]. Formation of triple modes of vibration, which are usually characteristic helices at these sites may be necessary for optimal for smaller molecular groups separated in space and gene expression [43]. A protein capable of binding therefore provide more specific details for the to dT·(dA·dT) triplex was discovered by Kiyama molecular structure. IR, on the other hand, while and Camerini-Otero [44]. This protein has a much having the same advantage of characteristic higher affinity of binding to triplex than vibrational modes, lacks VCDs stereosensitivity. In duplex dA·dT or to single-stranded dA or dT. NA molecules, the VCD signals are due to through- More recently, sequence-specific DNA and RNA space coupling of the normal vibrations of the recognition by specially designed oligonucleotides macromolecule and reflect the helical or ordered focused attention on the possible use of such structure of NAs [10,11]. The high sensitivity of the oligonucleotides as a new class of pharmacologi- VCD signals to changes in the coupled vibrations cally active compounds. These may allow for the leads to a high sensitivity of the spectra to changes in efficient treatment of different diseases with the geometry of the macromolecules. Since the first minimal side effects [45]. Homopyrimidine deox- application of VCD to NAs [12] it has been yoligonucleotides covalently linked to DNA cleav- successfully used to study different aspects of NA ing agents were synthesized, which cut duplex DNA structure [13–19]. in a sequence specific fashion via triplex formation In our laboratory, we have measured the VCD of [46,47]. These studies established the usefulness of several oligonucleotide and DNA complexes with such sequences as probes for chromosome mapping metal ions. These included structural transitions of and as anti-sense DNA for chemotherapeutic NAs between different conformations, namely, B to applications in which gene expression is regulated Z-form, duplex-triplex-single strand, DNA by triplex formation as demonstrated by in vitro condensation and aggregation, and interaction of inhibition of c-myc oncogene expression [48]. In our cis-diamminedichloroplatinum(II) (cis-DDP or study we demonstrated conformational transitions of cisplatin) with DNA. More details on these and other poly(rA)·poly(rU) induced by synergetic action of VCD investigations dealing with NA-metal ion Ni2þ ions and elevated temperatures [21]. The interaction can be found elsewhere [20–26]. poly(rA)·poly(rU) duplex disproportionates into a The natural conformation of DNA is a right- triple helix of poly(rU)·poly(rA)·poly(rU) and a handed B-form helix. However, the addition of single strand of poly(rA) [1]. The extra poly(rU) a number of metal ions to poly- or oligo(dG–dC) strand fits into the major groove of this double helix. V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 543

A very important aspect of metal ion effect on platinum coordination, the dihedral angle between DNA is the induction of DNA condensation and the guanine rings ranges between 76 and 878 aggregation. Divalent metal ions, especially at bending the DNA helix toward the major groove at increased temperatures, can induce DNA aggregation the lesion site, flattening and widening the [49–52]. Trivalent and higher valence ions as well as minor groove, and noticeably unwinding the divalent ions acting synergetically with other factors double helix. These distortions of the double can induce DNA condensation into highly condensed helix of d(CCTGGTCC)·d(GGACCAGG) and particles in vitro [6,53]. The condensation process d(CCTCTGGTCTCC)·d(GGAGACCAGAGG) can plays a very important role in DNA packing in living be detected distinctly by VCD [25,26]. Our main cells as well as in the process of gene delivery for gene objective for recording the VCD spectra of these therapy [6]. During condensation a DNA molecule two sequentially similar oligonucleotides was to transforms (or several molecules assemble) into very compare the results with those obtained by NMR dense higher ordered three-dimensional structures, spectroscopy and X-ray crystallography [60–62]. which enables DNA to reduce its volume 104–106 In the present publication we focus explicitly on times [6,54]. In the process of aggregation, DNA also the influence of Pt coordination on the overall forms three-dimensional structures, but in contrast to DNA structure as exemplified by the two analogous condensation these structures are not highly oligonucleotide sequences of different length, ordered and are relatively loose. In our study we one comprising a full helical turn and the other used Cr3þ ions to induce DNA condensation, and including an incomplete one. The results Mn2þ ions at elevated temperatures to induce DNA confirm that the octamer in contrast to the aggregation [22,23]. dodecamer appeared metastable even at 2 8C and Cis-diamminedichloroplatinum(II), otherwise slowly isomerized into other adducts after approxi- known as cisplatin, is a widely used anticancer drug mately 2 h. used for the treatment of metastatic testicular and ovarian tumors, various head and neck tumors, and advanced bladder cancer [55,56]. It cross-links DNA 2. Experimental forming various adducts. Enzymatic digestion studies revealed that the major product is 50-GpG or 50-ApG Sample preparations are described elsewhere and cross-linked forming 1,2-intrastrand adducts the experimental conditions are summarized in (G represents guanine, A adenine, and p a sugar- Table 1 [20–23,25,26]. Sodium cacodylate, NaCl, phosphate unit). Other adducts with fewer incidences MnCl2 £ 4H2O, NiCl2 £ 6H2O, CrCl3 £ 6H2O, 0 are 1,3-intrastrand adducts to 5 -GXG sequences, cis-[Pt(NH3)Cl2], calf thymus DNA, D2O (99.9%), where X is another base, as well as different and poly(rA)·poly(rU) were purchased from interstrand adducts [57]. It was thought that 1,2-intras- Sigma Chemicals and used without further trand cross-links are responsible for the carcinostatic purification. Desalted single stranded oligonucleo- activity of cisplatin, since these adducts have been tides (dG–dC)20, d(CCTGGTCC)·d(GGACCAGG) found in patients cured with this drug [58]. and d(CCTCTGGTCTCC)·d(GGAGACCAGAGG), Other reasoning supporting this assumption is that were obtained from the University of Calgary the geometrical isomer trans-DDP, which is clinically Core DNA Services. Ultra pure calf thymus DNA inactive, cannot form 1,2-intrastrand cross-links due used in the Cr3þ binding experiments was obtained to its stereochemistry [59]. Therefore, most of the from Prof. D. Lando, Institute of Bioorganic cisplatin related research has focused on these adducts Chemistry, Belarus National Academy of Sciences, aiming to elucidate cisplatin’s mechanism of action. Belarus [65]. Double distilled water was used for High resolution NMR and X-ray studies per- the preparation of all solutions in all experiments. formed on model DNA oligomers confirmed For the experiment with poly(rA)·poly(rU), double previous observations that platinum binds to N7 distilled water was additionally autoclaved. The Pt of two neighboring purine bases and the sugar modification of the octamer and dodecamer was adopts endo conformation [60–64]. As a result of achieved with the protocol described previously 544 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

Table 1 Experimental parameters

DNA/oligomer No. of Sample Metal Temperature pH No. of Path Rsltna base pairs concentration (8C) scans length (cm21) M (P) ac/dc (mm) Source Ion [Ion]/[P]

