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Research ArticleReceived: 27 October 2008 Accepted: 4 December 2008 Published online in Wiley Interscience: 4 February 2009

(www.interscience.wiley.com) DOI 10.1002/jrs.2192

Raman spectra and structure of a 25mer HCVRNAPedro Carmona,a∗ Marina Molinab and Arantxa Rodríguez-Casadoc

We have employed Raman spectroscopy to investigate the conformation of an (Hepatitis C virus) HCV RNA 25mer (1–25nucleotides) in solution. The principal findings of this study are (1) the A-form secondary structure involving C3′-endo/antiribofuranose pucker is predominant; (2) some uridine and guanosine nucleoside residues adopt the C2′-endo/anti and C3′-endo/syn conformations, respectively, which appear in looped nucleotide sequences; and (3) six out of nine guanine residuesare base-paired probably forming a stem. These results are interpreted as formation of a hairpin whose secondary structure isconsistent with that proposed on the basis of phylogenetic comparisons with other viral RNAs. Copyright c© 2009 John Wiley &Sons, Ltd.

Keywords: Raman spectroscopy; RNA; hepatitis C virus; secondary structure

Introduction

Hepatitis C virus (HCV) is an RNA virus that causes acute and chronicliver disease in humans, including chronic hepatitis, cirrhosis,and hepatocellular carcinoma.[1] The HCV genome is a single-stranded RNA molecule of positive polarity which contains ahighly conserved 5′ untranslated region (5′UTR) of 341 nucleotidesin length. The HCV structural proteins include the core protein,which forms the viral nucleocapsid and is involved in the assemblyand packaging of the viral genome.[2] The mechanisms of HCVassembly and formation of the nucleocapsid structure are not wellunderstood. As for many viruses, the assembly is believed to beinitiated by the binding of the core protein to a defined structure,often a stem-loop in the nucleic acid sequence.[3] This interactionfacilitates nucleation of the core protein and oligomerizationthat occur via specific protein–protein interactions. Some reportshave described the in vitro formation of nucleocapsid-like particles(NLPs) involving interactions of structured RNA stem-loops withthe core protein.[3,4]

It is well known that Raman spectroscopy can give informationabout the conformations and interactions of nucleic acids andproteins.[5] An advantage of Raman spectroscopy is that, inprinciple, information about both the protein and nucleic acidparts of a complex is obtained as these have clearly recognizablevibrations. The wavenumbers and intensities generated by thesevibrations are influenced by the macromolecular secondarystructures, hydrogen-bonding interactions, and the environmentof the molecular subgroups in question. With the aim of gettingthe said structural information, previous structural studies of theisolated interacting biomolecules are needed. Concerning the HCVgenome, the 5′UTR contains four domains numbered I–IV.[1,6]

Domains II, III, and IV constitute an internal ribosome entry site(IRES) which has been visualized by electron microscopy.[7,8] Onthe other hand, NMR spectroscopy has yielded atomic structuresof stem-loops IIIb,[9] IIId, and IIIe;[10] and X-ray crystallography hasbeen used to solve the structure of the IIIabc four-way junctionalong with eukaryotic translation initiation factor 3.[11] However,no detailed Raman (or infrared) studies have been reported onany of the domains I–IV constituting the conserved 5′UTR of HCV.

We carry out here the Raman analysis of an HCV RNA 25mer(1–25 nucleotides) comprising the domain I of the 5′UTR, inaqueous and heavy water solution. Data obtained from theseexperiments, combined with nucleotide sequence information,lead to a proposed secondary structure which is consistentwith an earlier proposed structure supported by phylogeneticcomparisons with other viruses.

Experimental

Materials

The following reverse-phase HPLC-purified oligonucleotide waspurchased from Eurogentec (Belgium): 5′-GCC-AGC-CCC-CUG-AUG-GGG-GCG-ACA-C-3′. The size and integrity of this RNAwas checked by polyacrylamide gel electrophoresis, and itsconcentration was determined by measuring the absorbanceat 260 nm and using 25 g−1 cm−1 L as extinction coefficient. ForRaman spectroscopy, this 25mer RNA was prepared at 3% w/wconcentration in a 50 mM Tris buffer (pH 7.4) containing 0.1 M NaCland 0.1 mM EDTA. For deuteration, 25mer RNA aqueous solutionsof the said concentration were allowed to dialyze against D2OTris buffer (50 mM containing 0.1 M NaCl and 0.1 mM EDTA, pD7.4). The oligonucleotide aqueous solutions were heated at 85 ◦Cfor 5 min then rapidly cooled and stored overnight at 4 ◦C beforespectral measurements.

