The temperature dependence of Raman intensities of DNA

155

Biochimica et Biophysica Acta, 361 (1974) 155--165 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98078 T H E T E M P E R A T U R E DEPENDENCE OF RAMAN IN T E N SIT IE S OF DNA EVIDENCE F O R P R E M E L T I N G CHANGES AND C O R R E L A T I O N S WITH U L T R A V I O L E T SPECTRA

LAJOS RIMAIa, VERONICA M. MAHERb, DAVID GILLa, IRVING SALMEENa and J. JUSTIN McCORMICKb aThe Ford Motor Company Scientific Laboratories, Dearborn, Mich. 48126 and bDivision of Biological Sciences, Michigan Cancer Foundation, Detroit, Mich. 48201 (U.S.A.)

(Received March 4th, 1974)

Summary A detailed study was made of the t e m p e r a t u r e dependence of the intensities o f certain bands in the Raman spectra of double-stranded calf t h y m u s DNA. The t e m p e r a t u r e d e p e n d e n t Raman bands were assigned to structural c o m p o n e n t s of the macromolecule by comparison with the behavior of temperature d e p e n d e n t intensities of simple model polymers and nucleotides. In addition to changes occurring near the melting temperature, complex changes were observed in Raman spectra at temperatures between 40 and 55°C, well below the melting t e m p e r a t u r e of this DNA. To determine the correlation between these Raman spectral changes and ultraviolet absorption changes in the pre-melting t e m p e r a t u r e range, we studied the t e m p e r a t u r e dependence o f ultraviolet difference spectra for calf t h y m u s DNA. Using a simple model we can a c c ount for the variety of Raman temperature dependences and qualitatively correlate these with the t e m p e r a t u r e dependences observed in the ultraviolet spectra. These new findings were obtained at a t e m p e r a t u r e range sufficiently low to be of significance for the study of DNA in vitro.

Introduction Base pair ratios and the secondary structure of double stranded nucleic acids are manifest in the t e m p e r a t u r e d e p e n d e n c e of the intensities of certain ultraviolet absorption bands [1,2]. The same factors also affect a n u m b e r of infra-red active vibrational modes. These infrared modes can be assigned to individual functional groups (e.g., specific bases or phosphodiester bonds) and systematic infrared studies on model systems can be interpreted in terms of

156 specific molecular arrangements [3]. The Raman scattering cross-section of a molecule involves directly both the electronic excited states (including those of the structure-sensitive ultraviolet absorption bands) and the vibrational states as in the infrared spectra [4]. Recent work by others,on the Raman spectra of model polynucleotides, RNAs, and calf t h y m u s DNA indicates a number of vibrational bands specific to bases and the phosphodiester group [5--20]. Studies of the temperature dependence of several Raman band intensities in double-stranded model polymers [5--7] indicate that at least a qualitative correlation exists between these temperature dependences and those observed in the ultraviolet spectrum. Furthermore, the temperature dependence of the optical rotatory dispersion and circular dichroism in the ultraviolet--visible region [21,22] and more recent work on the temperature-difference ultraviolet absorption spectra of T-phage DNA [23], reveal structure in the temperature profiles below the region of cooperative melting region (T m ). These changes in the spectra have a parallel in the pre-melting behavior of the viscosity of DNA solutions [ 24]. The present work was undertaken to investigate the temperature dependence of certain Raman intensities of a naturally occurring DNA polymer (calf thymus) in order to determine correlations which may exist between the temperature dependences of the Raman spectra and those of other optical properties of DNA, in particular, the ultraviolet absorbance. Special emphasis was given to changes occurring in the pre-melting temperature range. In addition to Raman spectra we have studied the temperature difference ultraviolet spectra of calf thymus DNA and found that, as reported for T-phage DNA [23], this t o o exhibits spectral changes in the pre-melting temperature range. We discuss the results in terms of a simple model which qualitatively can explain the variety of ultraviolet temperature difference profiles and Raman temperature profiles. This model assumes that two different mechanisms cause the temperature dependent conformational changes in DNA. One, (the cooperative base-stacking interaction), directly affects the electronic states of long segments of the DNA polymer. If acting alone, this mechanism would cause all the spectral phenomena to change abruptly at temperatures near the melting temperature. The other mechanism, (probably associated with hydrogen bonding), directly affects the vibrational states. This effect is localized and therefore, would not show the steep temperature dependence so characteristic of cooperative phenomena. Experimental procedure Type I calf thymus DNA (Sigma) was purified according to Marmur [25] with the addition of phenol for final deproteinization. The final traces of phenol were removed by 3--5 extractions with ethyl ether and final traces of ether removed by gentle bubbling of nitrogen. To minimize spectral interference caused by citrate, the DNA was dissolved at 10 to 30 mg/ml in 0.015 M NaC1 and 0.0015 M sodium citrate in H 2 0 or 2 H 2 0 pH 7.1--7.3. To prepare such highly concentrated samples, the DNA was sheared to approx. 106 mol. wt by repeated passage through a 27 gauge needle.