2þ (GC)20 40 0.085 MnCl2 £ 4H2OMn 0–8.5 25 6.8 ^ 0.2 7500/750 50 4 2þ Poly(rA)·poly(rU) – 0.18 NiCl2 £ 6H2ONi 0.4 23–90 (^0.5) 6.0–6.5 4000/400 50 8 2þ Calf thymus ,700 0.13 MnCl2 £ 4H2OMn 0–10 23–94 (^0.5) 6.5 ^ 0.5 4000/500 50 8 DNA 3þ Calf thymus ,850 0.1 CrCl3 £ 6H2OCr 0–3 23 ^ 1 6.5 ^ 0.5 7500/500 50 8 DNA Octamerb 8 1.9 £ 1023 – – – 2.0 ^ 0.1 6.25 7500/500 45 4 Octamer-Pt 8 1.9 £ 1023 Cisplatinc Pt2þ 0.081 2.0 ^ 0.1 6.25 2500/50 45 4 Dodecamerd 12 1.7 £ 1023 – – – 5.0 ^ 0.1 6.25 7500/500 45 4 Dodecamer-Pt 12 1.7 £ 1023 Cisplatinc Pt2þ 0.054 5.0 ^ 0.1 6.25 7500/500 45 4

a Rsltn, Resolution. b d(CCTGGTCC)·d(GGACCAGG). c cis-[Pt(NH3]2Cl2. d d(CCTCTGGTCTCC)·d(GGAGACCAGAG).

[25,26,61]. Gel electrophoresis showed that the the spectra of the solvent obtained at the same addition of cis-Pt was greater than 95%. DNA conditions. Noise estimates were recorded by melting curves obtained for the two platinated subtracting two successive VCD spectra collected at oligonucleotides indicated melting temperatures of the same conditions. 18 and 37 8C for the octamer and dodecamer, respectively. All solutions were prepared in cacodylic buffer. 3. Results and discussion NaCl was added as necessary. The pH of all solutions was adjusted as indicated in Table 1. Calf thymus 3.1. B–Z conformational transition of (dG–dC)20 DNA was sonicated to an average length of 700–850 induced by Mn2þ ions base pairs. Complete deuterium exchange was achieved by lyophilizing and redissolving all The absorption and VCD spectra of (dG–dC)20 2þ solutions three times in D2O. Samples were contained without Mn ions (Fig. 1) correspond to those in a demountable cell (International Crystal observed typically for the right-handed B-form of Laboratories, Inc.) composed of two BaF2 windows poly- and oligo(dG–dC) [10,13,15,19,67–74]. separated by a Teﬂon spacer. The temperature was The assignments below are based on previously maintained in a thermostat chamber connected to a published spectra and more recent high level ab initio circulating water thermostat (NESLAB Instruments calculations [20,74]. The strongest VCD couplet at Inc.) using an electronic thermometer with copper- 1691(2)/1678(þ)cm21 corresponds to the most constantan thermocouple (OMEGA Technology intense absorption at 1683 cm21 and arises from the Company Inc.). carbonyl stretch of guanine (G(6)yO, Table 2). All VCD and absorption spectra were measured in The less intense VCD couplet at 1663(þ )/ 21 21 21 the range of 1800–750 cm in D2O with the VCD 1654(2)cm (absorption at 1657 cm ) belongs instrument described elsewhere [66]. The number of to the same mode in cytosine (C(2)yO). The apparent scans required for optimal quality and for the absorption peak at 1649 cm21 does not appear to have resolution used are cited in Table 1. The VCD spectra a clear VCD counterpart. According to the ab initio were corrected for polarization artifacts by subtracting simulation its origin may be a coupled mode involving V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 545

2 arising from the symmetric stretch of PO2 and C–C stretch of deoxyribose at 1087 and 969 cm21, respectively [15]. The absorptions at 1075, 1053 and 1018 cm21 [72,73,76], which are also due to the sugar-phosphate backbone, do not display clearly distinguishable VCD counterparts. The VCD spectra of (dG–dC)20 at the highest concentration of Mn2þ we investigated (8.5 [Mn]/[P]) are particularly diagnostic for the left-handed Z-form of oligo- and poly(dG–dC) (Fig. 1) [10,13,15,70,71, 75]. The VCD couplet at 1691(2)/1678(þ)cm21 of the B-form is replaced by a couplet of reversed sign at 1671(þ)/1656(2)cm21 in the Z-form signaling the change in the helical sense of (dG–dC)20. The corresponding absorptions occur at 1683 and 1664 cm21, respectively, in the two conformations. The 1664 cm21 band in the Z-form remains the most prominent feature. Similarly, the absorption at 1633 cm21 and matching VCD couplet at 1637(2)/ 1627(þ)cm21 of the Z-form are shifted from 1657 cm21 and 1663(þ)/1654(2), respectively, of the B-form. The same sign reversal is observed also for the CyN stretch of cytosine with its VCD couplet at 1502(2)/1492(þ)cm21 in the Z-form compared to the couplet at 1506(þ)/1498(2)cm21 in the B-form. Similar changes are also seen in the sugar-phosphate Fig. 1. Absorption and VCD spectra (in D2O) and noise estimate of þ (dG–dC) in the nitrogen base and phosphate regions in the region. The couplets at 1089(2)/1070( )and 20 21 B-conformation (without Mn2þ) and in the Z-conformation (with 8.5 973(2)/958(þ)cm characteristic for the B-form [Mn]/[P]). are reversed to 1087(þ)/1075(2) and 969(þ)/ 946(2)cm21 in the Z-form.

CyC stretch of cytosine and CyO stretch of guanine 3.2. Double- to triple-helix transition with a computed absorption maximum at 1647 cm21 of poly(rA)·poly(rU) induced by Ni2þ and positive VCD peak at 1643 cm21 [74]. The broad ions at elevated temperature absorption at 1623 cm21 together with the corresponding negative VCD peak at 1627 cm21 were The absorption and VCD spectra of poly(rA)·po- reproduced computationally at 1618 and ly(rU) with 0.4 [Ni]/[P] at different temperatures are 1618(2 )cm21, respectively. The 1573(þ ), displayed in Figs. 2 and 3, respectively. The assign- 1570(þ)/1556(2), and 1527(þ)/1521(2) features ments for the main features in absorption and VCD corresponding to the 1579, 1564, and 1521 cm21 are listed in Table 3. The shapes and wavenumbers of absorptions are predicted to be due to C–N(D2) and the absorptions and the VCD features at 23 8C (trace a CyN stretch of guanine, and C–N(D2) bend of in Figs. 2 and 3)correspondtoadouble cytosine, respectively. The absorption at 1502 cm21 helical structure of poly(rA)·poly(rU) [17,18,77]. and its VCD couplet at 1506(þ)/1498(2)cm21 arise This spectrum is essentially identical to that of from CyN stretch of cytosine. In the phosphate poly(rA)·poly(rU) without addition of Ni2þ ions region, the VCD couplets at 1089(2)/1070(þ) and (spectrum not shown), indicating that the direct effect 973(2)/958(þ)cm21 are the only clearly discernible of the metal ions on the spectrum is negligible and that VCD features corresponding to the absorptions all changes in the spectra occur due to structural 546 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

