The Journal of
Physical Chemistry
0 Copyright, 1988, b y the American Chemical Society
VOLUME 92, NUMBER 23 NOVEMBER 17, 1988
LETTERS Raman Study of the Low-Frequency Vibrations of Polynucleotides T. Weidlich* and S. M. Lindsay Physics Department, Arizona State University, Tempe, Arizona 85287 (Received: August 5, 1988)
We have performed low-frequency Raman experiments on several polynucleotides with different sequences, conformations, and number of strands per helix. Below 200 cm-l we have observed three Raman active intrahelical modes in A-DNA and B-DNA. Within one class of structure the modes show little sequence dependence. The bridging hydrogen bonds between the two strands only have a small influence on the frequency of the modes. The modes are strongly dependent on conformation and may therefore be useful as marker bands for structures and intrahelical interactions.
Introduction Low-frequency vibrations of macromolecules involve rigidly bound subgroups, which are connected by weak bonds andfor weak nonbonded interactions. In DNA these large-scale motions occur at frequencies below -380 cm-',' and most experimental and theoretical studies have concentrated on the modes below 120 cm-1.2-23 Among the most interesting modes are soft modes, which
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have been predicted for both A-B and B-Z transition^,'^*^^ and vibrations which stretch the bridging hydrogen bonds between the h e l i ~ e s . ' ~The J ~ latter modes may be important for the melting of the and therefore for replication and transcription.22 Previously, three or four modes (depending on conformation) below 120 cm-' have been observed by Raman spectroscopy.5+6 (12) Weidlich, T.; Lindsay, S. M.; Lee, S.A.; Tao, N. J.; Lewen, G. D.; Peticolas, W. L.; Thomas, G. A. J . Phys. Chem. 1988, 92, 3315. (13) Weidlich, T.; Lindsay, S. M.; Rupprecht, A. Phys. Rev. Lett. 1988,
Van Zandt, L. L.; Saxena, V. K.; Schroll, W. K., to be published. Urabe, H.; Tominaga, Y . J . Phys. SOC.Jpn. 1981,50, 3543. Beetz, Jr., C. P.; Ascarelli, G. Biopolymers 1982, 21, 1569. Tsuboi, M.; Tominaga, Y . ;Urabe, H. J . Chem. Phys. 1983, 78,991. Urabe, H.; Tominaga, Y . ;Kubota, K. J . Chem. Phys. 1983, 78, 5937. (6).Urabe, H.; Hayashi, H.; Tominaga, Y.;Nishimura, Y.; Kubota, K.; Tsuboi, M. J . Chem. Phys. 1985, 82, 531. (7) De Marco, C.; Lindsay, S. M.; Pokorny, M.; Powell, J. W. Biopolymers (1) (2) (3) (4) (5)
1985, 25, 2035. (8) Wittlin, A.; Genzel, A.; Kremer, F.; Haeseler, S.; Poglitsch, A,; Rupprecht, A. Phys. Rev. A 1986, 34, 493. (9) Powell, J. W.; Edwards, G. S.; Genzel, L.; Kremer, F.; Wittlin, A,; Kubasek, W.; Peticolas, W. L. Phys. Rev. A 1987, 35, 3929.
(IO) Urabe, H.; Sugawara, Y . ;Tsukakoshi, M.; Ikegami, A.; Iwasaki, H.; Takahiro, K. Biopolymers 1987, 26, 963. ( 1 1) Lindsay, S. M.; Lee, S. A.; Powell, J. W.; Weidlich, T.; Demarco, C.; Lewen, G. D.; Tao, N. J.; Rupprecht, A. Biopolymers 1988, 27, 1015.
