ORIGINAL PAPER

Improving real-time measurement of H/D exchange usinga FTIR biospectroscopic probe

Pedro Carmona & Arantxa Rodríguez-Casado &

Marina Molina

Received: 23 September 2008 /Revised: 13 November 2008 /Accepted: 18 November 2008 / Published online: 4 December 2008# Springer-Verlag 2008

Abstract We describe the improvement of a novel ap-proach to investigating hydrogen/deuterium (H/D) ex-change kinetics in biomolecules using transmissioninfrared spectroscopy. The method makes use of a Fouriertransform infrared spectrometer coupled with a micro-dialysis flow cell to determine exchange rates of labilehydrogens. With this cell system, the monitoring ofexchange reactions has been studied here as a function ofsome cell characteristics such as: (a) dialysis membranesurface contacting both the H2O and D2O compartments;(b) molecular cutoff of dialysis membrane; and (c) distancebetween the cell-filling holes. The best improvement hasbeen obtained by increasing the dialysis membrane surfacefollowed by increase of molecular cutoff. However, notsignificant differences were found using various distancesbetween filling holes. The fastest exchange rate which canbe measured with the cell system used here is found to be k=0.41±0.02 min−1, that is, about threefold greater than theone got in a previous work. This microdialysis flow cell hasbeen used here for the study of H/D exchange in nucleic

acids with subsequent structural analysis by 2D correlationspectroscopy.

Keywords Fourier transform infrared spectroscopy . H/Disocopic exchange . Nucleic acids . Proteins

Introduction

The applications of vibrational spectroscopy to elucidatethe structures of biological molecules and their complexeshave continued to increase in number and diversity becausethe knowledge of biomolecular conformations has been oneof the major goals in biochemistry [1–3]. The complexity ofthe various biomolecular structural levels has challengedscientists in search of increasingly powerful analytical tools.The said molecular species such as proteins and nucleic acidscontain many bands arising from side chains of residues andfrom their corresponding backbones. Therefore, their vibra-tional spectra are complex and may be difficult to analyze andinterpret visually. In this connection, the use of 2D correlationanalysis can be useful to some extent. 2D correlationspectroscopy is a cross-correlation technique which is appliedto a previously measured set of perturbation-induced spectraas a function of two independent frequency positions. Thus,this technique generates in-phase and out-of-phase correla-tions between spectral intensity variations occurring atdifferent frequencies that are generated by the application ofan external perturbation of the molecular system in question[4]. This method offers several potential advantages. Firstly,it may simplify complex spectra like a resolution enhance-ment technique. For instance, two overlapping and indistin-guishable bands with different dynamic response maytheoretically be separated. In addition, it may allow theestablishment of band assignments. Indeed, by studying

Anal Bioanal Chem (2009) 393:1289–1295DOI 10.1007/s00216-008-2535-5

P. Carmona (*)Instituto de Estructura de la Materia (CSIC),Serrano 121,28006 Madrid, Spaine-mail: p.carmona@iem.cfmac.csic.es

A. Rodríguez-CasadoIMDEA Alimentación,28049 Madrid, Spain

M. MolinaDepartamento de Química Orgánica I,Escuela Universitaria de Optica,28037 Madrid, Spain

correlations between bands located in different spectralregions, it is possible, if their intensity variations aresynchronously correlated, to assign them to the vibration ofthe same structural element. Finally, this technique can beuseful to identify asynchronisms in a series of spectra. It isthen potentially possible to establish a sequence of eventsduring a physical or chemical process.

When the external perturbation is hydrogen/deuterium(H/D) exchange, the origins of asynchronous changes ofvarious structural elements may be, for example, theirdifferent solvent exposure and hydrogen bond interactionswhere they take part. In addition, combination of principalcomponent analysis (PCA) with 2D correlation spectrosco-py may enhance the spectral resolution [5]. This resultsfrom dividing the spectra into several deuterium exposuretime domains (PCA) and subsequent application of 2Dcorrelation analysis to several spectra in each time domain.

