VOL. 14, 247-264 (1975)
BIOPOLYMERS
Melting and Premelting Phenomenon in DNA by Laser Raman Scattering STEPHEN C. ERFURTH and WARXER L. PETICOLAS, Department of Chemistry, University of Oregon, Eugene, Oregon 97403
Synopsis Raman 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 a t 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 a t various temperatures, the IINA appears to remain in the B conformation until the melting point is reached, a t 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.
INTRODUCTION Experimental approaches such as hydrogen and the formaldehyde-DNA reaction5t6have shown that even a t low temperatures DNA is subject to local structural fluctuations, which result in frequent opening and closing of the structure. Since DNA functions in vivo far below thc T,, such structural fluctuations may have considerable biological significance in recombination, transcription, and replication processcs. Previous work with optical techniques, CD and ORD7-Ii and viscometric methodsi2 have indicated a considerable premelting phenomenon in DNA, poly d(AT), and poly d(G-C). This work was initiated in order to understand better the changes occurring in the geometry of the bases and the backbone conformations of DNA before and during the helix-to-coil transition. An advantage of 247
@ 1975 by John Wiley & Sons, Inc.
248
ERFURTH AND PETICOLAS
Raman spectroscopy in studying the melting of DKA is that one can observe changes in each base and separate them from changes in the dcoxyribose-phosphate backbone. Hon ever, onc of the major disadvantagcls of Raman spectroscopy is the relatively high concmtrations nceded t o obtain Raman spectra as compared to other optical techniques. Approximately 1.5 mg/ml of a polynucleotide are nceded t o obtain a high-resolution Raman spectrum. Raman spectra of ribonucleic acid and deoxj ribonuclcic acid structures have been shown t o be quite sensitive t o conformational ~ h : i n g e s . ' ~ - I~n~ particular, studies on self-stacking in polyadenylic and polycytidylic acids show t h a t the intensity of the Raman bands of certain in-plane ring vibrations decreases due t o stacking of the polynucleotidc bases.1g*" This phenomenon has been called Haman hypochromism.13 In t h r past, Raman hypochromism has been interpreted n ith thc simplifying assumption that the decrease in intensity in thc Raman dfect has thc same origin as the decrease in intensity in the ultraviolet absorption, and hmce that the intensity of the hypochromic Raman bands is largely derived from thc lowlying hypochromic ultraviolet absorption bands by means of a preresonance Raman effect.I3 l 9 The gcmeral corrwtncss of this assumption has recently been demonstrated by resonance Raman measurements on nucleotides using the frequency-doubled argon laser line a t 2572.5 8. Here it was found that hypochromic bands in adenine become truly resonant with laser light in this frequency while nonhypochromic bands, in general, do not.27 Thus i t can be argued that Raman bands t h a t show the true resonant Raman effect (RRE) at 270 rim probably show a strong preresonance Raman effect from this band as well. However, the Raman effect as distinguished from the R R E must derive its intensity from all of the excited states in the molecule, including the continuum, and consequently there will be other factors affecting the intensity besides the preresonance effect. I n this paper l i e show that ccrtain bands exhibit an increase in intensity n ith incrcxasc in temperature during the premelting region, although there is no corrtywnding change in the uv absorbance at 260 nm. The changes in the Raman intensity, like the changes in the CD, arc probably related t o subtle geometrical changes in base orientation t h a t do not affect the ultraviolet absorption band a t 260 nm. I n fact, each of the three different optical techniques, Ultraviolet hypochromism in thc 260-nm absorption band, CD, and the Raman effect, all derive their intensity changes from a summation over all of the excited states o f the medium, but in each case the summation involves a different set of matrix elements. It is not surprising that each of thew optical techniques has a different scnsitivity to these premelting changes so that 11 hile the uv hypochromism gives no premelting changes, CD givcls a prcmelting changc. opposittl to that 11 hich occurs in the melting region" and, as we shall see, Raman gives an effect in the same direction but smaller than the changcs that occur during the melting region.
MELTING .4ND PREMELTING IN DNA
249
The Raman spectra of DNA are found to be somewhat difficult to interpret due to the different basrs, which have scveral bands a t almost the same frequency. Although there has been a very thorough study of the Raman bands of ribonucleic acid derivatives,28a t the presrmt time very few Raman spectra of DXA or the deoxyribomononuclcotidcs have been published. I n order to make certain assignments for both vibrations of the backbonc and the bases in DKA, t i e have found it very useful t o take the Raman spectra of mixtures of d(.oxyribomononuclcotides with exactly the same concentration ratio as in calf thymus DKA.
MATERIALS AND METHODS Calf thymus Ka-DSA was obtained from Worthington Biochemical Corporation, lot IOA. Samples were prepared as 2% solutions (20 mg/ml) in 0.01 A l sodium cacodylate and 0.001 1l.I EDTA (c.thylcnedinitrilotetraacetic acid, disodium salt), pH 7.0. The total S a + Concentration was 0.07.5 M . Deuterated DNA was prepared by first lyophilizing from buffered DZO solutions and then prepared for study by adding the appropriate amount of D,O. All samples were washed in 70% ethanol before preparation in order to remove any cxccss XaCl prcwnt, n-hich w d d iricrcasc the melting tcmperaturc. In order to minimizcl any effects due. to degradation, new samples were used for each tcmpcraturc. All samples n-ercl allowed to eyuilibrate 10 min before starting the spectrum. The average time to obtain a spectrum xas 30 min. Samples of 2’-deoxyadenosincl .>‘-phosphate (dAAIP) lot OAA, 2’-deoxycytidine j’-phosphate (dCM1’) lot OAA, 2’-droxyguanosinc 5’-phosphate ( d G l I P ) lot ’BA, and thymidine .;’-phosphate (TAW) lot lKA, were all obtained from Worthington Biochemical Corporation. Aqueous solutions of deoxyribomonoriucleotides w r c . prepared by dissolving thc appropriatr amount in glass-disti1lc.d water and adjusting thc pH to 7.0. Dcutcnted samples were preparc.d by thc same method as the D S A samp1t.s and adjusted to p D 7.0. Appropriate pcwcntagc.s of t h r various bases were dctermincd from the data publishcd in thc Handbook of B i o c h e m a ~ t r y . ~ ~ Uv melting experiments were performe$ in a Gilford model2000 recording spectrophotometcr (quipped for the automatic measurement and recording of temperature. The uv melting curvm of scvcml samples may bt. determined simultaneously by arbitrarily adjusting on the absorbance. scale the initial starting point of the automatic recording pen. The platinum resistance thermomctw in this instrument is located within thc sample chamber controlled by the thermostat and is directly below the cuvettes. The samples were hmted from 23” to 100°C a t a constant rate of l”C/min. The Raman apparatus has bwn described p r e v i o u ~ l y2o. ~ ~The Raman spectra presented hcrc were taken u i t h a rcsolution of about 4 cm-I and a time constant of 1.0 SCC. An entirc scan took approximately 30 min. Each point is an average of six to eight scans.