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61, 1674. (14) Eyster, J. M.; Prohofsky, E. W. Biopolymers 1974, 13, 2505 and 2521. (15) Eyster, J. M.; Prohofsky, E. W. Biopolymers 1977, 16, 965. (16) Prohofsky, E. W.; Lu, K. C.; Van Zandt, L. L.; Putnam, B. F. Phys. Lett. A 1979, 70A, 492. (17) Gao, Y . ;Devi-Prasad, K. V.; Prohofsky, E. W. J . Chem. Phys. 1984, 80, 629 1. (18) Kim, Y . ;Devi-Prasad, K. V.; Prohofsky, E. W. Phys. Rev. B: Condens. Mutter 1985, 32, 5185. (19) Kim, Y . ; Prohofsky, E. W. J . Biomol. Struct. Dyn. 1986, 4, 437. (20) Gao, Y.; Prohosfky, E. W. J . Chem. Phys. 1983, 80, 2242. (21) Kim, Y.; Prohofsky, E. W. Phys. Rev. B Condens. Mutter 1986, 33, 5676. ( 2 2 ) Prohofsky, E. W. Phys. Rev. A 1988, 38, 1538. (23) Garcia, A. E.; Soumpasis, D. M., submitted for publication in Proc.
Nutl. Acad. Sci. U.S.A.
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Detailed assignments are still lacking, except for the mode near 25 cm-I, which appears to be an interhelical or lattice m ~ d e . ~ . ’ ~ A mode at 100 cm-I in B-form D N A softens upon heating and disappears at the highest temperatures2 and could therefore be the hydrogen bond stretch mode predicted near those frequen~ i e s . ’ ~ Recently *~’ this mode has also been observed in helically stacked aggregates of guanosine monophosphates.I0 The remaining two modes near 33 and 60 cm-I are intrahelical,6 but no further information has been obtained about these vibrations. Several far-infrared studies on RNA and DNA have been performed, and differences between calf thymus DNA and some polynucleotides have been o b ~ e r v e d . ~Powell * ~ . ~ et al. observed four sharp and temperature-dependent absorption bands in poly(dA).poly(dT) which were quite different from those in other polynucleotide^.^ Very little is known about the sequence and structure dependence of the Raman active modes of DNA and RNA. We present the initial results of a low-frequency Raman study of polynucleotides, homopolymers, and denatured DNA in concentrated solutions (50-150 mg/mL). It is the purpose of this study to exploit the structural polymorphism of polynucleotide^^^ to obtain more information about the nature of the low-frequency vibrations in DNA. A complete account of this study will be published later.
Sample Preparation and Characterization The polynucleotides poly(dA).poly(dT) (lot 640-88), poly(dAdT)*poly(dA-dT) (lot 00782-38), poly(dA-dU)*poly(dA-dU) (lot 005 17930), poly(dA-dC).poly(dG-dT) (lot 00527940), poly(dGdC).poly(dG-dC) (lot PD717910), poly(dG).poly(dC) (lot 00784-84), and poly(r1)-poly(rC) (lot 00324723) were purchased from PL Biochemicals, and poly(rC) (lot 67F-4026), poly(rG).poly(rC) (lot 56F-4021), and poly(rU).poly(rA)pAy(rU) (lot 97F-06661) were obtained from Sigma Chemical Co. All samples were obtained in double-helical form, with the exception of poly(rC) and poly(rU).poly(rA).poly(rU).Poly(dA).poly(dT) and poly(r1)-poly(rC) were dissolved in a buffer solution (10 mM Tris.HC1, 1 mM EDTA, and 200 mM NaC1, pH 7) at a concentration of -200 pg/mL, annealed at 45 OC, alcohol precipitated, washed with 95% ethanol, and dried over silica gel. They were redissolved in the same buffer for a final concentration of 70-1 50 mg/mL. All other polynucleotides were treated exactly the same way, with the exception of the annealing step. Poly(rC) was also dissolved in a slightly acidic buffer (pH 4.2). Calf thymus DNA (Sigma Chemical Co., lot 66F-9645) was dissolved in distilled water, and then brought to pH 13 in order to denature it. The DNA was alcohol precipitated, dried, and redissolved in a pH 13 solution for final concentrations between 40 and 200 