On the above basis, it is expected that, the more changesare resolved, the more detailed structural information canbe obtained. This involves the use of an appropriate probewhich allows measurements of H/D kinetics of the groupsin the studied biomolecules. In a previous work [6], weused a new accessory for real-time transmission infraredmeasurements of H/D exchange. This accessory resultedfrom the combination of two dialysis membranes and aconventional liquid cell having two cylinders containingD2O buffer. It can be used to study biomolecules havinggroups with exchange rate (k) up to 0.13±0.006 min−1. Thedeuteration kinetics of the aqueous solutions inside the cellwith the geometric characteristics used allowed us toresolve isotopic exchange kinetics of the protein secondarystructures for which k is smaller than the aforementionedvalue [7]. The aim of the present work has been to knowthe influence of the geometric characteristics of thisaccessory on the solvent exchange rate constant. In thisconnection, we have considered here the molecular cutoffof the dialysis membranes, magnitude of the dialysismembrane surface on which H/D exchange takes place,and the distance between the cell-filling holes. By using thebest geometric characteristics of the H/D cell, we reportalso the 2D correlation analysis of the structure of anoligonucleotide comprising the 1–25 nucleobase sequencein the viral RNA of the hepatitis C virus (HCV).

Experimental

Materials for Fourier transform infrared spectroscopy

To determine the performance characteristics of the Fouriertransform infrared biospectroscopic cell, we employed D2Obuffer effluent for deuterium exchange of the followingH2O buffer solutions: polycytidylic acid and the 1–25

nucleotide sequence of the HCV RNA. Polycytidylic acidand polyadenylic acid were acquired from Sigma and usedwithout further purification. The purified oligonucleotidewith the 1–25 nucleobase sequence of the HCV RNA wasobtained from Eurogentec S.A. (Belgium) and purified byhigh-performance liquid chromatography and polyacryl-amide gel electrophoresis. The said RNA sequence is 5′-GCCAGCCCCCUGAUGGGGGCGACAC-3′. D2O forH/D isotopic exchange was acquired from Aldrich, and itsminimum isotopic purity was 99.9 at.% D.

The nucleic acid solutions for spectroscopic measure-ments were prepared with 2% w/w concentration at pH 7.4in 50 mM Tris buffer containing 0.1 M NaCl. The solutionswere heated at 85 °C for 5 min then rapidly cooled andstored at 4 °C until spectral measurements.

Regenerated cellulose dialysis membranes from Pierce(SnakeSkin® Dialysis Tubing) were used with 3.5 and10.0 kDa molecular weight cutoff.

Spectroscopic measurements

The characteristics of the basic cell used for spectralmeasurements are described elsewhere [6]. However, inorder to improve the performance of the biospectroscopiccell, we have used here a microinjection syringe pump forcontinuous renewal of the D2O buffer inside the cellcylinders (Fig. 1). On the other hand, various distancesbetween the cell-filling holes and dialysis membranes with

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appropriate molecular cutoffs and with various areas of themembrane surface contacting the buffers were used asdescribed below.

The spectral measurements were carried out as describedin a previous work [6]. Briefly, the spectra were measuredevery minute while the hydrogen exchange progressed.With this aim, a Perkin-Elmer 1725X spectrometer with0.2 cm s−1 scan speed and deuterated triglycine sulfate(DTGS) spectral detector was used, and the spectra resultedfrom accumulation of eight scans at 2 cm−1 resolution overa range of 4,000–700 cm−1. The liquid samples were placedin a cell with ZnSe windows and with optical path length of12 μm. For nucleic acid study, the infrared spectra of buffersamples were recorded under the same conditions as themedia containing the said biomolecules. The spectrum ofthe buffer measured at a deuteration time point wassubtracted from the spectrum of the nucleic acid solutioncorresponding to the same time point of deuteration, so thatthe bands of H2O, DOH, and D2O molecular species areremoved. The resulting difference spectra were subsequent-ly smoothed with a seven-point Savitsky–Golay function toreduce the noise. This and other spectral treatments such asbaseline correction and solvent subtraction were carried outwith the GRAMS/AI software (Thermogalactic).