Table 2

Assignments for IR and VCD spectra of B-form and Z-form of (dG–dC)20

B-form (cm21) Z-form (cm21) Assignments

IR VCD IR VCD

1683 1691(2)/1678(þ) 1664 1671(þ)/1656(2) C(6)yO stretch (G) 1657 1663(þ)/1654(2) C(2)yO stretch (C) 1649 1651 1633 1637(2)/1627(þ) C(2)yO stretch (C) 1637(þ)CyC stretch (C) þ CyO stretch (G) 1623 1627(2) 1611

1579 1573(þ) 1581 C–N(D2) stretch (G) 1564 1570(þ)/1556(2) 1563 1561(2)/1540(þ)CyN stretch (G) 1534 1540(þ)/1535(2) 1537

1521 1527(þ)/1521(2) 1519 C–N(D2) bend þ def. (C) 1502 1506(þ)/1498(2) 1499 1502(2)/1492(þ)CyN (C) 1123 2 1087 1089(2)/1070(þ) 1087 1087(þ)/1075(2) Symmetric PO2 stretch 1075 Deoxyribose-phosphate 1053 1060 Deoxyribose C–O stretch 1018 1014 Deoxyribose ring 969 973(2)/958(þ) 969 969(þ)/946(2) Deoxyribose C–C stretch 927 Z-form marker 892 873 B-form marker

Refs. [20,74]. changes of the macromolecule. There are three distinct absorptions in the nitrogen base region. The band at 1692 cm21 and its VCD couplet at 1704(2)/1692(þ)cm21 were assigned to the C(2)yO vibration of uracile (U) [17,18,72,73]. The stronger absorption at 1669 cm21 (peak 2) and much stronger VCD counterpart at 1677(2)/1665(þ)cm21 (peaks 2 and 20) belong to the C(4)yOvibrationof U. The absorption at 1631 cm21 (peak 4) and the corresponding 1635(2 )/1627(þ)cm21 couplet (peaks 4 and 40) were assigned to the ring vibration involving CyCandCyN of hydrogen-bonded adenine (A). The weak absorption at 1569 cm21 and probably also the very weak negative VCD band at the same position could be assigned to a CyN vibration of A (position not numbered). Of the two signiﬁcant 2 VCD features in the sugar-phosphate (PO2 ) region, the one most intense is the couplet at 1098(2)/ 1087(þ)cm21 (peaks 6 and 60) arising from the absorption at 1091 cm21 (peak 6), which was 2 assigned to the PO2 symmetric stretching mode 21 [17]. An adjacent absorption at 1075 cm was not Fig. 2. Absorption spectra of poly(rA)·poly(rU) with 0.4 [Ni]/[P] at observed in this earlier report. Another distinct VCD different temperatures: (a) 23 8C, (b) 43 8C, (c) 45 8C, (d) 69 8C, (e) feature in this region is the negative peak at 72 8C, (f) 86 8C, (g) 90 8C, and (h) after 12 h of cooling to 23 8C. V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 547

1125 cm21 corresponding to the absorption at 1121 cm21 (peaks 5), which were assigned to the 0 C(2 )endo –O stretching mode of ribose [17]. Upon increasing the temperature from 23 to 43 8C, both the VCD and absorption spectra changed to represent an equilibrium mixture of double- and triple-stranded molecules (cf. traces a and b in Figs. 2 and 3). The starting temperature of the double- to triple-helical transition was signiﬁcantly lower than in the case without Ni2þ indicating stabilization by Ni2þ of the triple helical structure (spectra not shown). Further increasing the temperature to 45 8C essentially completed the double- to triple-helical transition (traces c in Figs. 2 and 3). The absorption and VCD spectra between 45 and 69 8C correspond to the triple-helical structure of poly(rU)·poly(rA)·poly(rU) [17,18,77] with the VCD spectrum displaying the (2þþ 2 þ ) pattern characteristic for the triplex [17,18]. The main change in the sugar-phosphate region upon transition from the double- to triple-helix is a gradual disappearance of the 1091 cm21 absorption (peak 6 in Fig. 2), which indicates some structural changes of the sugar-phosphate backbone as Fig. 3. VCD spectra of poly(rA)·poly(rU) with 0.4 [Ni]/[P] at different temperatures: (a) 23 8C, (b) 43 8C, (c) 45 8C, (d) 69 8C, (e) a result of the transition. The VCD spectrum in this 72 8C, (f) 86 8C, (g) 90 8C, and (h) after 12 h of cooling to 23 8C. region is also altered but to a lesser degree.

Table 3 Assignments for IR and VCD spectra of different forms of poly(rA)·poly(rU) þ 0.4 [Ni]/[P]

Absorption VCD Assignments

Index Wavenumber Index Wavenumber in Fig. 2 (cm21) in Fig. 3 (cm21)

Duplex Triplex Single-stranded Duplex Triplex Single-stranded (23 8C) (53 8C) (72 8C) (23 8C) (53 8C) (72 8C)

1 1692 1696 1689 1(2)/10(þ) 1704(2)/1692(þ) 1704(2)/1689(þ) – C(2)yO (U) 2 1669 1673 – 2(2)/20(þ) 1677(2)/1665(þ) 1681(2)/1673(þ) – H-bonded C(4)yO (U) 3 – 1658 1658 3(2)/30(þ) – 1658(2)/1650(þ) – Free C(4)yO (U) 4 1631 1627 1623 4(2)/40(þ) 1635(2)/1627(þ) 1631(2)/1615(þ) – Ring mode (A) 5 1121 1122 – 5(2)/50(þ) 1125(2)/1110(þ) 1125(2)/1114(þ) – C(20)–O ribose stretch 6 1091 – – 6(2)/60(þ) 1098(2)/1087(þ) 1098(2)/1083(þ) 1098(2)/1081(þ) Symmetric

PO2-stretch 7 1075 1079 1079 – – – – 0 8 863 863 – – – – – C(3 )endo ribose (A-form marker)

Ref. [12,17,19,71,73,76–79,90]. 548 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