157 For the Raman experiments a short section of 1 mm internal diameter glass capillary was filled with approx. 1 to 2 pl of the DNA solution (total DNA approx. 50 pg for the more concentrated samples). Spectra were obtained from the samples with incident laser power approx. 100 mW using excitation wavelengths of 488.0 and 514.5 nm with accumulation of 4 to 12 scans at speeds of 1 to 5 cm -~ • s-1 in the memory of a multi-channel analyser. A 5 cm -~ spectral slit width was used. The laser beam was focused onto the sample perpendicular to the axis of the capillary and collected at a 90 ° angle by the spectrometerphoton-counting system described previously [26]. The capillary was inserted into a copper tube around which heating coils were wound, and the temperature sensed by a thermocouple inserted into one end of the tube. The copper tube had slots for illumination and light collection; the section of the capillary containing the sample was brought into view through these slots. The temperature accuracy and stability were within + I°C which was more than adequate to demonstrate the major effects in this work. The ultraviolet absorption measurements were performed on samples (50 to 150 pg DNA/ml), in 1 cm path cells, using a Cary 14 spectrophotometer. The temperatures of both sample and reference cells were electronically regulated to + 0.2 ° C by controlling the current through heating coils outside of the cells. The sample temperatures were sensed by calibrated "Thermilinear" thermistor probes (Yellow Springs Lab., Yellow Springs, Ohio). For difference spectra, both cells contained an aliquot of the same DNA solution, the temperature of the reference cell was 30°C, and spectra were taken with the 0.1 absorbance full scale sensitivity at a very slow scan rate so as to average the noise. The spectrum obtained when the two cells were at 30°C had a small slope to it that could n o t be eliminated by electronic balancing. This was substracted from the difference spectra before they were replotted point by point. Results

Raman spectra of DNA at different temperatures Fig. 1 shows Raman spectra of calf t h y m u s DNA in the 650--850 and 1450--1850 cm -1 ranges at 6 different temperatures. The dashed line indicates the 1637 cm -1 bending vibrational band of water and the weak line at 1730 cm -~ originates from citrate in the solvent. R o o m temperature Raman spectra of calf t h y m u s DNA have been reported previously and a number of the major bands assigned to specific base vibrations or to the phosphodiester backbone [5,6,20] by comparing band frequencies with those from simple model polymers such as poly[d(A--T)], poly(A) • poly(U), etc., and ribonucleotides [5,6, 20,27]. The spectra at 25°C shown in Fig. 1 are similar to those reported by Small and Peticolas [5,6] and Ehrfurth et al. [20]. However, because of the differences in the recording conditions (e.g. an expanded scale over a narrow frequency range and the ability to accumulate repetitive scans) detailed structure is now evident in certain bands. This structural detail was confirmed and analyzed by studying the temperature dependence of the intensity of these bands. For example, the shape of the strongest band (around 800 cm -1) is markedly temperature dependent. This can be explained by assuming this to be

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Fig. 1. R a m a n s p e c t r a o f c a l f t h y m u s D N A d i s s o l v e d i n 0 . 0 1 5 M N a C I , 0 . 0 0 1 5 M s o d i u m c i t r a t e , 5 1 4 . 5 n m e x c i t a t i o n , at the i n d i c a t e d t e m p e r a t u r e s in t w o f r e q u e n c y r e g i o n s : A b o v e , f r o m 6 5 0 to 8 5 0 c m -1 S t o k e s s h i f t s a t 1 0 m g / m l ; B e l o w f r o m 1 4 5 0 t o 1 8 5 0 c m -1 S t o k e s s h i f t s a t 3 0 m g / m l . - . . . . . , H 2 O b e n d i n g b a n d p r o f i l e . T h e w e a k l i n e a t 1 7 4 0 c m -1 o r i g i n a t e s f r o m c i t r a t e .