250
ERFURTH AND PETICOLAS
RESULTS Since Raman spectroscopy requires high concentrations relative to optical methods, it seemed appropriate to run the uv melting curves a t high concentration in order to make certain that there is no effect of polymer concentration on the melting temperature. Figure 1 shows the uv melting curves for calf thymus DNA a t 0.03 mg/ml and 10 mg/ml. A 10-mm pathlength cell was used for the 0.05-mglml sample and a 0.05-mm path-lrngth cell for the 10-mg/ml sample. As can be seen, the melting curves are independent of concentration. The melting temperature is in agreement with the predicted melting point (Tm), 80°C, based on A T/G C ratios.30 Figure 1 indicates that uv absorption does not detect base unstacking until almost 70°C in native calf thymus DNA a t 0.075 M Na+. Thus we find that even a t very high concentrations no premelting changes are found in uv absorption a t 260 nm. I n general, the premelting effect and the intensity changes we have observed in the Raman effect for the various bands are the same for DNA run in D20 and H20 except for changes in frequencies due to deuteration. Deuterated DNA allows one to observr several very important bands, which are obscured in HrO solutions due to HzObending vibrations, and it is easier to separate the base vibrations from the backbone vibrations in DzO. For these reasons the majority of the paper will deal with deuterated solutions. I n order t o make the best assignments to the bands, solutions of various combinations of the deoxyribomononucleotides (dAMP, TMP, dCMP, dGMP) were run a t 2.5" and 75°C in both DZO and HzO a t the same concentration as found in the DNA solutions. No differences were found between the low- and high-temperature spectra of the pyrimidine bases, dCAfP, and
+
+
20 30 40 50 60 70 80 90 Temperature, 'C
Fig. 1. Uv melting curves at 260 nm for calf thymus Na-DNA in H20, pH 7.0. arbitrary base line was subtracted to bring the absorbance on scale.
An
MELTING AND PREMELTING I N DNA
25 1
Fig. 2. Various combinations of the deoxyribomononucleotides a t the appropriate percentages (molar ratios) found in calf thymus DNA; dAMP 28%, T M P 28%, dCMP 21%, and dGMP 22%, in HzO at 25°C and p H 7.0.
T M P . The low-temperature spectra are shown in Figures 2 and 3. Changes were found in the purine bases dGMP and d A J I P a t 75°C after several hours due to the slow deuteration of the C-8 proton in the purine rings. Figures 4 and 5 show the Rnman spectra of calf thymus D N A in both HzO and DZO at temperatures below and above the melting point. A list of frequencies, assignments, and changes observed in going from the ordered to disordered state at the melting point are shown for calf thymus D N A in both H20 and DzO solutions in Table I. All intensity measurements were made relative to the 1094-cm-' band in HzO and the 1091-cm-' band in DzO, which has been assigned t o the POZ-
ERFURTH AND PETICOLAS
252
4
Fig. 3. Various combinations of the deoxyribomononucleotides at the appropriate percentages (molar ratios) found in calf thymus DNA; dAMP 28%, T M P 28%, dCMP 21% and dGMP 22%, in 11~0at 25°C and pD 7.0.
dioxy symmetric s t r e t ~ h . 8 ~ 1This 3 ~ band has been found to be relatively independent of the conformation of polyribonucleotides arid DNA.16~*9~22 A study of thc temperature dependence of the POz- dioxy symmetric stretch relative to the internal standard sodium cacodylatc (610 cm-l) found a 10% decrease in intensity from 2:' t o 50°C with no significant changes above 50°C. To ensurc reliability of the measurements, tho intensities w r c also checked against the internal standard in cach spectrum. Thus no serious errors arc likely to be introduced in tho intensity measurements.
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253
4
Fig. 4. llaman spectra of calf thymus IINA a t 25", 84", and 98°C in H20, pH 7.0, 0.01 M in sodium cacodylate and 0.001 ill in EIITA. The total N a + concentration was 0.075 M .
Base Bands Showing Premelting The more intense bands of adenine that show premelting are found a t 716 ern-' and 1300 em-' in D20. Figure 6 is a plot of the intensity change of the 1300-cm-' band versus temperature in D20. It can be seen that there is a definite increase in the intensity prior to melting. Figure 3 shows that the 1300-em-' band contains contributions from both adenine and cytosine. The hypochromism observtd in this band is most probably due t o adenine since this band is found t o bc quite hypochromic in poly A but not in poly
2.54
ERFURTH AND PETICOLAS
Fig. ca
C.19 Figure 5 shows that the 734-em-' thymine band shifts slightly t o lower frequency and thus adds to the intensity of the adenine band a t 716 em-'. Thus there are a number of changes in the spectrum going on simultaneoously. Indeed in all of these spectra it is evident that yuitr a number of different changes arc occurring so that we are not measuring a single
MELTING AND PREMELTING I N DNA
255
change from one state (ordered) to another (disordered) but are seeing rather complex and subtle changes in the conformation. The band showing large changes upon melting in thymine occurs a t 1673 cm-' in D20. This band is best observed in D,O since an H 2 0 bending TABLE I Spectral Changes Observed on Melting of Calf Thymus DNA cm-1 in
czo
cm-1 in
HzO
Changes Observed Upon Melting (if any)
786
Small increase Decreases Large increase Shifts t o lower cm-l Large increase in intensity No change Small increase in intensity
828
835
Shifts t o lower cm-1
867 893
879
Decreases Decreases Decreases Decreases Decreases No change Decreases and shifts to a higher cm-1 No change Disappears Moderate increase Moderate increase Moderate increase Large increase Small increase Moderate increase No change Moderate increase Small increase Decreases Shifts to lower cm-1, deuteration of G 8 proton on A and G Moderate increase No change in intensity, shifts t o lower cm-1 No change in intensity, shifts t o lower cm-1 Moderate increase in intensity Large increase in intensity due mainly to G No change Large increase in intensity, shifts to lower cm-1
656 677 716 734 765 785
672 683 729 750
920 966 1013 1047 1091
1260 1300 1343 1375 1418
1015 1051 1094 1144 1186 1214 1225 1240 1259 1303 1340 1378 1421 1463
1484
1491 1501 1520
1521
1575
1534 1579
1620 1673
1660
Assignment
T G A T PO, diester symmetric stretch PO2 diester symmetric stretch and C PO2 diester antisymmetric stretch ? Deoxyribose-phosphate Deoxyribose Deoxyribose Deoxyribose C-0 stretch C-0 stretch C-0 stretch PO2- dioxy symmetric stretch Deoxyribose-phosphate T T A T c, A A A T, A A, G Deoxyribose-phosphate G, A
A
C? C=O of T
ERFURTH AND PETICOLAS
256
1.651
xe
!
1.45
Temperature, "c
Fig. 6. Plot of the Iiaman intensity of the 1300-cm-' adenine band (relative to the 1091-cm-' band) of calf thymus DNA in 1 1 2 0 vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and pI1 7.0.