The constituents of the above H2O and D2O buffers were freefrom RNAases and DNAases, and purchased from Fluka.

∗ Correspondence to: Pedro Carmona, Instituto de Estructura de la Materia (CSIC),Serrano 121, 28006 Madrid, Spain. E-mail: p.carmona@iem.cfmac.csic.es

a Instituto de Estructura de la Materia (CSIC), Serrano 121, 28006 Madrid, Spain

b Departamento de Química Organica I, Escuela Universitaria de Optica, 28037Madrid, Spain

c IMDEA Alimentacion, 28049 Madrid, Spain

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Instrumentation

The Raman spectra were recorded with a Renishaw Raman SystemRM2000 equipped with, a Pelletier-cooled CCD camera, and anotch filter to eliminate the elastic scattering. The samples wereexcited with the 785 nm laser line having an output laser powerof 5.0 mW, and the spectral resolution was 4 cm−1. Calibration ofthe Raman wavenumbers was carried out using the 520.5 cm−1

line of a silicon wafer. The intensity calibration was done usingthe response correction application of the spectrometer software.This correction is based on a calibrated standard light source forwhich accurate intensity information is available as a function ofwavenumber. Signals obtained were fed to a microcomputer forstorage, display, plotting, and processing; and the manipulationand evaluation of the spectra were carried out using the Grams/AIsoftware (Thermogalactic). The samples were placed in a 40-µlthermostatic quartz cell, and 30 scans were collected and averagedto yield spectra of suitable signal-to-noise ratio. The buffer spectrawere recorded with the same instrument settings employed foroligonucleotide solutions.

Data analysis

Buffer background was subtracted from the signal-averagedspectra by difference methods described elsewhere[12] usingthe above software. In difference spectra, only those peaksexceeding the noise level by a factor of two or greater areconsidered significant. On this basis, the said software was appliedto fit the 850–750 cm−1 region to a sum of Gaussians by anonlinear least-squares procedure. The mathematical solution tothis decomposition may not be unique, but if restrictions areimposed such as the maintenance of the initial band positions inan interval of ±1 cm−1, exclusion of bands with negative heights,and the preservation of the bandwidth within the expected limitsor the agreement with theoretical boundaries or predictions, theresult becomes, in practice, unique. Linear baseline correctionswere performed on the spectra in order to facilitate intensitycomparisons.

Results and Discussion

Figure 1 shows the Raman spectra of HCV RNA in H2O and D2Obuffers and Table 1 lists the wavenumbers and assignments of themost prominent Raman marker bands. The 1100–780 cm−1 rangegenerally reflects ribose-phosphate backbone conformation, andthe 1750–1600 cm−1 range is sensitive primarily to base pairing.

Backbone conformation

Two categories of Raman conformation markers have beendelineated,[13] namely, those relating to the conformationsof the sugar-phosphate backbone and those relating to theconformations of the constituent nucleosides. The occurrenceof unique backbone conformation markers for Z (745 ± 2 cm−1),A (807 ± 3 cm−1), and B (835 ± 7 cm−1) forms facilitates the useof Raman spectroscopy to distinguish the above forms from oneanother.

It is well established that the ratio of intensities of lines near 810and 1100 cm−1 in Raman spectra of polynucleotides (I810/I1100) in-dicates the degree of order in the ribose-phosphate backbone.[14]

The phosphate line near 1100 cm−1 (PO2− symmetric stretching

vibration) serves as an internal intensity standard while the line

600 800 1000 1200 1400 1600

0.002

0.004

0.006

0.008

629

1705

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an in

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ity/a

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. uni

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157514

8281278

372

466

8

1317

125010

98

1368

1416

1530

1599

1043

983

921

867

776

811

718

668

624

1097 15

77

1474

1501

1621

1663

1699

1315

1248

1260

1046

875

980

Figure 1. Raman spectra of the 25mer HCV RNA in the 1750–600 cm−1

range. Top: in H2O buffer. Bottom: in D2O buffer.