The VCD spectrum at 69 8C (trace d in Fig. 3) 1623 cm21 in the single-stranded form. The appear- decreased in intensity without any significant changes ance of this VCD couplet implies that single-stranded in shape indicating the beginning of distortion of the poly(rA) exists in some kind of ordered structure, helical structure. More pronounced changes occurred different from canonical single-stranded helical at 72 8C (trace e in Fig. 3). The C(2)yO absorption of structure of poly(rA) [12,17,18]. This structure may U significantly decreased in intensity and shifted to result from Ni2þ ion intercalation between 1689 cm21 (peak 1 of trace e in Fig. 2), whereas the neighboring adenine bases as well as by forming C(4)yO absorption further increased in intensity but metal ion-water cross-links between adenine bases remained at 1658 cm21 (peak3inFig. 2). that are further apart from one another, and between The absorption due to the ring vibration of A also the bases and the phosphate groups. Forming such a increased significantly in intensity and shifted to structure may require increased adenine base stacking 1623 cm21 (peak 4 of trace e in Fig. 2). These changes and make the whole structure more rigid and ordered correspond to a transition to the single-stranded thereby giving rise to the VCD signal. It is also form and to a possible helix-coil transition of possible that poly(rA) molecules condense to tight single-stranded molecules [12,17,18,72,73,77].At compact structures by forming metal ion-water 72 8C (trace e in Fig. 3) virtually no VCD signal cross-links among different chains or different remote remained in the nitrogen bases region. At the same parts of one chain, which would also lead to a VCD time, a distinct VCD signal remained in the sugar- signal. The ordered structure of poly(rA) induced by phosphate region showing that the ordered structure of Ni2þ ion was reproducible though slightly different the sugar-phosphate backbone of the triplexes is more every time, perhaps due to forming different networks resistant to thermal denaturation than the stacked of metal ion-water cross-links. This structure was structure of the nitrogen bases, which was also noted relatively unstable, since increasing the temperature previously [17]. The sugar-phosphate backbone is to 90 8C induced significant disruption of the structure well stabilized by neutralization of the negative and the transition of poly(rA) to a coil. charges on the phosphates by Ni2þ ions. Melting Cooling the single-stranded molecules formed in begins with the distortion of nitrogen bases stacking, the presence of Ni2þ ions at 90 8C for 12 h down to andwhenarelativelyhighnumberofthe 23 8C produced triple-helical instead of double-helical nitrogen bases become disordered, distortion of the molecules (traces h in Figs. 2 and 3). This was sugar-phosphate backbone occurs [17]. In the absence different than without Ni2þ (spectra not shown), of Ni2þ ions, the distortion of the sugar-phosphate further demonstrating the stabilizing effect of Ni2þ backbone of single helices occurred at a lower ions on the triplex structure. A hysteresis effect may temperature than the distortion of base pair stacking take place during the reverse transition of the (vide supra, spectra not shown). triple-stranded to the double-stranded form. A similar Further increasing the temperature for about 10 8C hysteresis was demonstrated for the triple helix of (80–83 8C) changed neither the absorption nor the r(UCU5C6)·d(AGA5G6)·d(C6T5CT) formed by vary- VCD spectra (not shown). However, the VCD ing the pH [78]. spectrum at 86 8C was significantly altered without concomitant changes in absorption (cf. Figs. 2 and 3). 3.3. Calf thymus DNA Interaction with Cr3þ ions— The absence of changes in absorption compared to VCD evidence of DNA condensation 72 8C indicates that poly(rA) and poly(rU) remained single-stranded, whereas the distinct change in VCD 3.3.1. Assignments of VCD and absorption bands indicates that the structure of the single-stranded of natural calf thymus DNA molecules has changed. The main feature in VCD at The absorption spectra [71,73,76,80,81,82] and 86 8C is a relatively strong couplet at 1635(2)/ VCD spectra [15,19] of calf thymus DNA are 1619(þ )cm21 (peaks 4/40 trace f in Fig. 3). characteristic for the DNA B-conformation (top traces The couplet at 1635(2)/1619(þ)cm21 arises from in Figs. 4 and 5, Table 4). At least two absorptions the ring mode of A corresponding to the absorption at contribute to the broad band at the highest wavenum- 1619 cm21 shifted from its normal position at ber. One appears as a shoulder at ,1693 cm21 and has V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 549

Fig. 4. Absorption spectra of DNA and DNA with Cr3þ ions at different [Cr]/[P] ratios. All spectra are plotted on the same scale.

3þ been assigned to the stretching mode of C(2)yO (T). Fig. 5. VCD spectra of DNA and DNA with Cr ions at different [Cr]/[P] ratios. All spectra are plotted on the same scale. The other appears as a peak at ,1682 cm21 and is due y y to mostly C(6) O (G) coupled with C(4) O (T) and 21 C(2)yO (C). Corresponding to these absorptions is a The broad absorption at 1573 cm is a strong VCD feature at 1698(2)/1665(þ)cm21 with a superposition of several bands arising from ring y slight negative shoulder at ,1689 cm21. It can be vibrations of G and A involving mainly the C N 21 interpreted as a superposition of several couplets bonds, while the 1499 cm absorption has been y arising from all of these carbonyl vibrations. The band assigned to a cytosine mode that also contains C N at 1647 cm21 has been assigned to overlapping stretching. Neither of these absorptions have clearly absorptions due mostly to C(2)yO (C) coupled with corresponding VCD features, although the cytosine C(4)yO (T) and C(6)yO (G). The absorption at mode gives rise to a distinctive couplet in oligomers 1623 cm21 likely consists of contributions from ring with high GC content that appears consistently near 21 modes of A and also T and C [76]. The apparent VCD 1502(þ)/1498(2)cm in the B-form and reverses couplet at 1638(þ)/1619(2)cm21 can be ascribed to signs in the Z-form [20,30,67]. two overlapping couplets. One of these is due to a ring In the sugar-phosphate region, the prominent mode of A, which occurs at 1635(2)/1627(þ)cm21 in absorption at 1086 cm21 with its corresponding strong poly(rA)·poly(rU) (Fig. 3 and Table 3) and at 1635(2)/ positive VCD couplet at 1089(2)/1068(þ)cm21 has 1625(þ)cm21 in poly(dA)·poly(dT) and poly(dA– been assigned to symmetric P–O stretching of the dT)·poly(dA–dT) [10]. The other arises from thymine, phosphate groups [19]. The broad positive VCD which is found at 1640(þ)/1625(2)cm21 in component of this feature also contains contributions d(CGCGTGCG)·d(CGCGTGCG) [67]. from a ring mode of deoxyribose, which shows in 550 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

