SynopsisRaman spectra of DNA from calf thymus DNA have been taken over a wide range of temperatures (25'-9.5') in both D20 and H,O. A study of the temperature dependence of the Raman spectra shows that the temperature profiles of the intensities and frequencies of the various bands fall into four different categories: 1) base bands that show a reversible increase in intensity prior to the melting region, i.e., a definite premelting phenomenon; 2) base bands that show little or no temperature dependence, 3) deoxyribose-phosphate backbone vibrations that show no temperature dependence up to the melting region, at which point large decreases in intensity occur; and 4) slow frequency changes in certain in-plane vibrations of guanine and adenine due to deuteration of the C-8 hydrogen of these purines in DZO.Certain Itaman bands arising from each of the four bases, adenine, thymine, guanine, and cytosine have been found to undergo a gradual increase in intensity prior to the melting region at which point large, abrupt increases in intensity occur. The carbonyl stretching band of thymine, involved in the interbase hydrogen bonding actually undergoes both a gradual shift to a lower frequency as well as an increase in intensity. These changes provide evidence that some change in the geometry of the bases relative to each other begins to occur around 5073, well below the melting region of 70"-8.i°C.From the spectra taken at various temperatures, the IINA appears to remain in the B conformation until the melting point is reached, at which time the 1)NA progresses into a disordered random-coil form. No A-form conformation is found either in the premelting or the melting region.

Raman spectra of fibers of DNA that have been prepared in the A, B, and C forms are presented and compared with Raman spectra of DNA and RNA in dilute solution. It is shown that the phosphate vibrations in the region 750-850 cm-' are very sensitive to the specific conformation of the phosphate group in the backbone chain and are virtually independent of all other factors. Thus, a very simple method for the determination of the specific conformation of the backbone chain of nucleic acids, at least so far as the sugar-phosphate chain is concerned, appears available. The method is applied to short oligomers and dimers of ribonucleosides. It is found that at low temperatures, at pH 7, the phosphate group goes into the geometry of the A conformation when the stacking forces between the bases are sufficiently strong.The most reliable method for the determination of the structure of nucleic acids and polynucleotide helical chains appears to be that of x-ray diffraction (1, 2). However, this method is only applicable to nucleic acids in highly concentrated fibrous form. In general, it is not applicable to dilute nucleic acid solutions, although meaningful progress in the interpretation of x-ray scattering from fairly concentrated solutions has recently been reported (3). Thus, it would seem helpful to have available a method that could be used to obtain structural information, both on fibers and on dilute solutions, where these materials naturally occur. In this paper, we wish to report the observation of several Raman bands that arise from the vibration of the sugarphosphate backbone, in both ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) whose frequencies and intensities are directly related to whether or not the material is in the A, B, or C form, as designated by the x-ray crystallographers (1, 2), and are virtually independent of all other parameters, such as the base composition, the presence or absence of the 2'-hydroxyl, etc. Furthermore, these bands can be observed in single-chain structures and oligomers, so that the geometry of the phosphate group in these substances can, under favorable circumstances, be determined.Recently, work in several laboratories has shown the existence of a Raman band at about 810-814 cm-' that is always present in ribonucleic acid structures, when these structures are in an ordered or partially ordered form (4-6). This band is plainly evident in the Raman spectrum of yeast transfer RNA shown in Fig. 1, and has also been observed in ribosomal RNA (5). Upon raising the temperature of the solution, so that the secondary structure vanishes, this band at 814 cm-' inevitably vanishes (4, 5). Since this band is completely independent of base composition and is present in all ordered ribo-structures, it may be due to the sugarphosphate diestersymmetric (4, 5) stretch or antisymmetric (6) stretch. The band at 814 cm-' is highly polarized, so that the former assignment seems somewhat more reasonable. Recent work in this laboratory (4) has shown that in aqueous solution, deox...

Low-frequency Raman bands (lower than 50 cm-') exist in certain proteins. They are dependent upon the conformation of the protein molecule, but are relatively independent of the form of the sample, i.e., whether it is a film or a crystal.Low-frequency Raman spectra were obtained from samples of a-chymotrypsin that had been prepared in several ways. A peak at about 29 cm-' was found for all samples except the one that had been denatured with sodium dodecyl sulfate. Such low frequency motions must arise from vibrations that involve all, or very large portions, of the protein molecule.In the past few years the technique of laser Raman spectroscopy has been used with considerable success to obtain the Raman active vibrations of several proteins (1-4). However, if one examines the published spectra, it is apparent that it has been impossible in the past to obtain the Raman bands in proteins that lie below 150 cm-'. The reason for this is that the scattering due to the Rayleigh component is too large for a double-grating monochromator to discriminate against. Recently, in this laboratory, we have shown how to obtain Raman spectra only a few wave numbers from the exciting line on synthetic polymers, such as polyethylene and poly-i-alanine, by the iodine filter technique (5-7) and a Spex double-grating monochromator. More recently, we have also found it possible to obtain these low frequency bands using a Cary triple-grating monochromator. For the work reported here, we have used both of these instruments and have obtained equivalent results on each. This has been of the greatest help in elimination of the possibility of experimental artifacts. From our work with these instruments it is possible to show, for the first time, that definite low-frequency motions exist in many common proteins and that these vibrations appear to be sensitive to the conformation of the protein. As we will discuss below, such low-frequency motions must arise from vibrations that involve either all or very large portions of the protein molecule. Thus, it appears from our measurements that large portions of the protein molecule are constantly undergoing a coherent periodic vibration. The existence of such vibrations is of considerable interest even though the exact assignment of the motion is not possible at present.Figs. la and b show the low-frequency Raman spectra of samples of a-chymotrypsin that were prepared in several ways. In every case, except the sample that had been denatured with SDS, a pronounced peak at about 29 cm-' is found. It is apparent that there is some splitting of the peak in the single crystal and also some slight change in the shape of the peak with deuteration and acylation. However, upon denaturation with SDS the peak at 29 cm-' vanishes. Rather intense Raman scattering throughout the region of 20-150 cm-' is observed on the denatured material, but it is broad and structureless-a fact that probably reflects the decrease in the order of the protein conformation.The fact that this low-frequency band is dependent ...

SynopsisBoth Haman spect.ra and X-ray diffraction patterns have been obtained front oriented fibers of sodium deoxyribonucleic acid (Sa-DKA) as a function of salt content and relative humidity. We have confirmed the previously reported X-ray results that, for oriented fibers, the A-form always exists between 7.5 and 927, relative humidity and that the conformation m-ill change to the B-form a t 927; relative huniidity only if an excess (3-5Yc of added salt is present. Oriented fibers containing low amounts of added salt remain in the A-type conformation at 92% relative humidity and higher. An exact correlation has been found between the familiar Aand B-type X-ray diffraction patterns of D?iA fibers and the Rantan spectra previously reported without X-ray verification from this laboratory for the Aarid B-forms. I n particular, a band at 807 c n -' was always present when a fiber showed the A-type diffraction pattern, and this band shifts to 790 cm-l in the R-form. Using the Raman spectrum to determine the specific conformation of DNA in samples less amenable to X-ray analysis, we have studied the A S Btransformation in unoriented fibrnus masses of I)KA and in concentrated, oriented gels. We find that in unoriented fibrous masses, the A S B transition always occurs a t 927; relative humidity even a t very low salt concentration (0-4Tc). IIowever, in oriented DNA gels at low salt, the A-form can persist as a metastable state t.o concentration as low as 20% DNA. The origin of the bands at 807 and 790 c n -l and the possible biological implications of these findings are discussed.