mg/mL. The conformation of the samples was confirmed by using the high-frequency Raman spectrum.25 The R N A polynucleotides and poly(dG).poly(dC) were in A-form, and the other DNA sequences were in B-form, except for poly(dA).poly(dT). The most recent X-ray data on this polynucleotide suggest that it belongs the B-DNA family, but that the bases are tilted and the minor groove is significantly n a r r o ~ e d .Poly(rC) ~ ~ ~ ~ ~at pH 7 forms a 6-fold single helix with 3.1 1-8, repeat distance, and the sugar puckers in the C3’-endo form.24 The structure of poly(rC) at pH 4 has not been solved yet, but most likely it forms a parallel double helix of the form poly(rC).poly(rC-H’) (rather than the normal Watson-Crick double helix which has antiparallel strands).24 Poly(rU).poly(rA).poly(rU) forms a triple helix with hydrogen bonding of both Watson-Crick and Hoogsteen types.24 Results for poly(rA)pdy(rU) are not presented here, because they (24) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: Berlin, 1984; and references therein. ( 2 5 ) For a recent review, see, for example: Peticolas, W. L.; Kubasek, W. L.; Thomas, G. A.; Tsuboi, M. In Biological Applications of Raman Spectroscopy: Vol. 1-Raman Spectra and Conformations of Biological Macromolecules; Spiro, T. G., Ed.; Wiley: New York, 1987; pp 81-133. (26) Lipanov, A. A,; Chuprina, V. P. Nucleic Acids Res. 1987, 15, 5 8 3 3 . ( 2 7 ) Park, H.-S.; Arnott, S.; Chandrasekaran, R.; Millane, R. P.; Campagnari. F. J . Mol. Biol. 1987, 197, 5 13.
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R A M A N SHIFT (cm-’) Figure 1. Low-frequency Raman spectra from -250 (Stokes) to +250 cm-l (anti-Stokes) of (a) poly(dA).poly(dT),(b) poly(dA-dT).poly(dAdT), (c) poly(dA-dU).poly(dA-dU),(d) poly(dA-dC).poly(dG-dT), (e) poly(dG-dC).poly(dG-dC), and (0 calf thymus DNA at pH 13. These are spectra of DNA and bound water. The contributions of free water has been subtracted using the 170-cm-I mode of water as normalization. DNA concentrations of 50-1 50 mg/mL were used, and typical laser powers were 70 mW.
were ambiguous, the sample containing an excess of -25% poly(rU).
Experimental Section Raman spectra were taken on a Jobin-Yvon UlOOO Raman double monochromator. The slit width for the low frequency spectra was 150 Hm, giving an instrumental fwhm of 1.5 cm-I. We used between 40 and 120 mW (at sample) of the 5 145-A line of a Spectra Physics 2020 argon ion laser. At these powers we did not observe changes in the transmitted beam pattern, so heating did not occur. All samples were contained in sealed capillary tubes (Kimax-51), and spectra were obtained from a 90’ geometry. Raman scattering of the glass capillaries is several orders of magnitude weaker than scattering from the sample and could therefore be neglected. For concentrations between 50 and 150 mg/mL Raman scattering from water is about 2-10 times stronger than the scattering from DNA, and long averaging times were needed to ensure good spectra. Spectra of the buffer solution were subtracted from the DNA and RNA solutions, using the 170-cm-I line of water for normalization.1° Only the background due to the free water can be subtracted by this method, since the scattering from bound water (the first and, probably, the second hydration shells) differs considerably from that of free water at all frequencies.28 Results In Figure 1 we present Raman spectra from -250 (Stokes) to +250 (anti-Stokes) cm-l of the sequences poly(dA).poly(dT), poly(dA-dT)*poly(dA-dT),poly(dA-dU).pOly(dA-dU), poly(dAdC).poly(dG-dT), poly(dG-dC)-poly(dG-dC), and denatured calf (28) Tao, N. J.; Lindsay, S. M.; Rupprecht, A. Biopolymers, in press.