Table 1. Assignments of the most prominent Raman marker bandsfrom aqueous solution of 25mer HCV RNA

Wavenumber (cm−1) Assignmentsa

H2O buffer D2O buffer

1705 w 1699 w νC O paired guanine

1663 w Nonpaired G; nonpaired U

1676 sh Nonpaired G; nonpaired U

1635 sh r(A)

1599 vw 1621 w R(C); r(A)

1321 sh 1321 sh r(G); C3′-endo/syn

317 m 1315 m R(G); C3′-endo/anti

1302 w 1303 w r(A); r(C), C3′-endo/anti;

1295 sh 1295 sh r(C); C3′-endo/anti

1250 m 1248 m r(C); C3′-endo/anti

1242 sh 1240 sh r(U); C2′-endo/anti

1230 sh 1233 sh (U); C3′-endo/anti

828 sh 824 sh νsO-P-O; C2′-endo/anti

812 vs 811 vs νsO-P-O; C3′-endo/anti

789 sh 789 sh r(U); C3′-endo/anti

783 vs 776 s r(C); C3′-endo/anti

668 w 668 w r(G); C3′-endo/anti

644 vw 641 w (G); C3′-endo/syn

Abbreviations: vs, very strong; s, strong; m, medium; w, weak; vw,very weak; sh, shoulder; r(A), riboadenosine; r(C), ribocytidine; r(G),riboguanosine; r(U), ribouridine.a Assignments carried out on the basis of the following Refer-ences [13–28].

near 810 cm−1 (diester OPO stretching vibration) is very sensitivein intensity and position. In solutions of RNA polynucleotides, thisintensity ratio (R) takes on values ranging from 0 to 1.64, depend-ing upon the percentage of nucleotide residues which are presentin segments of ordered secondary structure of the A-type.[15,16]

Consequently, an R value of 1.25 obtained here is an indicationthat around 76% of the bases in this HCV RNA are ordered (with theC3′-endo/anti ribofuranose pucker) under the conditions of ourspectral measurements. This number, which has been obtainedby measuring the 812 cm−1 band height on a baseline passingthrough the intensity minima at 834 and 797 cm−1, is higher than

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an expected value corresponding simply to the double helicalregion of the proposed structure. Therefore, the Raman data showthat not only nucleotides in base-paired regions, but also themajority of unpaired bases should exist in the same highly spe-cific backbone geometry that gives rise to the 812 cm−1 Ramanline. This is consistent with the fact that Raman spectra of polyri-bocytidylic acid and polyriboadenylic acid show the formationof ordered single-stranded structure at neutral pH with stackedbases and a backbone geometry of the A-type.[17,18] In addition,single-stranded heterogeneous sequences in RNA adopt this typeof backbone conformation.[29]

In the Raman spectrum of this HCV RNA a shoulder locatednear 828 cm−1 (Table 1) is visible which can be generated by the Bbackbone form on the basis of the above spectrum–structurecorrelation. This means that, although the A-form secondarystructure is found to be predominant in this oligonucleotide,some nucleotide residues can adopt nucleoside conformations(C2′-endo/anti) which are characteristic of the backbone B-form. InRNA, this occurs in critical regions so that the backbone chain foldsinto some complicated patterns of helices and loops.[19,30] Thus,in addition to the C3′-endo/anti ribofuranose pucker associatedwith ribose-phosphate backbone of A-type, C2′-endo/anti sugarpuckerings are also present which can be attributable to positionswhere chain foldings switch abruptly from helical to looped.Hence, these results are consistent with the presence of an A-form duplex and a looped single-stranded sequence forming ahairpin, as suggested in other works.[4,31] A question arises as tothe spectroscopic determination of the base pairs in the hairpinduplex, what can be addressed through spectral analysis of basepairing.

Base pairing

The interaction of polynucleotides to form helical structuresresult in characteristic changes in the carbonyl region of thevibrational spectrum. Although the interpretation of these bandsis complex,[20] these spectral features are used for determinationof the base pairing content. In the Raman spectrum of an RNAin D2O solution (Fig. 2), the 1699 cm−1 band can be assigned tothe C O stretching mode of guanine base in Watson–CrickGC base-pairs.[21] However, this vibrational mode appears inthe 1665–1675 cm−1 range for nonpaired guanine residues inheavy water buffer.[21,22] Another base which can contribute tothe spectral profile in the carbonyl stretching region is uracil.Although the content of this base is relatively small (8% residues)in this oligonucleotide, it can generate Raman signals in the1680–1650 cm−1 range in D2O buffer. Base-paired uracil showsa strong band near 1680 cm−1,[14] whereas the νC O intensitymaximum shifts toward 1665 cm−1 in nonpaired residues.[17,32]