Results and discussion

The efflux rate of D2O buffer is influenced by the effectivepermeable membrane surface and molecular weight cutoffas described below. The solvent exchange constant in eachcase was obtained after fitting the following equation to theexperimental data:

Θh tð Þ ¼ kt þ 1ð Þe�kt ð1Þwhere Θh(t) is the fraction of unexchanged protium at timet, and k is the rate constant. This equation involves second-order rate treatment of the kinetic process because micro-dialysis diffusion takes place on a relatively long time scale,usually of the order of minutes, and therefore, it isnecessary to consider the said treatment of the kineticprocesses involving the buffer effluent molecules and thebiomolecules. The exchange equation adopts the aboveform if the rate constant of isotopic exchange is the same asthat of the efflux of solvent in the microdialysis cell [6, 8].The spectral measurements have been carried out usingdialysis membranes having 3.5 and 10 kDa molecularweight cutoff. An increase in this membrane parameterresults in greater rate of diffusion and consequent increaseof the exchange rate, and the same can be said for thedialysis membrane surface contacting both isotopicallyexchanging buffers. Thus, when using 3.5 kDa membranecutoff, the rate of solvent exchange (kse, min−1) was found

to be 0.13±0.01 and 0.30±0.01 min−1 for 10 and 100 mm2

membrane surfaces contacting both exchanging buffers,respectively, and 0.19±0.03 and 0.41±0.02 min−1 for10 kDa dialysis membranes with the above contactsurfaces. One would suggest reducing the distance betweenthe cell-filling holes. However, the use here of 31 and27 mm distances did not result in significant differences ofthe exchange rate. As expected, the greatest rate of solventexchange (kse=0.41±0.02 min−1) is found here when usingthe greatest molecular weight cutoff and dialysis membranesurface contacting both isotopically exchanging buffers.The said kse value involves a rate constant improvement ofa 3-fold increase relative to that reached in a previous work[6]. The above greatest kse value represents an upper limit(i.e., fastest exchange rate) which can be measured with thepresent system. One can obtain sufficient time-resolvedspectra with 0.2 cm s−1 scan speed and DTGS spectraldetector used here to monitor the isotopic exchange process.Thus, the said greatest kse value (0.41±0.02 min−1) isdetermined by the time constant of the employed experi-mental cell having 10 kDa cutoff dialysis membrane and100 mm2 buffer contacting surface.

With the aim of investigating exchange rates of labileprotons in nucleic acids, we monitored the shift ofdeuterium-exchange sensitive infrared bands of some poly-ribonucleotides using the experimental system describedhere. Figure 2 shows the infrared spectrum of poly(rC) inaqueous and heavy water buffer. Exchange of the cytosinebase generates significant changes in the 1,700−1,600 cm−1

region, where the main bands at 1,661 and 1,603 cm−1 inaqueous solution shift to 1,656 and 1,617 cm−1, respective-ly, upon deuteration. Therefore, any of them can be used formonitoring the isotopic exchange in Watson–Crick non-

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Improving real-time measurement of H/D exchange 1291