Temperature,
OC
Fig. 7. Plot of the Raman intensity of the 1638-cni-' thymine band (relative to the 1091-cm-l band) of calf thymus DNA in DZO vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and pl) 7.0.
vibration occurs a t this frequency. Figure 7 is a plot of the 1638-cm-' band in D20 versus temperature. Again the plot shows a similar premelting behavior as observed in the adenine band (Figure 6). The band a t 1673 em-' in Figure 5 is particularly interesting sincc it is a C=O vibration of thymine, which is involved in interbase hydrogen bonding. Figure 3 indicates a small band a t 1660 cm-' when thymine is abscnt, n-hich is due to guanine. Comparison of the highly stacked double-helical poly G-poly C
MELTING AND PREMELTJNG IN DNA
257
with the single-stranded unstacked poly G indicates little discernible difference in the carbonyl region of the s p e ~ t r a . ~From ~ , ~this ~ we conclude that the intensity changes and shifts observed in DNA a t 1673 cm-’ are due entirely t o thymine. Figure 5 shows a large gradual shift of 14 cm-I with a concurrent increase in intensity for the thymine C=O vibration, in going from the low-tcmperature double-helical form (1673 cm-l) to the high-temperature random-coil form (1658 cm-l). Similar shifts upon melting for both carbonyl vibrations of uracil have been found in the ir spectra of the poly U double helix3and the poly (A 2U) triple helix3 in which both carbonyl groups of the uracil arc found t o be involved in hydrogen bonding. Significant differences have been found in the double-bond region for poly A-poly U and poly (A-U)poly (A-U) a t low temperature in both the ir35,3G and Raman20,3G spectra. The high-temperature spectra were found to be quke similar for each technique. These differences in the carbonyl region of the low-temperature spectra for the two polymers were attributed to changes in the vibrational coupling of the vertically stackcd bases.36 Thus it is difficult to distinguish between the base stacking and base pairing interactions in the carbonyl region. At 25°C the Raman spectrum of the double-helical form of the alternating copolymer of poly d(A-T) in D2019shows an intense carbonyl band a t 1669 cm-l, which is quite close in frequency to the low-temperature thymine carbonyl band a t 1673 cm-’ in DNA. However the carbonyl band of 5’TMP,35in which no interbase hydrogen bonding or base stacking is cxpected, occurs a t 1659 em-’, which is quite close t,o the high-temperature thymine carbonyl frequency of 1658 cm-l in DNA. Therefore the shift in the carbonyl frequency of DNA upon melting is related to both base unstacking and the breakage of the interbase hydrogen bonds. At this time, however, we cannot distinguish between hydrogen-bond breaking and base unstacking in the carbonyl region. Evidence for thc breaking of interbase hydrogen bonds in the premelting region has been given by Printz and von Hippel using the hydrogen exchange technique. 1.2 Poly d(A-T) is very similar in structure to B-form DNA.38 The Raman spectra of aqueous solutions of poly d(A-T)20below and above the melting point show nearly all of the same intensity changes that we have discussed in this work for adenine and thymine upon melting DNA. Studies on the premelting of poly d(A-T) have been made using the CD technique, which shows changes in the CD all the way down to the freezing point of the solution. However, within the error of our measurements, no changes are observed in the Kaman spectra below 50°C. This again illustrates the differences obtained with different optical techniques. The guanine band, which shows the greatest premelting, occurs a t 1575 cm-’ in DzO. A small adenine band also occurs a t this frequency (Figure 3) but i t is not hypochromic in either poly A-poly UZ0or poly A. l 9 A graph of the intensity changes in the 1575-cm-l band in DzO versus temperature is shown in Figure 8.
+
258
ERFURTH AND PETICOLAS
30405060708090 Temperature, C ' Fig. 8. Plot of the Raman intensity of the 1 5 7 5 - ~ m -guanine ~ band (relative t o the 1091-cm-' band) of calf thymus DNA in I>zOvs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and PI) 7.0.
Temperature,
"C
Fig. 9. Plot of the Raman intensity of the 765-cm-I cytosine band (relative to the 1091-cm-' band) of calf thymus DNA in D20 vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and PI) 7.0.
The only cytosine band in which we were able t o find premelting was the 765-em-' band in DZO. A graph of the intensity change of this band in DZO versus temperature is shown in Figure 9.
Bands That are Independent of Temperature Two bands occurring a t 785 and 1091 em-' in DzO have been found to be independent of temperature on comparison with the sodium cacodylate band a t 610 cm-l a t temperatures above 50°C. The band a t 785 em-' in DNA has been previously assigned to the 0-P-0 dicster symmetric stretchz0while the band a t 1091 em-' is assigned to thc 0-1'-0 dioxy symmetric stretch. 3 1 , 3 2 Although the POz diester symmetric stretch of the
MELTING AND PREMELTING I N DNA
259
DNA chains has never been positively identified, from normal coordinate calculations on model compounds there is some evidence that it lies near 790 cm-1 where it is obscured by base vibration^,^^^^^ particularly the cytosine vibration a t 786 em-' in HzO. In DzO solutions of DNA where the cytosine band is shifted to 765 cm-' (Figure 5), an intense band is still found a t 785 crn-l, while in the monomer solutions (Figure 3) only a very small band is found a t this frequency. Therefore this band at 785 cm-' is probably part of the deoxyribose-phosphate backbone. The intensities of two adenine bands found a t 1343 and 1520 em-' appear not to change with temperature.
Bands That Decrease With Increasing Temperature Several deoxyribose or deoxyribose-phosphate vibrations are found between 828 and 1047 cm-l as seen in Figure 5. All decrease in intensity except the band a t 867 em-', which decreases after 1 hr a t temperatures above the T,. These backbone vibrations of the polymeric deoxyribosephosphate chain give an indication of the changes observed in the backbone conformation upon melting. None of these backbone vibrations show any premelting. Thus the geometry of the deoxyribose-phosphate backbone seems to remain intact up to the melting point. It can be seen from Figure 3 that no base vibrations occur in this region. In order to obtain a handle on the backbone we have examined thc behavior of the Raman active vibration a t 1047 em-' in DzO, which has been The ~ tentatively assigned to the deoxyribose C-0 stretching v i b r a t i ~ n . ~ 1047-em-' band is found to get broader and weaker upon melting. A graph of the intensity change in the 1047-cm-I band versus temperature is shown in Figure 10. The graph indicates that no premelting is occurring in this backbone band.
.
Temper a ture *C
Fig. 10. Plot of the Itaman intensity of the 1047-cm-l backbone C-0 band (relative to the 109l-cm-' band) of calf thymus DNA in D 2 0 vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and PI) 7.0.