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R A M A N S H I F T (crn-') Figure 3. Symmetrized low-frequency Raman spectrum of poly(dGdC).poly(dG-dC) (dots) with fit (solid line through spectrum) and individual Raman bands (solid lines below spectrum). A background was added to the 100-cm-' mode to separate the individual lines. The water contributions were approximated by a central Lorentzian. Note that the modes are very broad compared to high-frequency Raman bands.
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R A M A N SHIFT (crn-') Figure 2. Low-frequency Raman spectra from -250 (Stokes) to +250 cm-l (anti-Stokes) of (a) poly(dG).ply(dC), (b) poly(rG).poly(rC),(c) poly(rI).poly(rC), (4 poly(ru).poly(rA).poly(rU), (e) poly(rC) at PH 4.2, and (f) poly(rC) at pH 7.5. Other parameters as in Figure 1.
thymus DNA. All have the free water background subtracted. Raman spectra of poly(dG)-poly(dC) and the five RNA sequences poly(rG).poly(rC), pol~(rI).pol~(rC), poly(rU).poly(rA).poly(rU), poly(&) at pH 4.2, and poly(rC) at pH 7.5 are shown in Figure 2. We summarize the results of these spectra: 1. The 25-cm-' interhelical is not observed at these high water contents in any samples. 2. The Raman spectra of the four B-DNA sequences (Figure lb-e) are very similar to each other and to spectra obtained from calf thymus D N A and a B-DNA oligonucleotide crystal.12 Nonlinear least-squares fits of B-DNA spectra taken at lower humidities yield excellent fits to the spectra (x2z 1.2) if three intrahelical modes plus the interhelical mode are used.29 We have used this three-mode model to fit the polynucleotide spectra, approximating the Raman spectrum of bound water by a single central Lorentzian. The spectral fit to the poly(dG-dC)-poly(dG-dC) spectrum plus the individual lines are shown in Figure 3. Fits with fewer modes do not give reasonable results, but a larger number of modes cannot be excluded. The three Raman bands obtained from the fit have peak positions near 95,63, and 32 cm-l, and the lines are broad compared to high-frequency Raman active modes. Although the average number of bridging hydrogen bonds per base pair varies between 2.0 and 3.0 for the different polynucleotides, we observe only small differences in the Raman spectra, and the Raman shifts of the modes vary by less than 3 cm-' in the three-mode model. 3. In A-form DNA and R N A (Figure 2a-c) we observe modes near 31, 70, and 117 cm-'. The mode near 70 cm-' appears to be a doublet separated by 12 cm-' but cannot be resolved in all spectra (we will therefore refer to it as the 70-cm-' mode). The Raman line near 3 1 cm-I is more intense than the Raman line at the same frequency in B-form DNA. The modes are only slightly sequence dependent.
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(29) Weidlich, T.; Lindsay, S. M.; Rupprecht, A., to be published.