Table 4 absorption occurs. According to the theory of Assignments for IR and VCD spectra of calf thymus DNA Keller and Bustamante [87,88], the c-type CD IR (cm21) VCD (cm21) Assignments spectra can be produced in certain circumstances by particles (molecular aggregates) within the 1693 1698(2) C(2)yO of thymine spectral region of an absorption band. 1682 1698(2)/1665(þ) C(6)yO (G) coupled These circumstances require that the particle is with C(2)yO (C) and more or less three-dimensional, its dimensions are y C(4) O (T) comparable to the wavelength of the incident light, 1647 1638(þ) ? Mostly C(6)yO (C); C(4)yO (T), coupled the particle density is sufﬁciently high, and the

with C6yO(G) particle possesses a large-scale chirality. 1623 1619(2) Ring mode of A Therefore, it can be suggested from the VCD 1573 – CyN ring (G,A) spectra that DNA condensates produced by Cr3þ 1499 – CyN ring (C) 2 ions in the conditions of this experiment represent 1086 1089(2)/1068(þ) Symmetric PO2 stretch chiral particles with regular arrangements of DNA 1053 1060(þ) C–O sugar double-helices inside a particle. Judging by the 1021 1029(2)? Deoxyribose ring wavelength of the absorption bands where the 969 971(2)/957(þ) C–C sugar stretch anomalous VCD occurs, these relatively dense and 938 – Sugar large particles are of the order of several mm 895 920(2)/895(þ)? Sugar, B-form marker 836 – Sugar, B-form marker (approximately 2–10 mm). Increasing the Cr3þ concentration to 1 and Refs. [12,15,19,71–73,76,82,90]. further to 2 [Cr]/[P] produced similarly shaped VCD couplets as with 0.6 [Cr]/[P], although the absorption at 1053 cm21 [19,73,83]. Other ring modes amplitude is significantly lower and only slightly of the sugar occur at 1021, 969, 895 and 836 [83]. increased (about 1.5 fold) compared to DNA Of these only the 969 cm21 absorption yields a without metal ions. Further increasing Cr3þ con- persistent couplet at 971(2)/957(þ)cm21. centration up to 3 [Cr]/[P] returned the intensity of both main VCD couplets to the values obtained for DNA without Cr3þ ions. The shape of the VCD 3.3.2. VCD evidence of Cr3þ induced DNA features, however, particularly in the base region condensation changed noticeably compared to the VCD signals 3þ Addition of the Cr ions up to 0.6 [Cr]/[P] induced of DNA without metal ions, which is due to relatively minor changes in VCD and absorption structural changes that occurred by Cr3þ binding to connected with metal binding to sugar-phosphate DNA. The gradual decrease and eventual disap- residues and nitrogen bases [7,9,22–24,69,85,86]. pearance of the c-type VCD features arise most The most drastic changes occurred in VCD at 0.6 probably because of the increasing size of the [Cr]/[P]. For comparison the absorption and VCD condensed particles. As the particle size grows, 3þ spectra are plotted for several key Cr concentrations the particle dimensions become significantly larger in Figs. 4 and 5, respectively. The intensity increased than the wavelengths within the absorption bands almost 4-fold for the main VCD couplets in the two and the favorable conditions of strong interaction spectral regions, whereas the corresponding absorption of the infrared light with the condensed particles in decreased by about 30%. the region of interest fades away [87–89]. This greatly increased VCD intensity in the The absorption spectra indicate that the secondary favorable experimental conditions for DNA structure of DNA before and after the condensation condensation can be attributed to the c-type VCD remains within the B-form family [9,71,82,90]. It can spectra [87–89]. In addition to the significant therefore be confirmed unambiguously and without increase of VCD amplitudes, ‘tails’ can be seen needing any additional tools and experiments that the in the VCD spectra at wave numbers higher then DNA secondary structure of the c-type condensates 1700 cm21 (Fig. 5), i.e. in a region where no remains in the B-form. It can be further stated that V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 551 based on absorption [71,73,82,86,90] or VCD spectra correspond to those of B-form DNA [15,19,22]. The [17,23] no noticeable DNA denaturation has occurred. strong VCD signals in both spectral regions as well as the presence of the sugar absorption at 1053 cm21 3.4. VCD as a tool for monitoring DNA thermal indicate that no DNA denaturation has taken place at denaturation this temperature [15,19,21,22,71,72,73,86,90]. The absorption spectrum at 90 8C corresponds to that of VCD signals in NAs arise from vibrational mostly denatured DNA [73]. The significant shift of coupling when macromolecular constituents are the carbonyl absorption from 1681 cm21 at 23 8Cto arranged (stacked) in a regular array in the helical 1659 cm21 at 90 8C heralds the disruption of Watson- structure. Loss of stacking, which usually occurs Crick hydrogen bonding between G, C and T bases. during DNA denaturation, i.e. loss of the regularly The shift of the absorption at 1623 cm21–1621 cm21, ordered arrangement among the nucleobases assigned to a ring mode of A, and its increase in and among sugar-phosphate residues of the intensity indicate a breaking of the hydrogen bonds backbone, obliterates VCD signals in both spectral between the complementary bases A and T [17,21,73, regions [17,23]. 77]. The significant increase in intensity of all The absorption spectra of DNA at 23 8C absorption bands in the base region occurs because of correspond to those of the B-form (Fig. 6) as indicated strong hyperchromicity and means that stacking by the marker bands at 938, 896 and 836 cm21, and between the bases is lost [73,86].Theshiftor by the absence of either A-form markers at 898, disappearance of the B-form marker bands demon- 865, and 810 cm21, or the Z-form marker at 800 cm21 strates the absence of the B-conformation. The shift of [9,71,82,90]. The VCD spectra at 23 8Calso the sugar band at 1053–1064 cm21 and the significantly decreased intensity of the absorption bands in the sugar-phosphate region are also characteristic for the denaturation of the double-helical structure [73,86]. The VCD spectrum at 90 8C can therefore be identified with confidence to that of denatured DNA. Due to the loss of the ordered structure and disruption of the vibrational coupling, this spectrum does not show any reliable VCD features and largely represents random noise [17,20]. Therefore in contrast to ECD where DNA spectra are modified but not completely obliterated during denaturation, VCD can be used to determine unambiguously DNA helix-coil transition.