260
ERFURTH AND PETICOLAS
Since no changes are seen in the various deoxyribose-phosphate vibrations until the melting point is reached, this would indicate that the deoxyribosephosphate backbone conformation is not changing in the premelting region. Previous studies on fibers of calf thymus DNA have shown that Raman spectroscopy is quite sensitive to the backbone conformation of the DNA, i.e., the A family and B family of conformations.22 From the spectra taken at various temperatures, the DNA seems to remain in the B conformation until the melting point is reached, at which time it appears the DNA progresses into a disordered random-coil form. No evidence for an Aform conformation is found either in the premelting or,melting region due to the lack of an 807-em-' band. This is in agreement with the results of Gennis and Cantor." One base band due t o guanine, found at 677 cm-l in DzO, was found to decrease in intensity with increasing temperature. A similar effect has been observed in t-RNA for a guanine band at 670 cm-' upon melting.25
Reversibility Changes resulting from high temperature were completely reversible as long as the temperature remained below t h r T,. Increasing the tcmperature beyond the T , value and then rapidly cooling the sample back t o 23°C do not result in return t o the well-ordered low-temperature spectra. Spectra of calf thymus DNA in HZO and DZO were taken after the samples were at 9S"C for about 1 hr and were then rapidly cooled to 23°C. The samples were allowed t o cool for 10 min before the spectra were taken. An initial rapid decrease in the baseband intensities occurred, indicating a number of the bases are immediately stacking presumably with reforming of a number of interbase hydrogen bonds. 3Iost base bands did not completely decrease back t o their original height, indicating that the dcnaturation process (melting out) is irreversible under the procedurcs used here. Thus some bases remain unstacked probably due to mismatching of homologous segments, formation of loops, and from segments that remain single-stranded. It is quite interesting t ha t the deoxyribose-phosphate backbone vibrations (825-1061 cm-') reappeared a t almost their full intensity and appropriate frequency. This appears to indicate that at low temperatures the deoxyribose-phosphate backbone tends t o go into a stable conformation representing a potential energy minimum, which has the same geometry as the double helix (B type).
Deuteration of the C-8 Protons of Adenine and Guanine The slow isotopic exchange of the C-S proton of purincs has been studied by a number of techniques. Using nuclear magnetic resonance Bullock and Jardetzdy*l and Schmeizer e t have shown that the C-8 proton of purines exchanges with the deuterium of DzOa t tcmperaturrs near 100°C. Shelton
261
MELTING AND PREMELTING I N DNA
2% dGMP
I
In c m
w
pD7.0
n (I
04. C
( 3 (3
25. C (3 (3
(I
(3
Fig. 11. Itaman spectra of 2c0dGMP in II2O a t 25" and 84°C. The top spectrum was taken after being a t 84°C for 4 hr.
and Clark43using tritium labeled water concluded that only the purine nucleosides exchange and t h a t the half-time for exchange a t 100°C was about 1 hr. Doppler-Bernardi and F e l ~ e n f e l dhave ~ ~ described an exchange method for producing tritium labeled native DNA in vitro with minimal physical damage to the DNA. At temperatures above 70"C, we have observed a large shift to lower frequency of a base band a t 1484 cm-lin DKA (Figure 5 ) , which is assigned to adenine and guanine ring vibrations. The spectra in Figure 5 a t 75", 80", and 90°C were equilibrated for 2 hr a t the corresponding temperatures to show the changes in the 14S4-cm-l band. At 84°C this band shifts from 1484 t o 1457 em-' in about 4 hr. A study of the same band in d G N P (Figure 11) and dAMP (Figure 12) in D,O a t S4"C produced a similar shift in 4 hr.45 I n d G N P (Figure 11) the 1480 cm-' band shifts t o 1457 cm-' while in d A 3 P (Figure 12) the band shifts from 1481 to 1465 cm-'. YO changes were found for DNA, dGMP, or d A N P in aqueous solutions under the same conditions. These changes are not reversible upon lowering the temperature to 23°C. From these observations, we have assigned this frequency shift from 1484 t o 1457 cm-I in DNA to the slow druteration of the C-8 protons of adenine and guanine. Quantitative measurements of the rate process have been made and will be reported elsewhere.
262
ERFURTH AND PETICOLAS
Fig. 12. Rama.n spectra of 2 7 , dAMP in 1 1 2 0 at 25" and 84°C. The top spectrum was taken after being at 84°C for 4 hr.
CONCLUSIONS Certain Raman bands arising from each of the four bases, adenine, thymine, guanine, and cytosine undergo a gradual increase in intensity prior to the melting point. These changes indicate that disruption of vertical base-base stacking interactions occurs beginning a t about 50"C and increasing up until the melting point. However, observation of the various deoxyribose-phosphate backbone vibrations between 828 and 1060 em-' shows that the DNA backbone. does not undergo any significant conformational changc. prior to the melting point. This indicates that the backbone conformation of the helix is not disordered by the premeltirig phenomenon. As long as the temperature remains below the T , the changes arc completely reversible upon rapid cooling. Temperatures above the T , permit breaking of hydrogen bonds and unstacking of t h r bases rtlsulting in a t least partial separation of the double helix and formation of a random-coil form, which is irreversible in terms of reformation of the original orderrd duplex. However, rven aftw melting out, the deoxyribose.-phosphatc backbont. in DNA found a t low temperature is a very stable potential energy minimum,
MELTING AND PREMELTING I N DNA
263
which seems to be fully recovered after melting and then partial renaturation of the helices. Beginning a t about 50°C the carbonyl stretching band of thymine, involved in the interbase hydrogen bonding, was found to undergo a large gradual shift of 14 em-' to a lower frequency, with a concurrent increast. in intensity. Similar shifts to lower frequency with concurrent intensity changes have been found in the ir and Raman spectra of polyribonucleotidcs containing uracil.20 33-36 I'rintz and von Hippel' have shown that DNA is subject t o local structural fluctuations in the prcmelting region, u hich results in frequent opening and closing of the structure a t the level of the nucleotide pair. Thus both interbase hydrogen-bond breaking and bas0 unstacking appear to be occurring in the premelting region. At this time, however, we cannot distinguish between hydrogen-bond breaking and base unstacking in the carbonyl region. By comparing the spectra of DNA a t various temperatures to those of previously published spectra of known backbone conformation, i.e., the A and B forms,22it is possible to sec that DNA does not go into the A conformation either in the premelting or melting region. Rather the DNA remains in the B-form geometry up to the melting point, a t which time it goes into a disordered random-coil form. Finally, a large shift from 1484 to 14.57 cm-I has been observed in an inplane ring vibration assigned to guanine and adenine at temperatures above 70°C. At 84°C this band in deuterated DNA, dGJIP, and dAJI1' was found to shift in about 4 hr. This frequency shift has been tcntatively assigned to the slow deuteration of the C-S protons of adenine and guanine. This same shift has recently been observed by Thomas.46
References 1. Printz, M. P. & von Hippel, P. H. (1965) Proc. Nut. Acad. Sci. U.S. 53,363-370. 2. von Hippel, P. H. & Printz, M. P. (1965) Fed. Proc. 24, 14.18-1463. 3 . McConnell, B. & von Hippel, P. H. (1970) J . Mol. Biol. 50, 297-316. 4. McConnell, B. & von Hippel, P. 13. (1970) J . Mol. Riot. 50, 317-332. 5 . Utiyama, H. & I h t y , P. (1971) Riochemistry 10, 1254-1264. 6. von Hippel, P. H. & Wong, K. Y . (1971) J . d f o l . Biol. 61,587-613. 7. Ts'o, P. 0. P. & Helmkamp, G. (1961) Tetrahedron 13, 198-207. 8. Fresco, J. R. (1961) Tetrahedro,L 13, 183-197. 9. Brahms, J. & Mommaerfs, W. F. H. M. (1964) J . Mol. Biol. 10.73-88. 10. Ts'o, P. 0. P., Itappaport, S. A. & Bollrim, F. J . (1966)Riochemistry5,41.?S-4170. 11. Gennis, It. B. & Cantor, C. It. (1972) J . Mol. 13iot. 65, 381-399. 12. Freund, A. & Bernardi, G. (1963) i\'ature 200, 1318-1319. 13. Tomlinson, B. L. & Peticolas, W. L. (1970) J . Chem. Phys. 52,2154-2136. 14. Peticolas, W. L., Nafie, L., Stein, P. & Fanconi, B. (1970) J . Chem. Phys. 52, 1576-1384. 15. Thomas, Jr., G. J., Mederios, G. I>. & Hartman, K. A. (1971) Biochem. Biophys. Res. Cornmu?,.44, 587-592. 16. Aglward, N. N. & Koenig, J. I,. (1970) Macromolecules 3 , 570-596. 17. Thomas, Jr., G. J. & Hartman, K. A. (1973) Riochim. B i o p h y s . Acta 312,311-322. 18. Thomas, Jr., G. J., Chen, M. C. & Hartman, K. A. (1973) Riochim. Biophys. Acla 324,3749.