4. The DNA sequence poly(dA)-poly(dT) (Figure l a ) has Raman lines near 32, 71, and 95 cm-'. This Raman spectrum is different from typical Raman spectra of both A- and B-form DNA, in good agreement with recent far-infrared r e s ~ l t s . ~ 5. The triple helix poly(rU).poly(rA)-poly(rU) (Figure 2d) has four intrahelical vibrations with frequencies 29, 56, 79, and 123 cm-I. The bands near 56 and 79 cm-' are somewhat sharper than the 70-cm-' mode in the A-form polynucleotides. 6. The spectra obtained from poly(rC) at pH 7.5 (Figure 2f) and denatured DNA (Figure If) differ considerably from spectra of double-helical DNA. There is a Raman background from ca. 30 to >lo0 cm-' which is not due to scattering from water or background due to elastically scattered light. Observations on fibers of single-stranded DNAz9suggest that the 95-cm-I mode in B-DNA has softened and broadened, but unique peak positions could not be obtained from fits to these spectra. Clearly, the Raman active modes do not disappear upon denaturation, as previously reported.z 7. Poly(rC) at pH 4.2 (Figure 2e) has a Raman active mode near 147 cm-' and a strong unresolved central background. Discussion The frequencies of the modes vary strongly with structure but are nearly independent of the sequence within one class of structures. Several lattice dynamics calculations have predicted the influence of hydrogen bonding on the vibrations of DNA. The model of Powell et al. has predicted that modes above -50 cm-' should be split into Davydov pairs separated by a few cm-' due to the bridging hydrogen bond interactions between the two helices? One would therefore expect that changing the average number of hydrogen bonds ((nH)) would have a similarly small influence. Prohofsky and co-workers have predicted that even hydrogen bond stretching modes should be nearly independent of ( nH).'' One would expect that other modes would be influenced to a lesser degree. We observe that, within one structure, the modes between 30 and 120 cm-' are constant to within a few cm-I and do not depend strongly on (nH), which is in good agreement with predictions of both models. Eyster and Prohofsky have performed lattice dynamics calculations on poly(rU), poly(rA), and their double helical complex poly(rA).poly(rU).14 They have shown that the formation of the double helix has a relatively small influence on the phonon spectrum below 450 cm-', shifting vibrations by not more than 10 cm-' compared to the phonon spectrum for two single helices. Their calculations are difficult to confirm, since both poly(rA) and poly(rU) have solution structures which differ considerably from the 1 1-fold single helices used in their calculations.24 We have used these predictions to explain the results of the triple-
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helical RNA poly(rU).poly(rA).poly(rU), since its structure resembles a regular A-form double helix with an extra A-form strand inserted in the major groove (some small structural change is involved, mainly in the b a ~ k b o n d ) . The ~ ~ 70-cm-l mode observed in all A-form double helices splits into two narrower modes, and the other two modes (near 30 and 120 cm-]) shift by just a few cm-’ compared to double-helical-RNA sequences. These changes and splittings are similar to those observed by Eyster and Prohofsky which suggests that stacking of bases belonging to different strands and the formation of hydrogen bonds have a small influence on the phonon spectrum The observed modes are therefore likely to occur also in 1 1-fold single-helical DNA. The differences in the spectra are most likely due to differences in the secondary structures of the molecules. Poly(dA).poly(dT) belongs to the B-form family but shows some distinct features, e.g., a hydration spine and a much narrower minor groove compared with “normal” B-DNA.26,27 Indeed, the low-frequency Raman spectrum of poly(dA).poly(dT) is anomalous compared with the other B-form spectra. This is not due to the high AT content since the spectrum of poly(dA-dT).poly(dA-dT) is like that of any other B-form double helix. The 63-cm-] mode in the B-form polynucleotides shifts to about 70 cm-’ and becomes considerably more intense whereas the other modes do not shift considerably compared to spectra of “normal” B-DNA. This effect cannot be explained with the narrowing of the minor groove alone since C-DNA spectra are nearly identical with those of B-DNA,29 although the minor groove has n a r r ~ w e d . ~ ’ Another example for the influence of the secondary DNA structure on the low-frequency vibrations is poly(rC). At pH 7 poly(rC) forms a single helix with 6 bases per turn, whereas at pH 4 it forms a parallel double helix (probably with 12 base pairs per turn). The most prominent mode in the double-helical form is at 147 cm-I, whereas in the single-helical form it is well below 100 cm-I. These differences are not due to the formation of hydrogen bonds (see above) and must therefore represent the changes in RNA conformation. These observations can, so far, not be related to specific interactions within the DNA, or to “simple” motions of the single or the double helix. Torsional modes, for example, should be
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(30) Arnott, S.; Bond, P. J.; Selsing, E.; Smitch, P. J. C. Nucleic Acids Res. 1976, 3, 2459.