3.5. DNA interaction with Mn2þ ions at elevated temperatures—VCD evidence of DNA aggregation

Changes in absorption and VCD spectra of DNA with 2.4 [Mn]/[P] at different temperatures (Fig. 7) provide the richest information about DNA condensation and aggregation induced by the combined action of Mn2þ ions and elevated temperature. More details on other aspects of Mn2þ interaction with DNA at room and elevated temperatures and at various metal ion concentration can be found elsewhere [23]. Fig. 6. Absorption (bottom set) and VCD (top set) spectra of calf Whereas the absorption spectra exhibit relatively thymus DNA at different temperatures. monotonous changes towards those that are 552 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

Fig. 7. Absorption spectra (left) and VCD spectra (right) of DNA with 2.4 [Mn]/[P] at different temperatures. All spectra in each set are plotted on the same scale.

characteristic for denatured DNA (Fig. 7), signifi- temperature increase to 60 8C. The appearance of cant and varied changes can be seen in the VCD the c-type VCD spectra can be attributed to DNA spectra. First, a significant increase of amplitude of condensation, i.e. formation of large dense particles the main VCD couplets in the base and phosphate with ordered arrangement of DNA molecules. DNA regions occurs at 55 8C. This increased VCD denaturation can be deduced from the absorption amplitude cannot be ascribed to increased absorp- spectra only at temperature 60 8C and higher. tion. In fact, the absorption intensity diminishes Therefore, DNA exists in a double-helical form slightly at 55 and 58 8C. In addition, a long ‘tail’ during condensation, which is one of the prerequi- appears in the non-absorbing region above sites for forming c-type particles [22,91,92]. 1700 cm21. These changes together with overall Furthermore, judging by the absorption spectrum band shapes are reminiscent of those described for at 55 8C the secondary structure of DNA in the DNA-Cr3þ complexes where they are ascribed to c- condensed particles corresponds to the B-form type VCD spectra. Similar features remain as the family in agreement with previous data [22,93,94]. temperature is increased to 58 8C, although the The relatively small increase of the VCD amplitude amplitudes slightly decrease. The VCD spectrum (about 2-fold) compared to the much greater becomes again significantly different upon a further amplitude increase induced by Cr3þ can be related V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 553 to a lower density of the particles formed by Mn2þ upon disruption of hydrogen bonding become ions due to the lower charge on the ion. In the linked to neighboring strands by metal ion bridges. present conditions the divalent Mn2þ ions probably In this way, an extensive network forms of metal barely neutralize the 90% of the negative charges on ion cross-linked DNA molecules. A similar the phosphates required for condensation [6,95]. explanation of DNA aggregation due to interaction The regular structure of ordered particles becomes of partially melted regions on different strands of distorted, i.e. particles melt, below the melting DNA was also suggested by other authors [96]. temperature of DNA [91].Thec-type VCD Therefore, the shape of VCD spectra, which spectra disappeared upon just a slightly increased appeared at temperatures close to or above the temperature most likely due to melting of the melting (Fig. 7), can be ascribed to DNA aggregation. condensed particles followed by melting of the In agreement with previous investigations [97], double-stranded DNA. the characteristic VCD spectra do not disappear A further temperature increase changed the when the temperature is raised well above the melting absorption spectrum towards that of denatured temperature. VCD, therefore, appears to be sensitive DNA. In contrast, the VCD spectrum at 60 8C not only to DNA condensation but also to DNA became significantly different from that correspond- aggregation and can distinguish between these two ing to native B-form DNA, condensed DNA, or processes of DNA collapse. This ability of VCD is melted DNA. This spectrum consists of relatively superior to many other techniques, which cannot strong and indistinct features, comparable in intensity easily distinguish between these two and refer to both to the VCD couplets of native DNA. The overall as DNA ‘compaction’. band shape does not significantly change when the It should be noted that for all other Mn2þ temperature is increased to 70 8C, which is well concentrations except 2.4 [Mn]/[P] no c-type spectra above the DNA melting temperature for this Mn2þ were observed at any temperature. However, intense concentration. and indistinct VCD features associated with DNA It has been shown that elevating the tempera- aggregation appeared upon raising the temperature ture of DNA in the presence of most divalent above the melting point for DNA complexes with metal ions including Mn2þ induces DNA aggrega- other Mn2þ concentrations between 2.4 and 10 tion [50,52,96,97]. DNA aggregation in the [Mn]/[P] (spectra not shown). This indicates that presence of transition metal ions has been found while DNA aggregation induced by Mn2þ ions at to occur in the thermal denaturation region of elevated temperature occurs in a relatively wide DNA. For most divalent metals, aggregation starts range of experimental conditions, DNA conden- as DNA denaturation begins, and upon complete sation, while possible in general, occurs in a very DNA denaturation the aggregates disappear [97]. narrow range. However, in the case of Mn2þ ions the aggregates that appeared in the thermal denaturation region did not disappear at temperatures well above the 4. Cis-platin complexes melting temperature even up to 100 8C possibly due to very stable cross-links formed by Mn2þ 4.1. IR absorption and VCD spectra ions [97]. Based on the theory of DNA aggrega- of the unmodified duplexes tion in the thermal denaturation region [49],a model has been proposed for describing tempera- The IR spectra (Fig. 8 for the octamer, I, and ture induced aggregation of DNA in the presence Fig. 10 for the dodecamer, II) as well as the VCD of divalent metal ions [50,52,97], which suggests spectra (Figs. 9 and 11, respectively) show the typical that a combination of metal–base interaction and features of B-form DNA [25,26 and citations therein]. heating disrupts the base pairing within the DNA The IR spectra of the two duplexes are remarkably duplex. This allows the divalent metals to bind to similar in shape. The slightly different peak positions additional sites on the DNA bases during the of the most intense bands reflect the different G·C vs. melting process. DNA strands whose bases open A·T contents of the two oligonucleotides. Similarly, 554 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

Fig. 9. VCD spectra of unplatinated (bottom) and platinated (top) Fig. 8. Absorption spectra of unplatinated (bottom) and platinated d(CCTGGTCC)·d(GGACCAGG) in D2O. (top) d(CCTGGTCC)·d(GGACCAGG) in D2O. the VCD couplets reﬂect the different stacking The C4yO stretching vibration of thymine is environment. The characteristic positions are in represented by the couplet at 1642(þ )/ the typical range (Table 5). The C(2)yO stretch of 1633(2)cm21 in I and at 1644(2)/1634(þ)cm21 thymine of I appears at 1718(þ)/1707(2)cm21 with in II. The ring in-plane absorption band of adenine in a corresponding absorption at 1712 cm21.The the octamer appears as a shoulder at 1618 cm21 but is analogous VCD feature of II is located at 1714(2)/ obscured in VCD. The same mode in the dodecamer ,1702(þ)cm21 and the absorption at 1706 cm21. cannot be clearly seen in absorption but gives rise to a Another VCD feature also assigned to C(2)yO weak VCD feature at 1627(þ)/1616(2)cm21. stretching of thymine is found at 1700(þ)/ Some differences are also observed in the region 1689(2)cm21 with its absorption at 1691 cm21 in where the phosphodiester backbone and various sugar I, and ,1702(2)/1695(þ)cm21 with its absorption modes absorb. The main absorption at 1085 and 1697 cm21 in II. Two VCD couplets with their 1086 cm21 in I and II, respectively, are associated absorptions representing CyO stretching of almost identical. The main VCD couplet at guanine occur at 1687(þ)/1682(2) and 1671(2)/ 1089(2)/1082(þ)cm21 in I is narrower, while the ,1659(þ)cm21 in I, but only one corresponding couplet at 1095(2)/1080(þ)cm21 in II is broader. couplet appears in II at 1689(2)/1679(þ)cm21. The VCD features arising from other vibrations of the The CyO stretch of cytosine occurs in I at sugar-phosphate backbone at 1079(2)/1072(þ) and ,1657(2)/1647(þ)cm21 in VCD 1652 cm21 in 1054(þ)/1045(2)cm21 appear to be more resolved absorption, with the analogous features in II at in I than the related couplets at 1074(þ)/1060(2) and 1660(þ)/1654(2) and 1656 cm21, respectively. 1054(þ)/1044(2)cm21 in II. V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 555