paired cytosine bases. In order to compare and relate thekinetics of deuteration of poly(rC) with the change in H/Dcomposition of the solvent during efflux, it is necessary tomeasure simultaneously the intensity of an isotope-sensitiveinfrared band of the solvent. For this purpose, we haveemployed the integrated intensity of the δD2O bandappearing near 1,210 cm−1. On the basis of normalizedintensities of the cytosine (1,603 cm−1) and solvent bands(1,210 cm−1), we have measured the rate constants of thesaid kinetics. As shown in Fig. 3, the progress ofpyrimidine exchange is nearly as rapid as deuterium efflux,yielding the apparent rates of kse=0.41±0.02 min−1 (sol-vent) and kC=0.39±0.01 min−1 (cytosine base). The latterrate constant is very close to the time-resolution limit of themethod and evidently reflects the rapid exchange ofcytosine protons. This is consistent with the more rapidexchange which has been measured by stop-flow UVspectroscopy on similar model polynucleotide [9]. Thus,exchange measurements by this UV technique on poly(rC)at 25 °C and pH 7 indicate an exchange rate (kC=12.6 s−1)which significantly exceeds the upper limit of detection ofthe present experimental system. Similar results have beenfound here for the kinetics of deuteration of poly(rA), therate constant of which (kA=40.3±0.02 min−1) and that ofthe solvent (kse=0.41±0.02 min−1) are indistinguishablekinetically. Previous studies using stop flow UV spectros-copy showed that the exchange rates for adenine base inpoly(rA) and poly(rA)·poly(rU) duplex were ∼3 min−1 and∼1.5 min−1, respectively, at 20 °C [8, 10]. In the samestudy, the exchange rate constants of uracil base in poly(rU)and poly(rA)·poly(rU) were found to be ∼1,000 min−1 and∼67 min−1, respectively. However, uracils of packaged

RNA and protein subunits of bean pod mottle virus exhibitsignificant strong retardation of the NH exchange (kU=0.18±0.02 min−1) in comparison to solvent or poly(rU) or poly(rA)·poly(rU) [11]. On the other hand, the exchange ratemeasured for guanine residue can vary widely as, forinstance, from 1.2±0.2 min−1 in some double-strandedRNAs to ∼0.16 min−1 in poly(rG)·poly(rC) [8, 12]. Themore rapid guanine exchange in some double-strandedRNAs may reflect the random distribution of G·C base pairswithin the genome RNA in question, which may facilitatetransient base pair openings. The above data suggest that themicrodialysis infrared cell used here can be effective inmonitoring H/D exchange reactions of nucleic acids withrates of k=0.41±0.02 min−1 or less. In practice, this permitsto measure proton exchanges of purine and pyrimidine baseswhen specific protection is afforded, for instance, bysecondary structure as in the case of Z DNA [13, 14],tertiary and quaternary structures as well as nucleic acid–protein interactions [11]. Resolution of exchange kinetics isexpected to result in the appearance of asynchronous peaksin 2D correlation spectroscopy. Thus, the asynchronous map,shown below, of the oligonucleotide sequence of HCV RNAstudied here comprises characteristic peaks of guanineresidues showing relatively slow exchanging. This may bedue to the presence of contiguous G·C base pairs whichhinder transient base pair openings. The spectral analysis ofthis biomolecule is described in the following.

The 1,800–1,500 cm−1 region of the 1-25 sequence ofHCV RNA consists of absorption bands originating fromthe in-plane double-bond vibrations of the bases (Fig. 4).The strong band at 1,666 cm−1 in the spectrum of thesample measured in H2O buffer stems from overlappingcontribution of the νC2=O2 vibration of free and base-paired cytosines and adenines [15–18] and the spectral

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Fig. 3 The time course of protein exchange in poly(rC) solutionmonitored by the relative intensity of the 1,603 cm−1 band of cytosinebase. The solid line (solvent) and the dashed line (polynucleotide)represent the exchange profiles calculated by Eq. 1 with kse=0.41±0.02 and kC=0.39±0.01 min−1, respectively. A sample cell was usedwith the 10 kDa and 100 mm2 membrane parameters

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Fig. 4 Infrared spectra of HCV RNA in aqueous (upper) and heavywater buffer (lower). The sample concentration was 2% w/w in50 mM Tris buffer containing 0.1 M NaCl at pH 7.4