observed at lower frequencies4 and do not have the right sequence dependence31to explain any of the observed modes. Recent results by Urabe et a1.I0 indicate that base stacking plays an important role in the “lOO-cm-l mode” of DNA (95 cm-’ in B-form, 117 cm-’ in A-form), whereas the backbone should only play a minor role. Base stacking energies depend very strongly on the AT and GC content, whereas the 100-cm-’ mode only shows a very small dependence on the sequence. Also, the most prominent mode in the poly(rC).poly(rC-H+) appears at 147 cm-I, compared with 95-120 cm-I in DNA and stacks of guanosine and monop h o s p h a t e ~ . ~These ~ * ~ ~results suggest that base stacking interactions only have a minor influence on the frequency, but a large influence on the intensity of this mode. The relatively large intensity of this mode suggests that it might be a libration of the bases.
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Conclusions We have performed low-frequency Raman spectroscopy on concentrated solutions of polynucleotides with different sequences. The Raman spectra depend very strongly on secondary structure, but, within one conformation, the modes depend only slightly on sequence. The bridging hydrogen bonds between the strands has a small influence on the modes. The observations on poly(dA).poly(dT) show that even relatively small changes in the secondary structure can lead to relatively large frequency and intensity changes in some modes. A study of the low-frequency vibrations of other poly- and oligonucleotides with well-understood structures should lead to a better understanding of the intrahelical interactions of DNA and RNA. Acknowledgment. We thank Prof. W. L. Peticolas, Prof. E. W. Prohofsky, Prof. J. W. Powell, Prof. S. A. Lee, Dr. A. E. Garcia, Dr. G. A. Thomas, Prof. R. M. Wartell, and Prof. L. L. Van Zandt for helpful suggestions, discussions, and sharing of unpublished results. This work was supported in part by O N R Contract No. NOOO14-87-K-0478 and N S F Grant BBS 8615653. T.W. gratefully acknowledges an Arizona Board of Regents graduate scholarship. (31) Millar, D. P.;Robbins, R. J.; Zewail, A. H. J . Chem. Phys. 1981, 74, 4200. (32) Nielsen, 0 . F.; Lund, P. A,; Peterson, S. B. J. Am. Chem. Soc. 1982, 104. 1991.
Critical Slowing Down in Periodically Perturbed Chemical Oscillations G . Dewel*,+and P. Borckmanst Service de Chimie-Physique, CP 231 -Campus Plaine, UniversitP Libre de Bruxelles, B- 1050 Bruxelles, Belgium (Received: May 2, 1988; In Final Form: August 17, 1988)
We analyze recent experimentson the nonlinear relaxation (critical slowing down, phase slippage, frequency pulling) of chemical oscillators subjected to low-amplitude forcing in the vicinity of the transition from entrained behavior to quasi-periodic motion using a simple model. The phase slippage staircase is reinterpreted in terms of a plateau behavior analogous to that occurring near the hysteresis limits
Periodic perturbations of an oscillator system of the limit cycle type, with frequency wo, can give rise to various dynamical behaviors.lI2 According to the value of the perturbation amplitude ( e ) , frequency (w&, and initial conditions, the response approaches a periodic, quasi-periodic, or chaotic trajectory. When the amplitude is weak, only two kinds of situations are known to emerge. ‘Research Associates at the Belgium National Science Foundation (F. N.R.S.).
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(1) Entrainment occurs around special frequencies satisfying wp/wo = R, where R is a rational number. In particular, the band corresponding to R = 1 is called the fundamental entrainment band. (2) Outside these bands, the response produces quasi-periodic variations of the state variables. ( I ) Rehmus, P.; Ross, J. In Oscillafions and Trauelling Waues; Field, R. J., Burger, M., Eds.; Wiley: New York, 1985; Chapter 9, p 287. ( 2 ) Schneider, F. W. Ann. Reo. Chem. 1985, 36, 347.
0 1988 American Chemical Society