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ERFURTH AND PETICOLAS
19. Small, E . W. & Peticolas, W. L. (1971) Biopolymers 10, 69-88. 20. Small, E. W. and Peticolas, W. L. (1971) Biopolymers 10, 1377-1416. 21. Thomas, Jr., G. J. (1970) Biochim. Riophys. Acta 213,417-423. 22. Erfurth, S. C., Kiser, E. J . & Peticolas, W. L. (1972) Proc. -\at. Acad. Sci. U.S. 69,938-941. 23. Brown, K. G., Kiser, E. J . & Peticolas, W. L. (1972) Hiopolymers 11, 1855-1869. 24. Lafleur, L., Rice, J. & Thomas, Jr., G. J. (1972) Biopolymers 11, 2423-2437. 25. Small, E. W., Brown, K. G. & Peticolas, W. L. (1972) Biopolymers 11, 1209-1215. 26. Hartman, K. A., Clayton, N. & Thomas, Jr., G. J. (1973) Biochem. Biophys. Res. Commuii. 50,942-949. 27. Pezolet, M. & Peticolas, W. L. (in preparation). 28. Lord, R . C. & Thomas, Jr., G. J. (1967) Speclrochim. Acta25A, 2551-2591. 29. Sober, H. A,, Ed. (1970) Haudbook of Biochemistry, 2nd ed., Chemical Rubber Co., Cleveland, Ohio. 30. Mandel, M. & Marmur, J. (1968) dtethods Euzymol. 12B, 195-206. 31. Schimanouchi, T., Tsuboi, M. & Kyogoku, Y. (1964) Advan. Chem. Phys. 7,435498. 32. Tsuboi, M. (1957) J . Amer. Chem. SOC.79,1351-1354. 33. Bode, I)., Heinecke, M. & Schernau, U. (1973) Biochem. Biophys. Res. Commun. 52,1234-1240. 34. Miles, H. T . & Frazier, J. (1964) Biochem. Biophys. Res. Commun. 14,21-28. 35. Morikawa, K., Kyogoku, Y . & Tsuboi, M. (1969) cited in Tsuboi, M., Appl. Spectrosc. Rev. 3 , 4 5 9 0 . 36. Morikawa, K. Tsuboi, M., Takahashi, S., Kyogokn, Y., Mitsui, Y . , Iitaka, Y. & Thomas, Jr., G. J. (1973) Biopolymers 12,799-816. 37. Erfurth, S. C. & Peticolas, W. L., unpublished results. 38. Davies, 1). It. & Baldwin, It. L. (1963) J. Mol. Biol. 6,251-235. 39. Brown, E. & Peticolas, W. L., unpublished results. 40. Tsuboi, M. (1969) Appl. Spectros. Rev. 3 , 4 5 9 0 . 41. Bullock, F. J. & Jardetzky, I). (1964) J. Org. Chsm. 29, 1988-1970. 42. Schweizer, M. P., Chan, S. I., Helmkamp, G. K. & Ts’o, P. 0. P. (1964) J. Amer. Chem. SOC. 86,696-700. 43. Shelton, K. It. & Clark, J. M. (1967) Biochemistry 6,2735-2739. 44. Doppler-Bernardi, F. & Felsenfeld, G. (1969) Biopolymers 8,733-741. 4.5. Kiser, E. J., Yu. T . J. & Peticolas, W. L. (in preparation). 46. Thomas, G. T., private communication.
Received August 13, 1974 Accepted September 25, 1974
BIOPOLYMERS
Melting and Premelting Phenomenon in DNA by Laser Raman Scattering STEPHEN C. ERFURTH and WARXER L. PETICOLAS, Department of Chemistry, University of Oregon, Eugene, Oregon 97403
Synopsis Raman 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 a t 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 a t various temperatures, the IINA appears to remain in the B conformation until the melting point is reached, a t 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.
INTRODUCTION Experimental approaches such as hydrogen and the formaldehyde-DNA reaction5t6have shown that even a t low temperatures DNA is subject to local structural fluctuations, which result in frequent opening and closing of the structure. Since DNA functions in vivo far below thc T,, such structural fluctuations may have considerable biological significance in recombination, transcription, and replication processcs. Previous work with optical techniques, CD and ORD7-Ii and viscometric methodsi2 have indicated a considerable premelting phenomenon in DNA, poly d(AT), and poly d(G-C). This work was initiated in order to understand better the changes occurring in the geometry of the bases and the backbone conformations of DNA before and during the helix-to-coil transition. An advantage of 247
@ 1975 by John Wiley & Sons, Inc.