Table 5 IR and VCD (cm21) of d(CCTGGTCC)·d(GGACCAGG) and d(CCTCTGGTCTCC)·d(GGAGACCAGAGG) free and bound to cisplatin

Assignment Octamer free (bottom) Octamer-Pt complex (top) Dodecamer free (bottom) Dodecamer-Pt complex (top)

IR VCD IR VCD IR VCD IR VCD (Fig. 8) (Fig. 9) (Fig. 8) (Fig. 9) (Fig. 10) (Fig. 11) (Fig. 10) (Fig. 11)

CyC base 1727 1733(þ)/1724(2) 1729(þ)/1718(2) stretch C(2)yO 1712 1718(þ)/1707(2) 1706 1714(2)/,1702(þ) stretch (T) 1691 1700(þ)/1689(2) 1691 1697(þ)/1687(2) 1697 ,1702(2)/1695(þ) 1697 1700(2)/1695(þ) C(6)yO 1685 1687(þ)/1682(2) 1681(þ)/1675(2) 1689(2)/1679(þ) 1666 1687(2)/1679(þ) stretch (G) 1666 1671(2)/,1659(þ) 1666 1668(þ)/1662(2) 1672(þ)/1666(2) CyO stretch 1652 ,1657(2)/1647(þ) 1652 1654(2)/1643(þ) 1656 1660(þ)/1654(2) 1654 1660(þ)/1654(2) (C) C(4)yO 1648 1642(þ)/1633(2) 1641(þ)/1635(2) 1639 1644(2)/1634(þ) 1637(2)/1631(þ) stretch (T) Ring mode 1618 1623 1631(þ)/1619(2) 1627(þ)/1616(2) 1623 1622(þ)/1614(2) (A) 1607(2)/1602(þ) C–N(D2) 1589 ,1598(þ)/1585(2) 1587 1594(þ)/1585(2) 1600(þ)/1587(2) stretch (G) 1575 ,1575(2)/1562(þ) 1569 1571(2)/1564(þ) 1579 1583(2)/1575(þ) 1579 1583(2)/1570(þ) CyN stretch 1562 1553(þ)/1544(2) 1550(þ)/1542(2) 1567 1571(2)/1564(þ) ,1560 1579(2)/1565(þ) (G) 1560(2)/1554(þ) Ring mode 1531 1540(þ)/1525(2) 1532 1538(þ)/1530(2) 1533 1532(þ)/1523(2) 1530 1537(þ)/1523(2) (C) CyN stretch 1502 1511(2)/1494(þ) 1506 1510(þ)/1502(2) 1500 1510(2)/?(þ) 1502 (C) Symmetric 1118 1120(2) 1122 1127(2)/1118(þ) 2 PO2 plus C–O stretch 1104 1109(þ)/1101(2) 1110 1114(þ)/1104(2) Symmetric 1085 1089(2)/1082(þ) 1089 1091(2)/1081(þ) 1086 1095(2)/1080(þ) 1087 1089(2)/1079(þ) 2 PO2 stretch Phosphodiester 1075 1079(2)/1072(þ) 1079 1079(2)/1071(þ) 1074(þ)/1060(2) 1074 1076(2)/1068(þ) backbone coupled to sugar modes 1062a 1066(2)/1058(þ)a 1050a 1054(þ)/1047(2)a 1056 1060(þ)/1045(2) ,1056 1054(þ)/1044(2) 1054 1058(þ)/,1048(2) 1043a 1045(þ)/1039(2)a 1037(þ)/1027(2) Sugar modes 1016 1016 1018 1016 971 981(2)/964(þ)a 969 971 975(2)/962(þ) 970 914 919 924(2)/915(þ) 919 890 890 896 895 877 866

Refs. [22,74,76,84,99]. a Assignments/designations uncertain.

4.2. IR and VCD spectra of the Pt-coordinated stretch in guanine, and 1403 cm21 in I (Fig. 8). duplexes The absorption spectrum of II seems less affected by the formation of the Pt complex, although several The absorption and VCD spectra of the platinated changes are still evident (Fig. 10). These include the duplexes change appreciably upon coordination. decreased intensity of the 1697 cm21 band, which Specifically the relative intensity is significantly appears now as a shoulder, the greater intensity of the increased for the bands at 1623 cm21 arising from bands at 1654 and 1623 cm21, and the significant 21 2 the ring mode of adenine, 1587 cm due to C-N(D2) intensity increase of the symmetric PO2 vibration at 556 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560

Chart 1.

1087 cm21. The VCD spectrum of the platinated octamers has changed to a much greater extent (Fig. 9 vs. Fig. 11). Although almost all of the VCD couplets have changed either their peak positions or intensities, the most radical changes have occurred for the bases that are directly related to the Pt lesion, namely Gp4-C13 and Gp5-C12 in I (Chart 1), and Gp6-C19 and Gp7-C18 in II (Chart 2) [25,26]. Platinum coordination pulls the guanine bases toward the major groove thereby disrupting the stacking between them and the adjacent thymine rings [60,61]. Indeed, the 1718(þ)/1707(2)and 1714(2)/1702(þ )cm21 couplets attributed to stacking interaction between neighboring 50TpG bases, viz. T3pGp4 and Gp5pT6 in I and T5pGp6 and Gp7pT8 in II, are missing in the VCD spectra of the platinated duplexes suggesting destacking of the bases [25,26]. Another feature in the VCD of the free octamer affected by Pt coordination is the couplet Fig. 10. Absorption spectra of unplatinated (bottom) and platinated at 1671(2)/1659(þ)cm21 associated with stacking (top) of d(CCTCTGGTCTCC)·d(GGAGACCAGAGG) in D2O. of Gp4pGp5(Fig. 9). In the platinated duplex the couplet appears 1668(þ)/1662(2 )cm21 with reversed sign suggesting changed stacking conditions. A similar picture also emerges for the dodecamer, where a weak negative couplet occurs at 1672(þ)/ 1666(2)cm21 attributed to C(6)yO stretching of Gp6 and Gp7, which is not observed for the unmodiﬁed duplex (Fig. 11). The slight shift of this couplet and the accompanying sign reversal observed likely originate from the changed stacking environment of Gp6 and Gp7 upon platination. The most striking features of I appear in the range 1633–1598 cm21, where no VCD couplets were detected in the spectrum of the free octamer (Fig. 9). Two prominent couplets emerge in VCD of the platinated octamer as a result of its distorted conformation. The ﬁrst is located at 1631(þ)/1619(2)cm21, and the second at 1607(2)/ 1602(þ)cm21. The former couplet and its absorptioncm21 were assigned unambiguously to stacking interactions between two adenines [25]. Fig. 11. VCD spectra of unplatinated (bottom) (top) platinated (top) This assignment is based on the appearance of d(CCTCTGGTCTCC)·d(GGAGACCAGAGG) in D2O. of the same couplet in several sequences containing V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 557