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profile above 1,670 cm−1 can be attributable to overlappedabsorption of guanines (νC6=O6) and uracils (νC4=O4)[15, 17, 19]. The band apparent at 1,601 cm−1 is assigned toring stretching vibration of free cytosine and an overlappingmedium intensity ring band of adenine [20]. This vibra-tional mode upshifts towards higher frequencies upon base-pairing or deuteration, and its intensity decreases upon basestacking [16, 17]. The RNA spectrum in heavy waterprovides an intense band near 1,650 cm−1, which has beenassigned to the νC2=O2 vibration of cytosines plus theνC4=O4 vibration of uracils [15, 17, 21]. The oligonucleo-tide deuteration involves intensity increasing of the band at1,567 cm−1 and its shoulder near 1,575 cm−1 which are dueto the strong C=N ring vibrations of guanine [20]. Theinfrared intensities of these guanine bands have beenreported to increase upon duplex melting [19].

The bands of backbone-sugar vibrations are locatedbetween 1,500 and 750 cm−1. They appear at the expectedfrequencies for the C3′-endo conformation of ribosescharacteristic of A-form geometry [21, 22]. The antisym-metric stretching vibration of the nonbridging PO2

− appearsat 1,240 cm−1 which is indicative of the A-type form havingthe said C3′-endo sugar puckering. The band near1,088 cm−1 in the RNA spectrum in H2O buffer has beenassigned to the νsPO2

− mode coupled with the C5′–O5′vibrations [23]. Several interesting changes were observedupon deuteration in the ribose vibrations falling in thespectral region between 1,100 and 900 cm−1. The vibrationof the ether group of the ribose residue appears at1,062 cm−1 in H2O buffer. On deuteration, the δ2′ODvibration is observed near 1,035 cm−1. Further, one can seethat the 1,062 cm−1 ether vibration is shifted, on couplingwith the δ2′OD vibration, towards higher frequencies andmerges into the absorption band located near 1,075 cm−1.The fact that the νsCOC ether vibration and the δ2′ODvibration couple proves that the 2′OH group is linked withthe neighboring ribose residue via hydrogen bonding,which has been found to occur in double-helical regionsof 23 S RNA and tRNAPhe [24]. The bands at 994 and970 cm−1, which are absent in D2O buffer, have beententatively assigned to a ribose vibrational modes involvingthe 2′OH group and O5′–C5′H groups [25–27]. Thespectral region below 900 cm−1 contains well-knownmarker bands for the sugar conformation. The phospho-diester backbone vibrations coupled to the sugar motionsgenerate the prominent bands at 865 and 809 cm−1 whichreflect predominant C3′-endo conformation of the ribosesinvolved in the RNA A-form [23].

One of the goals of our infrared measurements isenhancing spectral resolution and determining the timesequence of events during H/D exchange by using 2Dcorrelation spectroscopy. In this way, we can either detectthe atomic groups that are hydrogen bonded or know the

extent to which some functional groups are solventexposed. The H/D exchange of this RNA 1–25 sequencewas carried out with a dialysis membrane of 3,500molecular weight cutoff due to the relatively low molecularweight of this oligonucleotide, namely, 8.02 kDa. With theuse of this infrared cell, the exchange kinetics of the freecytosine bases measured through the 1,601 cm−1 band (kC=0.29±0.03 cm−1) is indistinguishable from that of thesolvent (kse=0.30±0.02 min−1; Fig. 5). This result indicatesagain that the measurement of the exchange rate of cytosineprotons in this HCV RNA is limited by the time constant ofthe cell system used here. In addition, this result isconsistent with the far more rapid exchange rates whichhave been measured by stop-flow UV spectroscopy onbase-paired and free cytosine residues [9, 11, 12]. On thisbasis, not unexpectedly, this band varies synchronouslywith part of the intensity of the 970 cm−1 band (Fig. 6)attributable to ribose–phosphate backbone [28], which issolvent exposed in duplex sequences [29]. The sequencesof RNA where the sugar–phosphate backbone is exposedtend to twist the corresponding bases towards the center ofhelical regions, and vice versa [30]. Thus, RNA foldingleads to conformations in which the backbone is buried, butthe bases may be exposed. This can explain the fact that thefree, solvent exposed, cytosine bases generate an asynchro-nous peak at (970, 1,605 cm−1) (Fig. 7) with part of theintensity of the 970 cm−1 band, the intensity change of thisband occurring after the changes located near 1,605 cm−1.The same conclusion can be drawn by considering the994 cm−1 ribose band which is involved in the asynchro-nous peak at (994, 1,605 cm−1).