248
ERFURTH AND PETICOLAS
Raman spectroscopy in studying the melting of DKA is that one can observe changes in each base and separate them from changes in the dcoxyribose-phosphate backbone. Hon ever, onc of the major disadvantagcls of Raman spectroscopy is the relatively high concmtrations nceded t o obtain Raman spectra as compared to other optical techniques. Approximately 1.5 mg/ml of a polynucleotide are nceded t o obtain a high-resolution Raman spectrum. Raman spectra of ribonucleic acid and deoxj ribonuclcic acid structures have been shown t o be quite sensitive t o conformational ~ h : i n g e s . ' ~ - I~n~ particular, studies on self-stacking in polyadenylic and polycytidylic acids show t h a t the intensity of the Raman bands of certain in-plane ring vibrations decreases due t o stacking of the polynucleotidc bases.1g*" This phenomenon has been called Haman hypochromism.13 In t h r past, Raman hypochromism has been interpreted n ith thc simplifying assumption that the decrease in intensity in thc Raman dfect has thc same origin as the decrease in intensity in the ultraviolet absorption, and hmce that the intensity of the hypochromic Raman bands is largely derived from thc lowlying hypochromic ultraviolet absorption bands by means of a preresonance Raman effect.I3 l 9 The gcmeral corrwtncss of this assumption has recently been demonstrated by resonance Raman measurements on nucleotides using the frequency-doubled argon laser line a t 2572.5 8. Here it was found that hypochromic bands in adenine become truly resonant with laser light in this frequency while nonhypochromic bands, in general, do not.27 Thus i t can be argued that Raman bands t h a t show the true resonant Raman effect (RRE) at 270 rim probably show a strong preresonance Raman effect from this band as well. However, the Raman effect as distinguished from the R R E must derive its intensity from all of the excited states in the molecule, including the continuum, and consequently there will be other factors affecting the intensity besides the preresonance effect. I n this paper l i e show that ccrtain bands exhibit an increase in intensity n ith incrcxasc in temperature during the premelting region, although there is no corrtywnding change in the uv absorbance at 260 nm. The changes in the Raman intensity, like the changes in the CD, arc probably related t o subtle geometrical changes in base orientation t h a t do not affect the ultraviolet absorption band a t 260 nm. I n fact, each of the three different optical techniques, Ultraviolet hypochromism in thc 260-nm absorption band, CD, and the Raman effect, all derive their intensity changes from a summation over all of the excited states o f the medium, but in each case the summation involves a different set of matrix elements. It is not surprising that each of thew optical techniques has a different scnsitivity to these premelting changes so that 11 hile the uv hypochromism gives no premelting changes, CD givcls a prcmelting changc. opposittl to that 11 hich occurs in the melting region" and, as we shall see, Raman gives an effect in the same direction but smaller than the changcs that occur during the melting region.
MELTING .4ND PREMELTING IN DNA
249
The Raman spectra of DNA are found to be somewhat difficult to interpret due to the different basrs, which have scveral bands a t almost the same frequency. Although there has been a very thorough study of the Raman bands of ribonucleic acid derivatives,28a t the presrmt time very few Raman spectra of DXA or the deoxyribomononuclcotidcs have been published. I n order to make certain assignments for both vibrations of the backbonc and the bases in DKA, t i e have found it very useful t o take the Raman spectra of mixtures of d(.oxyribomononuclcotides with exactly the same concentration ratio as in calf thymus DKA.
MATERIALS AND METHODS Calf thymus Ka-DSA was obtained from Worthington Biochemical Corporation, lot IOA. Samples were prepared as 2% solutions (20 mg/ml) in 0.01 A l sodium cacodylate and 0.001 1l.I EDTA (c.thylcnedinitrilotetraacetic acid, disodium salt), pH 7.0. The total S a + Concentration was 0.07.5 M . Deuterated DNA was prepared by first lyophilizing from buffered DZO solutions and then prepared for study by adding the appropriate amount of D,O. All samples were washed in 70% ethanol before preparation in order to remove any cxccss XaCl prcwnt, n-hich w d d iricrcasc the melting tcmperaturc. In order to minimizcl any effects due. to degradation, new samples were used for each tcmpcraturc. All samples n-ercl allowed to eyuilibrate 10 min before starting the spectrum. The average time to obtain a spectrum xas 30 min. Samples of 2’-deoxyadenosincl .>‘-phosphate (dAAIP) lot OAA, 2’-deoxycytidine j’-phosphate (dCM1’) lot OAA, 2’-droxyguanosinc 5’-phosphate ( d G l I P ) lot ’BA, and thymidine .;’-phosphate (TAW) lot lKA, were all obtained from Worthington Biochemical Corporation. Aqueous solutions of deoxyribomonoriucleotides w r c . prepared by dissolving thc appropriatr amount in glass-disti1lc.d water and adjusting thc pH to 7.0. Dcutcnted samples were preparc.d by thc same method as the D S A samp1t.s and adjusted to p D 7.0. Appropriate pcwcntagc.s of t h r various bases were dctermincd from the data publishcd in thc Handbook of B i o c h e m a ~ t r y . ~ ~ Uv melting experiments were performe$ in a Gilford model2000 recording spectrophotometcr (quipped for the automatic measurement and recording of temperature. The uv melting curvm of scvcml samples may bt. determined simultaneously by arbitrarily adjusting on the absorbance. scale the initial starting point of the automatic recording pen. The platinum resistance thermomctw in this instrument is located within thc sample chamber controlled by the thermostat and is directly below the cuvettes. The samples were hmted from 23” to 100°C a t a constant rate of l”C/min. The Raman apparatus has bwn described p r e v i o u ~ l y2o. ~ ~The Raman spectra presented hcrc were taken u i t h a rcsolution of about 4 cm-I and a time constant of 1.0 SCC. An entirc scan took approximately 30 min. Each point is an average of six to eight scans.
250
ERFURTH AND PETICOLAS
RESULTS Since Raman spectroscopy requires high concentrations relative to optical methods, it seemed appropriate to run the uv melting curves a t high concentration in order to make certain that there is no effect of polymer concentration on the melting temperature. Figure 1 shows the uv melting curves for calf thymus DNA a t 0.03 mg/ml and 10 mg/ml. A 10-mm pathlength cell was used for the 0.05-mglml sample and a 0.05-mm path-lrngth cell for the 10-mg/ml sample. As can be seen, the melting curves are independent of concentration. The melting temperature is in agreement with the predicted melting point (Tm), 80°C, based on A T/G C ratios.30 Figure 1 indicates that uv absorption does not detect base unstacking until almost 70°C in native calf thymus DNA a t 0.075 M Na+. Thus we find that even a t very high concentrations no premelting changes are found in uv absorption a t 260 nm. I n general, the premelting effect and the intensity changes we have observed in the Raman effect for the various bands are the same for DNA run in D20 and H20 except for changes in frequencies due to deuteration. Deuterated DNA allows one to observr several very important bands, which are obscured in HrO solutions due to HzObending vibrations, and it is easier to separate the base vibrations from the backbone vibrations in DzO. For these reasons the majority of the paper will deal with deuterated solutions. I n order t o make the best assignments to the bands, solutions of various combinations of the deoxyribomononucleotides (dAMP, TMP, dCMP, dGMP) were run a t 2.5" and 75°C in both DZO and HzO a t the same concentration as found in the DNA solutions. No differences were found between the low- and high-temperature spectra of the pyrimidine bases, dCAfP, and
+
+
20 30 40 50 60 70 80 90 Temperature, 'C
Fig. 1. Uv melting curves at 260 nm for calf thymus Na-DNA in H20, pH 7.0. arbitrary base line was subtracted to bring the absorbance on scale.
An
MELTING AND PREMELTING I N DNA
25 1
Fig. 2. Various combinations of the deoxyribomononucleotides a t the appropriate percentages (molar ratios) found in calf thymus DNA; dAMP 28%, T M P 28%, dCMP 21%, and dGMP 22%, in HzO at 25°C and p H 7.0.