Concerning the Raman bands below 1650 cm−1, these can beassigned to cytosine and adenine bases.[14,17] Given that the1699 cm−1 band corresponds to base-paired guanine residues,the intensity of this band relative to the νsPO2

− intensity at1098 cm−1 can provide the proportion of guanine residuesthat are Watson–Crick base-paired. In order to estimate asaccurately as possible maxima and intensities of the bands inthe 1725–1600 cm−1 region, a band decomposition is presentedin Fig. 2. The intensity ratio I1699/I1098 is 0.35 ± 0.02 in the Ramanspectra of poly(rG)·poly(rC) duplexes recorded by us and otherauthors.[14] On the other hand, the intensity of the 1699 cm−1 bandfrom the HCV RNA oligonucleotide relative to the νsPO2

− intensitycorresponding to the mole fraction (9/25) of guanine residues

Figure 2. Curve fitting of the D2O buffer Raman spectrum of the 25merHCV RNA in the 1730–1590 cm−1 range.

is 0.23 ± 0.01. The ratio between the said relative intensities(0.23/0.35) is 0.65, which is near the fraction (6/9) of guanineresidues that can be base paired. This result is consistent with theformation of a duplex stem involving the sequences 5′-GCCCCC-3′

and 5′-GGGGGC-3′ in the oligonucleotide. This folding would resultin a tetraloop containing the 5′-UGAU-3′ sequence which is U richand consequently the tetraloop is thermodynamically stable.[33]

Some of the tetraloop residues adopt nucleoside conformationsother than C3′-endo/anti, namely, C2′-endo/anti and C3′-endo/syn,as described below.

Nucleoside conformation marker bands

Nucleoside conformation markers originate primarily from certainbase ring vibrations that are weakly coupled via the glycosylbond to motions of the furanose ring atoms.[23] These markersare sensitive to either furanose pucker, or glycosyl orientation, orboth. On this spectroscopic basis, guanosine residue in tetraloopshas been reported to adopt C3′-endo/syn conformation.[19,34]

The Raman spectrum of this HCV RNA oligonucleotide (Table 1,Fig. 3(a)) seems to provide another case of RNA hairpin containinga guanine residue with the said nucleoside conformation. Thisis supported by the band located at 1321 cm−1 which has beenassigned to this structure on the basis of the Raman spectra ofpoly(rG-rC) in the Z-form[24] and some tetraloops showing a strongband at this wavenumber.[19,34] This band is more visible andbetter resolved in D2O buffer because the contiguous 1317 cm−1

band is downshifted to 1315 cm−1 upon deuteration (Fig. 3(a)).Moreover, for similar reason the weak Raman band observed at644 cm−1 in aqueous spectra is more visible at 641 cm−1 in D2Osolution spectra (Fig. 3(b)). This band is also a marker of the C3′-endo/syn orientation of the guanine residue in the loop, because itswavenumber value corresponds well to that observed in Z-forms ofRNAs.[24] The medium-intensity band at 1317 cm−1 (Fig. 3(a)) canbe attributable to the C3′-endo/anti conformation of other guanineresidues in either the duplex stem or the single-stranded segments,because generally speaking guanosine residues in duplexesand single-strands are known to adopt the said nucleoside

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conformation.[23] On the other hand, in the 1300–1200 cm−1

range the dominant 1250 cm−1 band can be reliably assignedto cytidine residues with the C3′-endo/anti conformation. Thisband is accompanied by a clear shoulder at 1242 cm−1 whichis characteristic of uridine residue C2′-endo/anti structure.[19,25,26]

Another shoulder is observed near 1233 cm−1 which can beascribed to uridine residue C3′-endo/anti conformation on thebasis of the Raman spectra of poly(rA)·poly(rU) duplexes inaqueous solution.[14,20] On A → Z conformational transition inpoly(rA-rU), a wavenumber downshifting from 788 to 780 cm−1

was observed in the ring-breathing mode of uracil appearing nearthese wavenumbers.[27] This wavenumber shifting is also predictedon the basis of theoretical calculations for uridine residue havingthe C3′-endo/anti and C2′-endo/anti nucleoside structures whichare present in the A and Z nucleic acid forms, respectively.[25]