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Fig. 5 Hydrogen–deuterium exchange of HCV RNA monitored bythe 1,601 cm−1 band which is generated mainly by cytosine base. Thesolid line (solvent) and the dashed line (polynucleotide) represent theexchange profiles calculated by Eq. 1 with kse=0.30±0.01 and kC=0.29±0.03 min−1, respectively. A sample cell was used with the3.5 kDa and 100 mm2 membrane parameters

Improving real-time measurement of H/D exchange 1293

Guanine performs a key role in nucleic acid folding dueto its capacity to form numerous hydrogen bonds andstacking interactions [31, 32]. The secondary and tertiaryforms of RNA molecules used to show hydrophobic groupssuch as guanine bases oriented towards the interior ofhelices [30]. Thus, the protection of some guanine residuesthrough hydrogen bonding and/or their orientation towardsthe interior of RNA molecules makes them to be distin-guished by their slow exchange kinetics, as occurs, forinstance, in base-paired guanine bases. This is consistentwith the asynchronous peaks involving part of the 1,567 cm−1

guanine band and the bands of other bases and ribose–phosphate backbone (Fig. 7). The said peaks suggest that theisotopic exchange in the corresponding guanine bases isslower than those of the other residues involved in the aboveasynchronous peaks, which is probably due to duplexformation involving the sequence of the contiguous guaninebases in this oligonucleotide. This is supported by the factthat the kG value obtained by measuring the 1,567 cm−1 band(0.17±0.02 min−1) is very close to the kG values obtained forthis type of duplexes, as described above (∼0.16 min−1). Inaddition, the formation of the said duplex segment is alsosupported by characteristic Raman bands for this nucleic acidstructure (unpublished results).

Conclusions

We have described a microdialysis sample flow cell for usein conjunction with transmission infrared spectroscopy to

investigate hydrogen-isotope exchange reactions of biomo-lecules such as nucleic acids. The real-time measurement ofthis isotopic exchange can be subsequently analyzed by 2Dcorrelation spectroscopy to get structural information onnucleic acids and proteins. The system requires onlymicrolitervolumes of the initial substrate and perturbing effluentsolutions. The dependence of the solvent efflux rates on someparameters such as the effective permeable membrane surface,the membrane molecular weight cutoff, and the distancebetween the cell-filling holes suggests increasing the first twoparameters with the aim of improving the time resolution ofthe H/D exchange kinetics in various structures and/or groups.We have obtained a solvent exchange or efflux rate of k=0.41±0.02 min−1 with the greatest dialysis membrane surface andmolecular weight cutoff used here, namely, 10 kDa and100 mm2, respectively. H/D-exchange processes with halflives of the order of a few minutes can be resolved using theabove cell, as may occur when specific protection of purineand pyrimidine bases is afforded, for instance, by secondary,tertiary, and quaternary structures as well as by nucleic acid–protein interactions. Concerning proteins, the α-helix, β-sheet, and random coil secondary structures whose exchangerate k values are nearly 10–2, 5×10–4, and 0.5 min−1, res-pectively, can certainly be kinetically distinguished [6, 33]because the first two are lower than the fastest exchange rate(k=0.41±0.02 min−1) which can be measured with the cellsystem used here. Finally, the use of this microdialysis cellhas two advantages: (a) avoiding the use of solid, previouslylyophilized substances, which risk being aggregated (dena-tured) for subsequent measurement in the exchanging solvent,

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and (b) avoiding the sample dilution (and subsequent signalloss) involved in the known stopped-flow method.

Acknowledgments The authors gratefully acknowledge financialsupport from the Spanish Ministerio de Ciencia e Innovación (projectCTQ2006-04161/BQU).