Chart 2. neighboring adenine, viz., d (A4 TATCATTGG)· the platinated octamer (Fig. 9 and Table 5). The main 2 d(CCAATGATAT4) [98], the anti-parallel hairpin couplet due to the symmetric PO2 mode at 1089(2)/ 21 d(T8C4A8) [98], the triple helices of poly(dA)·poly(dT) 1082(þ)cm shifted slightly to 1091(2 )/ [16], and poly(rA)·poly(rU) double and triple helices 1081(þ)cm21. The second component of the main [17]. Such A–A stacking in I (Chart 1) can be couplet at 1079(2)/1072(þ)cm21 in I also remains accomplished between A11-A14 when the Gp4-C13 unchanged. One noticeable difference is the changed p 0 and G 5-C12 base pairs are pulled out toward the major shape and position of the C(2 )endo sugar pucker [99]. groove and the whole DNA structure is kinked to The 1054(þ)/1047(2)cm21 couplet has shifted such an extent that both adenine bases are sufficiently upwards to produce the broad feature at 1060(þ)/ close to interact with one another. The feasibility 1045(2)cm21. of such excessive conformation was not discussed The major VCD couplet of platinated II in the before. One reasonable explanation is that one phosphate region also closely resembles that of the observes the beginning of the slow isomerization, of unplatinated dodecamer (Fig. 11). The negative lobe is which the first signs have been detected in the NMR the slightly downshifted, and is significantly increased in following day [60]. intensity. Although some intensity increase is also No signs for isomerization were observed in the observed for the same couplet in I (Fig. 9), here this VCD spectra of the dodecamer, nor were any feature is more pronounced. It is generally accepted reported in solution or crystalline states [61,62]. that an increased VCD intensity of the couplets arises Evidently, the longer DNA duplexes can adopt from a tighter helical order. The remarkable feature for conformations that are stable enough to absorb the the dodecamer is an intensity increase of the VCD 2 strain induced by cis-DDP. couplet associated with the symmetric PO2 vibration, Moving beyond the Pt coordination site, the and a decrease of the VCD intensities in the carbonyl distortion diminishes on both sides of the oligonucleo- region. The reduced VCD intensities in the carbonyl tides. This is manifested in the octamer by the minor region might be connected with the unwinding of the downshifts of the couplets at 1700(þ)/1689(2) and helix and the increased average rise per base pair by 1687(þ)/1682(2)cm21 whicharerelatedtothe approximately 0.78 per residue reported for stacking between C2–G15 and T3-A14 and the the dodecamer [61,62]. Alternatively, the increased same stacking at the other end of the sequence. intensity of the VCD couplet associated with the Equivalent but smaller wavenumber shifts for the backbone vibrations is consistent with the observation analogous couplets are observed in the VCD of the that the overall helical shape is closer to the A rather dodecamer. The couplet at ,1702(2)/1695(þ)cm21 then the B-form [61,62]. has shifted to 1700(2)/1695(þ )cm21 and at 1689(2 )/1679(þ) to 1687(2)/1679(þ )cm21 suggesting little deformation of the DNA duplex 5. Summary beyond the Pt lesion. At the first base step, the distortion of the duplex decreases as indicated by The fact that circular dichroism should be stacking of the terminal cytosines in the octamer observable also at long wavelengths has not escaped (1657(2)/1647(þ) and 1654(2)/1643(þ)cm21 for the attention of scientists in the past. Of particular the unplatinated and platinated oligomers, respect- fundamental and historic interest pertaining ively). The dodecamer structure remains intact and no to polymeric substances are the theoretical wavenumber shift is observed. investigations of Deutsche and Moscowitz who The principal VCD features in the phosphate demonstrated in 1968 that circular dichroism of region of I are clearly reproduced in the spectrum of vibrational origin should be measurable for idealized 558 V. Andrushchenko et al. / Journal of Molecular Structure 661-662 (2003) 541–560 model helical polymers [100,101]. They also realized methods for studying DNA condensation and aggre- that such measurements should be feasible for gation. The very visible effects of cisplatin coordi- polymers of biological significance. Another 20 nation on the VCD spectra of the selected years would pass before technology caught up with oligonucleotides suggest another and quicker method the theoretical demonstration of vibrational circular for detecting damage to the secondary structure of dichroism. The feasibility of measuring VCD spectra DNA. It is interesting to note in this connection that of DNA, RNA, and various oligomers was then antitumor drugs, which are effective because of their amply proven through the work of T.A. Keiderling ability to distort the helical structure of DNA by and M. Diem and their coworkers in the late 1980s intercalation, also give characteristically distinctive [12,13 for example]. On the basis of these early signatures in VCD [67]. The burgeoning possibility to successes, we focused since the early 1990s on verify interpretations of VCD spectra by high level exploring just how useful and definitive VCD could quantum mechanical simulations will be to characterize the DNA structure. We were eventually replace qualitative assignments and interested in particular in the effects of NA sequences, thereby enhance the diagnostic power of the method various structural forms of DNA, and interactions [74]. Lastly, once the VCD signals are better under- with some agents specifically metal ions and selected stood and characterized, VCD can become a partner drug molecules [67,98,102] on the VCD Spectra with other methods for examining protein-DNA compared to the absorption spectra. interactions. When dealing with large systems such as DNA or even with much shorter oligonucleotides one will naturally expect complicated absorption spectra and References even more so VCD spectra. Trying to unravel and interpret these convoluted signatures one is con- [1] W. Saenger, Principles of Nucleic Acid Structure, Springer- fronted with the daunting task of making reasonable Verlag, New York, 1984. assignments despite the apparent higher resolution in [2] M.A. Semenov, T.V. Bol’bukh, V.A. 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