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Fig. 2. C a l i b r a t i o n o f t h e i n t e n s i t y o f t h e b e n d i n g m o d e o f H 2 0 a t 1 6 3 7 c m -1 r e l a t i v e t o t h e i n t e n s i t y o f t h e 9 8 0 c m -1 s y m m e t r i c s t r e t c h i n g m o d e o f S O 4 2 - . T h e s u l f a t e is a n i n t e r n a l s t a n d a r d , d i s s o l v e d as ( N H 4 ) 2 S O 4 in water. The trace s h o w s the s u l f a t e line (left) a n d the w a t e r line (right) at t w o t e m p e r a tures, excited at 514.5 nm. The insert shows the temperature profile of the ratio of peak intensities (no change of line w i d t h ) e x c i t e d at 4 8 8 . 0 n m and 5 1 4 . 5 n m .

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Fig. 3. T e m p e r a t u r e d e p e n d e n c e of t h e r e l a t i v e i n t e n s i t i e s o f several R a m a n lines in calf t h y m u s D N A dissolved in 0 . 0 1 5 M NaCl, 0 . 0 0 1 5 M s o d i u m citrate. T h e R a m a n i n t e n s i t i e s w e r e m e a s u r e d r e l a t i v e to t h e i n t e n s i t y of t h e 1 6 3 7 c m -1 w a t e r b e n d i n g line.

a multi-band complex with the various components having different temperature dependences. By locating the frequencies within the 800 cm -~ region at which we obtain the largest temperature dependent intensity change, we can estimate the most probable frequencies of the contributing vibrations. For Raman intensity measurements, as a function of some parameter (e.g. temperature), an internal reference is necessary. It is well accepted that the SO42- symmetric stretching vibrational line of (NH4)2 SO4 is temperature independent and can serve as an internal reference [11]. We have found that the intensity of the 1637 cm -~ water band, relative to that of the SO42-, is constant (see Fig. 2). Therefore, the 1637 cm -1 Raman line of water (and accordingly the 1207 cm -1 line of 2 H 2 0 ) i s also a satisfactory intensity standard for temperature measurements [28]. Using either line as our internal standard, we have determined the temperature dependence of the intensities of a number of Raman bands in DNA. These are illustrated in Fig. 3. Similar measurements of the temperature dependence of equivalent concentrations of nucleotides failed to show the significant marked temperature dependence observed for DNA indicating that the temperature dependence of the intensities of the Raman spectrum of DNA is an intrinsic property of the polymer in agreement with findings by Small and Peticolas [5,6]. Table I lists assignments for the DNA lines in the regions of interest. Many of these assignments were taken from published data. However, in some instances, viz., the 665--680 cm -1 regions, the 778--800 cm -~ complex, and the 1674 cm -1 band, our assignments in Table I differ from published ones. This is because, in our analysis, we made use of data we obtained from the Raman spectra of thymidine monophosphate and also of a comparison between the temperature dependences of the Raman intensities of calf t h y m u s DNA (present work) and those of poly[d(A--T)] given by Small and Peticolas [5,6]. It was particularly useful to obtain detailed spectra for thymidine since the presence of methyl group causes alterations in the spectral contributions from the pyrimidine ring vibrations and from the associated carbonyl groups [27].

[1.5]

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1670

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D a t a o b t a i n e d in p r e s e n t i n v e s t i g a t i o n . F o r T M P , w e also f i n d t h e f o l l o w i n g s t r o n g b a n d s : 1 2 4 0 [ 3 ] , 1 3 8 0 [ 6 ] a n d t h e P O 2- s t r e t c h a t 9 8 0 c m -1. D a t a t a k e n f r o m r e f . 6. In a d d i t i o n to t h e P O 2- s t r e t c h , 1 0 9 5 c m -1, t h e r e a r e a n u m b e r o f t e m p e r a t u r e - i n d e p e n d e n t lines b e t w e e n 1 2 0 0 a n d 1 4 0 0 c m -1. These references contain certain results which were pertinent to the assignment of the particular DNA band(s).