In addition, in some mononucleotide crystals having the uridinenucleoside C3′-endo/anti structure, the above uracil mode appearsnear 790 cm−1 whereas it is located near 780 cm−1 for crystalshaving the uridine C2′-endo/anti conformation.[28] On the otherhand, in the spectra of the 25-mer HCV RNA a shoulder at789 cm−1 both in H2O and D2O buffer is apparent (Fig. 3(b)). The783 cm−1 band shifts to 776 cm−1 upon deuteration, however the789 cm−1 shoulder remains practically at the same wavenumber.Interestingly, in synthetic polynucleotides the cytosine bandappearing near 785 cm−1 downshifts by about 10 cm−1 upondeuteration, unlike the corresponding pyrimidine ring vibration ofuracil which downshifts by 2 cm−1 at the most.[14] On the abovebasis, the shoulder at 789 cm−1 in the Raman spectrum of thisviral RNA oligonucleotide (Fig. 3(b), Table 1) can be attributableto the uridine residue C3′-endo/anti structure. Moreover, in thisoligonucleotide the mole fraction of cytidine residues is five-fold greater than uridine ones. Consequently, the majority ofthe intensity of the very strong band located at 783 cm−1

can be attributable to cytidine. Resonance Raman spectra ofpoly(rA-rU) and poly(rA)·poly(rU) in aqueous solutions showwavenumber upshifting for a ring vibration of adenine from 718 to724 cm−1 on an anti/syn riboadenosine nucleoside reorientation [A(C3′-endo/anti)→ Z (C3′-endo/syn) conformational transition].[27]

Therefore, the presence of two riboadenosine bands at 724 and719 cm−1 in the Raman spectra of this HCV RNA sequencecould be ascribed to two different nucleoside conformations.However, the lack of nonresonance Raman marker bands thatare unambiguously related to the C3′-endo/syn structure inriboadenosine does not permit exact detection of this nucleosideconformation in the loop.

Conclusions

Raman spectroscopy has permitted in the HCV RNA 25mer(1–25 nucleotides), the detection of some ribonucleosides thatassume unusual C2′-endo/anti and C3′-endo/syn conformations,particularly in uridine and guanosine residues, attributable topositions where chain foldings switch abruptly from helical tolooped. Analysis of the D2O solution Raman spectrum by fittingthe spectral envelope, in the double-bond region, to a sum ofGaussians representing paired and unpaired structures indicatesthat six out of nine guanine residues are Watson–Crick base-paired.The said spectral data suggest that this HCV RNA 25mer sequenceadopts a hairpin form with the structural details described above.

The spectroscopic results are consistent with the hairpinsecondary structure that has been proposed in earlier works for

600 700 800

0.003

0.004

0.005

0.006

0.007

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1295

1302

1258

1242

1230

1384

1315

1321

126012

48

1303

133712

4012

33

1367

1381

Figure 3. Raman spectra of the 25mer HCV RNA in the 1200–1425 cm−1

range (a) and 600–850 cm−1 range (b). Top: in H2O buffer. Bottom: inD2O buffer.

UGA

U3′-C A CA G C G G G G G

5′-G C C A G C C C C C

Figure 4. Schematic secondary structure of the HCV RNA domain I aspredicted from viral phylogenetic comparisons (References [6,31]).

this sequence in the HCV RNA.[4,6] This secondary structure (Fig. 4)has been proposed on the basis of assigning similar structuralcontexts to regions of the HCV 5′NTR which show significantprimary nucleotide sequence identity to other viral sequences.

Acknowledgment

The authors gratefully acknowledge financial support from theSpanish Ministerio de Ciencia e Innovacion (Project CTQ2006-04161/BQU).

References

[1] F. Penin, J. Dubuisson, F. A. Rey, D. Moradpour, J. M. Pawlotsky,Hepatology 2004, 39, 5.

[2] C. Lin, B. D. Lindenbach, B. M. Pragal, D. W. McCourt, C. M. Rice, J.Virol. 1994, 68, 5063.

[3] N. Majeau, V. Gagne, A. Boivin, M. Bolduc, J. A. Majeau, D. Ouellet,D. Leclerc, J. Gen. Virol. 2004, 85, 971.

[4] M. Kunkel, M. Lorinczi, R. Rijnbrand, S. M. Lemon, S. J. Watowich, J.Virol. 2001, 75, 2119.

www.interscience.wiley.com/journal/jrs Copyright c© 2009 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2009, 40, 893–897

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7

Raman spectra of the 25mer HCV RNA

[5] G. J. Thomas Jr, Annu. Rev. Biophys. Biomol. Struct. 1999, 28, 1.[6] E. A. Brown, H. Zhang, L. H. Ping, S. M. Lemon, Nucleic Acids Res.