T M P . The low-temperature spectra are shown in Figures 2 and 3. Changes were found in the purine bases dGMP and d A J I P a t 75°C after several hours due to the slow deuteration of the C-8 proton in the purine rings. Figures 4 and 5 show the Rnman spectra of calf thymus D N A in both HzO and DZO at temperatures below and above the melting point. A list of frequencies, assignments, and changes observed in going from the ordered to disordered state at the melting point are shown for calf thymus D N A in both H20 and DzO solutions in Table I. All intensity measurements were made relative to the 1094-cm-' band in HzO and the 1091-cm-' band in DzO, which has been assigned t o the POZ-
ERFURTH AND PETICOLAS
252
4
Fig. 3. Various combinations of the deoxyribomononucleotides at the appropriate percentages (molar ratios) found in calf thymus DNA; dAMP 28%, T M P 28%, dCMP 21% and dGMP 22%, in 11~0at 25°C and pD 7.0.
dioxy symmetric s t r e t ~ h . 8 ~ 1This 3 ~ band has been found to be relatively independent of the conformation of polyribonucleotides arid DNA.16~*9~22 A study of thc temperature dependence of the POz- dioxy symmetric stretch relative to the internal standard sodium cacodylatc (610 cm-l) found a 10% decrease in intensity from 2:' t o 50°C with no significant changes above 50°C. To ensurc reliability of the measurements, tho intensities w r c also checked against the internal standard in cach spectrum. Thus no serious errors arc likely to be introduced in tho intensity measurements.
MELTING AND PREMELTING I N DNA
253
4
Fig. 4. llaman spectra of calf thymus IINA a t 25", 84", and 98°C in H20, pH 7.0, 0.01 M in sodium cacodylate and 0.001 ill in EIITA. The total N a + concentration was 0.075 M .
Base Bands Showing Premelting The more intense bands of adenine that show premelting are found a t 716 ern-' and 1300 em-' in D20. Figure 6 is a plot of the intensity change of the 1300-cm-' band versus temperature in D20. It can be seen that there is a definite increase in the intensity prior to melting. Figure 3 shows that the 1300-em-' band contains contributions from both adenine and cytosine. The hypochromism observtd in this band is most probably due t o adenine since this band is found t o bc quite hypochromic in poly A but not in poly
2.54
ERFURTH AND PETICOLAS
Fig. ca
C.19 Figure 5 shows that the 734-em-' thymine band shifts slightly t o lower frequency and thus adds to the intensity of the adenine band a t 716 em-'. Thus there are a number of changes in the spectrum going on simultaneoously. Indeed in all of these spectra it is evident that yuitr a number of different changes arc occurring so that we are not measuring a single
MELTING AND PREMELTING I N DNA
255
change from one state (ordered) to another (disordered) but are seeing rather complex and subtle changes in the conformation. The band showing large changes upon melting in thymine occurs a t 1673 cm-' in D20. This band is best observed in D,O since an H 2 0 bending TABLE I Spectral Changes Observed on Melting of Calf Thymus DNA cm-1 in
czo
cm-1 in
HzO
Changes Observed Upon Melting (if any)
786
Small increase Decreases Large increase Shifts t o lower cm-l Large increase in intensity No change Small increase in intensity
828
835
Shifts t o lower cm-1
867 893
879
Decreases Decreases Decreases Decreases Decreases No change Decreases and shifts to a higher cm-1 No change Disappears Moderate increase Moderate increase Moderate increase Large increase Small increase Moderate increase No change Moderate increase Small increase Decreases Shifts to lower cm-1, deuteration of G 8 proton on A and G Moderate increase No change in intensity, shifts t o lower cm-1 No change in intensity, shifts t o lower cm-1 Moderate increase in intensity Large increase in intensity due mainly to G No change Large increase in intensity, shifts to lower cm-1
656 677 716 734 765 785
672 683 729 750
920 966 1013 1047 1091
1260 1300 1343 1375 1418
1015 1051 1094 1144 1186 1214 1225 1240 1259 1303 1340 1378 1421 1463
1484
1491 1501 1520
1521
1575
1534 1579
1620 1673
1660
Assignment
T G A T PO, diester symmetric stretch PO2 diester symmetric stretch and C PO2 diester antisymmetric stretch ? Deoxyribose-phosphate Deoxyribose Deoxyribose Deoxyribose C-0 stretch C-0 stretch C-0 stretch PO2- dioxy symmetric stretch Deoxyribose-phosphate T T A T c, A A A T, A A, G Deoxyribose-phosphate G, A
A
C? C=O of T
ERFURTH AND PETICOLAS
256
1.651
xe
!
1.45
Temperature, "c
Fig. 6. Plot of the Iiaman intensity of the 1300-cm-' adenine band (relative to the 1091-cm-' band) of calf thymus DNA in 1 1 2 0 vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and pI1 7.0.
Temperature,
OC
Fig. 7. Plot of the Raman intensity of the 1638-cni-' thymine band (relative to the 1091-cm-l band) of calf thymus DNA in DZO vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and pl) 7.0.
vibration occurs a t this frequency. Figure 7 is a plot of the 1638-cm-' band in D20 versus temperature. Again the plot shows a similar premelting behavior as observed in the adenine band (Figure 6). The band a t 1673 em-' in Figure 5 is particularly interesting sincc it is a C=O vibration of thymine, which is involved in interbase hydrogen bonding. Figure 3 indicates a small band a t 1660 cm-' when thymine is abscnt, n-hich is due to guanine. Comparison of the highly stacked double-helical poly G-poly C
MELTING AND PREMELTJNG IN DNA
257
with the single-stranded unstacked poly G indicates little discernible difference in the carbonyl region of the s p e ~ t r a . ~From ~ , ~this ~ we conclude that the intensity changes and shifts observed in DNA a t 1673 cm-’ are due entirely t o thymine. Figure 5 shows a large gradual shift of 14 cm-I with a concurrent increase in intensity for the thymine C=O vibration, in going from the low-tcmperature double-helical form (1673 cm-l) to the high-temperature random-coil form (1658 cm-l). Similar shifts upon melting for both carbonyl vibrations of uracil have been found in the ir spectra of the poly U double helix3and the poly (A 2U) triple helix3 in which both carbonyl groups of the uracil arc found t o be involved in hydrogen bonding. Significant differences have been found in the double-bond region for poly A-poly U and poly (A-U)poly (A-U) a t low temperature in both the ir35,3G and Raman20,3G spectra. The high-temperature spectra were found to be quke similar for each technique. These differences in the carbonyl region of the low-temperature spectra for the two polymers were attributed to changes in the vibrational coupling of the vertically stackcd bases.36 Thus it is difficult to distinguish between the base stacking and base pairing interactions in the carbonyl region. At 25°C the Raman spectrum of the double-helical form of the alternating copolymer of poly d(A-T) in D2019shows an intense carbonyl band a t 1669 cm-l, which is quite close in frequency to the low-temperature thymine carbonyl band a t 1673 cm-’ in DNA. However the carbonyl band of 5’TMP,35in which no interbase hydrogen bonding or base stacking is cxpected, occurs a t 1659 em-’, which is quite close t,o the high-temperature thymine carbonyl frequency of 1658 cm-l in DNA. Therefore the shift in the carbonyl frequency of DNA upon melting is related to both base unstacking and the breakage of the interbase hydrogen bonds. At this time, however, we cannot distinguish between hydrogen-bond breaking and base unstacking in the carbonyl region. Evidence for thc breaking of interbase hydrogen bonds in the premelting region has been given by Printz and von Hippel using the hydrogen exchange technique. 1.2 Poly d(A-T) is very similar in structure to B-form DNA.38 The Raman spectra of aqueous solutions of poly d(A-T)20below and above the melting point show nearly all of the same intensity changes that we have discussed in this work for adenine and thymine upon melting DNA. Studies on the premelting of poly d(A-T) have been made using the CD technique, which shows changes in the CD all the way down to the freezing point of the solution. However, within the error of our measurements, no changes are observed in the Kaman spectra below 50°C. This again illustrates the differences obtained with different optical techniques. The guanine band, which shows the greatest premelting, occurs a t 1575 cm-’ in DzO. A small adenine band also occurs a t this frequency (Figure 3) but i t is not hypochromic in either poly A-poly UZ0or poly A. l 9 A graph of the intensity changes in the 1575-cm-l band in DzO versus temperature is shown in Figure 8.