[1]

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AND DNA RAMAN

Frequency ( c m -1 )

POLY[d(A--T)]

Poly[d(A--T)] c

MONOPHOSPHATE,

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THYMIDINE

TABLE I

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a,6 5,6,8,27 5,6,7,26,27 a,6

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Table I lists the frequencies and relative intensities of lines we found in the spectrum of TMP (Column 1) as well as those reported [5,6] for poly[d(A--T)] at two different temperatures (Columns 2 and 3}. From the comparison of the frequencies and the relative intensity distributions of the spectra of poly[d(A--T)] and DNA, especially at high temperatures (where additivity of c o m p o n e n t bases can be considered to occur), with those found for TMP, we assign the 665--670 and 750 cm -1 bands to t h y m i n e and the strong complex 790 cm -1 band principally to the phosphate backbone of DNA. It is also concluded that the 1674 cm -1 band has a major contribution from the t h y m i n e carbonyls because the intensity of the carbonyl bands in the free guanine and cytosine nucleotides and their polynucleotides is an order of magnitude weaker than t h y m i n e or uracil or polynucleotides containing these bases [16]. A comparison of the temperature dependence of the spectra of poly[d(A--T)] with that of DNA in this region supports such a conclusion [5,6]. Such an assignment is further supported by the Raman spectra of calf t h y m u s DNA in 2 H 2 0 shown in Fig. 4 in which the 1674 cm -~ carbonyl stretching vibrational band is clearly seen to be strong at room temperature but markedly decreased in intensity at 46.1°C. The data of Fig. 3 illustrate four categories of Raman lines based on their temperature dependence: (A) temperature-increasing lines, e.g., the 730 cm -~ adenine line and the 798 cm -~ shoulder, which show a sharp intensity increase in the region of DNA melting (Tm ~ 73°C as determined by the 260 nm melting curve, Fig. 6, see also ref. 29); (B) temperature-decreasing lines which show a decreasing temperature dependence, distributed over a wide range, and have intensities which go to zero in the high temperature region, e.g., the t h y m i n e line at 750 cm -~ and the 1674 cm -~ carbonyl stretch; (C) lines with a n o n m o n o t o n i c (zigzag) temperature dependence, but which show a sharp increase somewhere in the melting region, e.g., 778 cm -~ and 835 cm-Z; and (D) temperature-independent lines such as the 790 cm -~ band. (There are other temperature-independent lines in the DNA spectra; see F o o t n o t e d. Table I).

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Fig. 5. T h e n e a r u l t r a v i o l e t t e m p e r a t u r e difference spectra of a calf thymus DNA solution approx. 65 Pg/ml in 0.015 M NaCl, 0.0015 M sodium citrate obtained with a Gary 14 using an 0.1 full scale absorbance sensitivity and extremely slow scans to average out the noise. The number pair associated with each trace indicates the sample temperature and reference temperature (30°C). No attempt was made to correct for thermal expansion of the solution, even though this would slightly enhance the differences in absorbance, because these were clearly indicated by direct measurement.

The correlation of these varied temperature profiles with the ultraviolet results is presented in the next sections.

Ultraviolet temperature-difference spectra The fact that the Raman spectra of DNA change extensively in the temperature region below that of the cooperative melting (the pre-melting region) suggested that identical physical changes might be responsible for both the Raman spectral changes in this region and the ultraviolet pre-melting changes [23]. In order to examine this possibility, a study was made of the temperature dependence of the ultraviolet temperature difference spectrum of calf t h y m u s DNA in the pre-melting region at a number of temperatures (see Fig. 5) using 30°C as the reference temperature. Fig. 6 shows the temperature dependence of the absorbance difference at 293, 274 and 260 and 250 nm taken from the spectra shown in Fig. 5. The complete 260 nm melting curve of this sample is also shown for comparison. The results are qualitatively similar to those obtained for a number of T-phage DNAs [23]. In the pre-melting region below 50°C, there are two positive bands at 293 and 274 nm and one negative band at 250 rim. As the temperature is increased above 50°C, and the cooperative melting region approached, we observe the increased absorption of the 260 nm band, whose increase in intensity is responsible for the apparent disappearance of the other three bands. The data in Fig. 5 indicate that the changes in the intensities of the 274 and 293 nm bands tend to level off between 50 and 55°C. This is clearly shown by the corresponding temperature profiles plotted in Fig. 6. The negative band at 250 nm indicates a ultraviolet transition at this wavelength which markedly decreases in intensity when the temperature is