References

1. Dostal L, Chen CY, Wang AHJ, Welfle H (2004) Biochemistry43:9600–9609

2. Jung C (2008) Anal Bioanal Chem 392:1031–10583. Benevides JM, Overman SA, Thomas GJ Jr (2005) J Raman

Spectrosc 36:279–2994. Noda I, Ozaki Y (2004) Two-dimensional correlation spectrosco-

py. Applications in vibrational and optical spectroscopy. Wiley,Chichester, UK

5. Wu Y, Murayama K, Ozaki Y (2001) J Phys Chem B 105:6251–6259

6. Rodríguez-Casado A, Molina M, Carmona P (2006) Anal BioanalChem 385:134–138

7. Raussens V, Ruysschaert JM, Goormaghtigh E (2004) ApplSpectrosc 58:68–82

8. Tuma R, Thomas GJ Jr (1996) Biophys J 71:3454–34669. Nakanishi M, Tsuboi MJ (1978) Mol Biol 124:61–71

10. Mandal C, Kallenbach NR, Englander SW (1979) J Mol Biol135:391–411

11. Li T, Johnson JE, Thomas GJ Jr (1993) Biophys J 65:1963–197212. Teitelbaum H, Englader SW (1975) J Mol Biol 92:55–7813. Markovits J, Ramstein J, Roques BP, Le Pecq JB (1985) Nucleic

Acids Res 13:3773–378814. Basu HS, Shafer RH, Marton LJ (1987) Nucleic Acids Res

15:5873–5886

15. Shimanouchi T, Tsuboi M, Kyogoku Y (1964) Infrared spectra ofnucleic acids and related compounds. In: Duchesne J (ed) Thestructure and properties of biomolecules and biological systems.Advances in Chemical Physics. Interscience, London, pp 435–498

16. Miles HT, Frazier J (1978) Biochemistry 17:2920–292717. Liquier J, Taillandier E, Klinck R, Guittet E, Gouyette C, Huynh-

Dinh T (1995) Nucleic Acids Res 23:1722–172818. Rodríguez-Casado A, Bartolomé J, Carreño V, Molina M,

Carmona P (2006) Biophys Chem 124:73–7919. Sarkar M, Dornberger U, Rozners E, Fritzsche H, Strömberg R,

Gräslund A (1997) Biochemistry 36:15463–1547120. Lindqvist M, Gräslund A (2001) J Mol Biol 314:423–43221. Taillandier E, Liquier J (1992) Methods Enzymol 211:307–33522. Ouali M, Letellier R, Sim JS, Akhebat A, Adnet F, Liquier J,

Taillandier E (1993) J Am Chem Soc 115:4264–427023. Taillandier E, Liquier J, Taboury JA (1985) In: Clark RH, Hester

RE (eds) Advances in infrared and Raman spectroscopy, vol. 12.Wiley-Heyden, New York, pp 65–114

24. Herbeck R, Zundel G (1976) Biochim Biophys Acta 418:52–6225. Liquier J, Akhebat A, Taillandier E, Ceolin F, Huynh-Dinh T,

Igolen J (1991) Spectrochim Acta A 47:177–18626. Letelier R, Ghomi M, Taillandier E (1989) J Biomol Struct Dyn

6:755–76827. Dohy D, Ghomi M, Taillandier E (1989) J Biomol Struct Dyn

6:741–75428. Banyay M, Sarkar M, Gräslund A (2003) Biophys Chem

104:477–48829. Saenger W (1984) Principles of nucleic acid structure, chap. 10.

Springer, New York30. Burrows CJ, Rokita SE (1994) Acc Chem Res 27:295–30131. Chastain M, Tinoco I (1991) Prog Nucleic Acids Res Mol Biol

41:131–17732. Westhof E, Masquida B, Jaenger L (1996) Folding Design 1:R78–

R8933. Raussens V, Ruysschaert JM, Goormaghtigh E (2004) Appl

Spectrosc 58:68–82

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