1992, 20, 5041.[7] L. P. Beales, D. J. Rowlands, A. Holzenburg, RNA 2001, 7, 661.[8] C. M. Spahn, J. S. Kieft, R. A. Grassucci, P. A. Penczek, K. Zhou,

J. A. Doudna, J. Frank, Science 2001, 291, 1959.[9] A. J. Collier, J. Gallego, R. Klinck, P. T. Cole, S. J. Harris, G. P. Harrison,

F. Aboul-Ela, G. Varani, S. Walker, Nat. Struct. Biol. 2002, 9, 375.[10] P. J. Lukavsky, G. A. Otto, A. M. Lancaster, P. Sarnow, J. D. Puglisi,

Nat. Struct. Biol. 2000, 7, 1105.[11] J. S. Kieft, K. Zhou, A. Grech, R. Jubin, J. A. Doudna, Nat. Struct. Biol.

2002, 9, 370.[12] B. J. M. Verduin, B. Prescott, G. J. Thomas Jr, Biochemistry 1984, 23,

4301.[13] G. J. Thomas Jr, A. H. J. Wang, in Nucleic Acids and Molecular Biology,

vol. 2, (Eds: F. Eckstein, D. M. J. Lilley), Springer: Berlin, 1988, p 1.[14] L. Lafleur, J. Rice, G. J. Thomas Jr, Biopolymers 1972, 11, 2423.[15] G. J. Thomas Jr, M. C. Chen, K. A. Hartman, Biochim. Biophys. Acta

1973, 324, 37.[16] G. J. Thomas Jr, B. Prescott, M. G. Halmiton, Biochemistry 1980, 19,

3604.[17] B. Prescott, R. Gamache, J. Livramento, G. J. Thomas Jr, Biopolymers

1974, 13, 1821.[18] C. H. Chou, G. J. Thomas Jr, Biopolymers 1977, 16, 765.[19] N. Leulliot, V. Baumruk, M. Abdelkafi, P. Y. Turpin, A. Namane,

C. Gouyette, T. Huynh-Dinh, M. Ghomi, Nucleic Acids Res. 1999, 27,1398.

[20] K. Morikawa, M. Tsuboi, S. Takehashi, Y. Kyogoku, Y. Mitsui, Y. Iitaka,G. J. Thomas Jr, Biopolymers 1973, 12, 799.

[21] J. M. Benevides, G. J. Thomas Jr, Nucleic Acids Res. 1983, 11, 5747.[22] J. Rice, L. Lafleur, G. C. Medeiros, G. J. Thomas Jr, J. Raman Spectrosc.

1973, 1, 207.[23] J. M. Benevides, S. A. Overman, G. J. Thomas Jr, J. Raman Spectrosc.

2005, 36, 279.[24] M. O. Trulson, P. Cruz, D. Puglisi, I. Tinoco Jr, R. A. Mathies,

Biochemistry 1987, 26, 8624.[25] N. Leulliot, M. Ghomi, H. Jobic, O. Bouloussa, V. Baumruk,

C. Coulombeau, J. Phys. Chem. B 1999, 103, 10934.[26] V. Reipa, G. Niaura, D. H. Atha, RNA 2007, 13, 108.[27] A. Tomkova, L. Chinsky, P. Miskovsky, P. Y. Turpin, J. Mol. Struct.

1994, 318, 65.[28] P. Carmona, M. Molina, R. Escobar, J. Raman Spectrosc. 1991, 22,

559.[29] H. Fabian, S. Bohm, R. Misselwitz, H. Welfle, W. Holzer, W. Carius,

V. V. Filimonov, Int. J. Biol. Macromol. 1987, 9, 349.[30] W. Saenger, Principles of Nucleic Acid Structure, Springer-Verlag: New

York, 1984.[31] M. Honda, L. H. Ping, R. C. A. Rijnbrand, E. Amphlett, B. Clarke,

D. Rowlands, S. M. Lemon, Virology 1996, 222, 31.[32] M. C. Chen, G. J. Thomas Jr, Biopolymers 1974, 13, 615.[33] P. C. Bevilacqua, J. M. Blose, Annu. Rev. Phys. Chem. 2008, 59, 79.[34] M. Abdelkafi, N. Leulliot, V. Baumruk, L. Bednarova, P. Y. Turpin,

A. Namane, C. Gouyette, T. Huynh-Dinh, M. Ghomi, Biochemistry1999, 37, 7878.

J. Raman Spectrosc. 2009, 40, 893–897 Copyright c© 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jrs