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Fig. 6. Temperature dependence of the absorbance difference at 4 different wave lengths in the near range ultraviolet taken from the spectra shown in Fig. 5. Left: plot of absorbance difference referred to a sample at the reference temperature, 30 ° C; Right: the c o m p l e t e melting curve at 260 nm.

increased from 30 to 50°C. The subsequent increase of absorbance above 50°C is due to the rapid increase of the 260 nm band with the onset of cooperative melting (see Fig. 6). Thus, these premelting difference spectra exhibit at least 3 ultraviolet bands with temperature dependences radically different from that of the 260 nm band. The present data and those reported by Sarocchi and Guschlbauer [23] show that the 260 nm band is insensitive to temperature changes in the pre-melting region. Discussion The fact that there are ultraviolet absorption bands with both increasing and decreasing temperature dependences in the pre-melting region is important for the interpretation of the Raman temperature profiles. The total Raman scattering cross-section can be expressed as the square of a sum of terms, each having the form of an absorption intensity involving a given vibrationalelectronic excited state [4,30,31]. If the only possible temperature dependence for an absorption intensity resembled that of the 260 nm band, all temperature dependent Raman intensities would exhibit a sharp increase around Tin, and be constant below Tm. That this is not the case is clearly shown by the data of Fig. 3. These data tell us that the sum used to calculate certain Raman intensities must contain terms with temperature dependences in the pre-melting region which are hyperchromic and/or hypochromic. Therefore, when a sum is squared, the result (to which the Raman intensity is proportional) may contain temperature-increasing, temperature-decreasing, or even zig-zag components (cf. the temperature dependence of bands 778 or 835 cm -1 in Fig. 3). Even though the amplitude of such premelting changes in the ultraviolet absorption

164 spectrum is only approximately 1% of the total 260 nm absorption change observed upon full melting, such "pre-melting" terms may well dominate the Raman intensity for certain bands. Because either the initial or the final molecular state in a Raman scattering process involves some vibrational excitation, the selection rules for the terms in the sum for this process are different from those for absorption bands. Furthermore, the Raman intensities increase rapidly with the length of homopolymeric segments containing the base which contributes to the given Raman line [33]. Thus, even if these segments make up only a small fraction of the polymer, their contribution to the Raman spectrum could be large, while that to the ultraviolet spectrum relatively small. The presence of the non-cooperative (localized) pre-melting changes in these spectra can be qualitatively explained using the following simple model: The analysis of Raman or infrared vibrational spectra of nucleic acids shows that the vibrational bands arise mainly from excitation localized in small molecular regions (e.g., a C=O bond or a PO42- group). Thus, they should be especially sensitive to localized (non-cooperative) structural changes such as breakage of hydrogen bonds. On the other hand, the interpretation of the 260 nm melting curve in terms of cooperative base stacking interactions implies that the electronic states of the molecule extend over a long range making them especially sensitive to cooperative interactions. Since each absorption band intensity (other than a O--O transition) involves both an electronic and a vibrational factor, and also since every term in the Raman intensity must involve a vibrational factor, this model is consistent with the general character of the experimental data we have obtained if one assumes that the premelting changes in the Raman reflect the temperature dependence of the vibrational factors in the Raman intensities, and also that the premelting changes observed for the absorption intensities of the 250, 274, and 293 nm bands reflect the presence of a vibrational factor. As a consequence, we are led to interpret the latter 3 bands as corresponding to vibro-electronic transitions rather than O--O. Conclusion In this study of the temperature dependence of certain Raman intensities in calf t h y m u s DNA, we have found a variety of profiles and evidence for pre-melting structural changes in the polymer. We have experimentally correlated this evidence with data obtained from a study of the temperature dependence of ultraviolet difference spectra and proposed a simple model which, by incorporating both localized and distributed features, qualitatively can explain the observed temperature dependences of Raman and ultraviolet spectral bands within both the premelting and melting temperature ranges.

Acknowledgements The excellent technical assistance of Rudolf Kilponen, Mary Ellen Heyde and Deborah Douville are gratefully acknowledged. This investigation was supported by National Institutes of Health USPH Contract No 1-CP-33226, Grant CA 13058 and by an Institutional Grant to the Michigan Cancer Foundation from the United Foundation of Greater Detroit.

165

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