+
258
ERFURTH AND PETICOLAS
30405060708090 Temperature, C ' Fig. 8. Plot of the Raman intensity of the 1 5 7 5 - ~ m -guanine ~ band (relative t o the 1091-cm-' band) of calf thymus DNA in I>zOvs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and PI) 7.0.
Temperature,
"C
Fig. 9. Plot of the Raman intensity of the 765-cm-I cytosine band (relative to the 1091-cm-' band) of calf thymus DNA in D20 vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and PI) 7.0.
The only cytosine band in which we were able t o find premelting was the 765-em-' band in DZO. A graph of the intensity change of this band in DZO versus temperature is shown in Figure 9.
Bands That are Independent of Temperature Two bands occurring a t 785 and 1091 em-' in DzO have been found to be independent of temperature on comparison with the sodium cacodylate band a t 610 cm-l a t temperatures above 50°C. The band a t 785 em-' in DNA has been previously assigned to the 0-P-0 dicster symmetric stretchz0while the band a t 1091 em-' is assigned to thc 0-1'-0 dioxy symmetric stretch. 3 1 , 3 2 Although the POz diester symmetric stretch of the
MELTING AND PREMELTING I N DNA
259
DNA chains has never been positively identified, from normal coordinate calculations on model compounds there is some evidence that it lies near 790 cm-1 where it is obscured by base vibration^,^^^^^ particularly the cytosine vibration a t 786 em-' in HzO. In DzO solutions of DNA where the cytosine band is shifted to 765 cm-' (Figure 5), an intense band is still found a t 785 crn-l, while in the monomer solutions (Figure 3) only a very small band is found a t this frequency. Therefore this band at 785 cm-' is probably part of the deoxyribose-phosphate backbone. The intensities of two adenine bands found a t 1343 and 1520 em-' appear not to change with temperature.
Bands That Decrease With Increasing Temperature Several deoxyribose or deoxyribose-phosphate vibrations are found between 828 and 1047 cm-l as seen in Figure 5. All decrease in intensity except the band a t 867 em-', which decreases after 1 hr a t temperatures above the T,. These backbone vibrations of the polymeric deoxyribosephosphate chain give an indication of the changes observed in the backbone conformation upon melting. None of these backbone vibrations show any premelting. Thus the geometry of the deoxyribose-phosphate backbone seems to remain intact up to the melting point. It can be seen from Figure 3 that no base vibrations occur in this region. In order to obtain a handle on the backbone we have examined thc behavior of the Raman active vibration a t 1047 em-' in DzO, which has been The ~ tentatively assigned to the deoxyribose C-0 stretching v i b r a t i ~ n . ~ 1047-em-' band is found to get broader and weaker upon melting. A graph of the intensity change in the 1047-cm-I band versus temperature is shown in Figure 10. The graph indicates that no premelting is occurring in this backbone band.
.
Temper a ture *C
Fig. 10. Plot of the Itaman intensity of the 1047-cm-l backbone C-0 band (relative to the 109l-cm-' band) of calf thymus DNA in D 2 0 vs. temperature, 0.01 M sodium cacodylate, 0.001 M EDTA, and PI) 7.0.
260
ERFURTH AND PETICOLAS
Since no changes are seen in the various deoxyribose-phosphate vibrations until the melting point is reached, this would indicate that the deoxyribosephosphate backbone conformation is not changing in the premelting region. Previous studies on fibers of calf thymus DNA have shown that Raman spectroscopy is quite sensitive to the backbone conformation of the DNA, i.e., the A family and B family of conformations.22 From the spectra taken at various temperatures, the DNA seems to remain in the B conformation until the melting point is reached, at which time it appears the DNA progresses into a disordered random-coil form. No evidence for an Aform conformation is found either in the premelting or,melting region due to the lack of an 807-em-' band. This is in agreement with the results of Gennis and Cantor." One base band due t o guanine, found at 677 cm-l in DzO, was found to decrease in intensity with increasing temperature. A similar effect has been observed in t-RNA for a guanine band at 670 cm-' upon melting.25
Reversibility Changes resulting from high temperature were completely reversible as long as the temperature remained below t h r T,. Increasing the tcmperature beyond the T , value and then rapidly cooling the sample back t o 23°C do not result in return t o the well-ordered low-temperature spectra. Spectra of calf thymus DNA in HZO and DZO were taken after the samples were at 9S"C for about 1 hr and were then rapidly cooled to 23°C. The samples were allowed t o cool for 10 min before the spectra were taken. An initial rapid decrease in the baseband intensities occurred, indicating a number of the bases are immediately stacking presumably with reforming of a number of interbase hydrogen bonds. 3Iost base bands did not completely decrease back t o their original height, indicating that the dcnaturation process (melting out) is irreversible under the procedurcs used here. Thus some bases remain unstacked probably due to mismatching of homologous segments, formation of loops, and from segments that remain single-stranded. It is quite interesting t ha t the deoxyribose-phosphate backbone vibrations (825-1061 cm-') reappeared a t almost their full intensity and appropriate frequency. This appears to indicate that at low temperatures the deoxyribose-phosphate backbone tends t o go into a stable conformation representing a potential energy minimum, which has the same geometry as the double helix (B type).
Deuteration of the C-8 Protons of Adenine and Guanine The slow isotopic exchange of the C-S proton of purincs has been studied by a number of techniques. Using nuclear magnetic resonance Bullock and Jardetzdy*l and Schmeizer e t have shown that the C-8 proton of purines exchanges with the deuterium of DzOa t tcmperaturrs near 100°C. Shelton
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MELTING AND PREMELTING I N DNA
2% dGMP
I
In c m
w
pD7.0
n (I
04. C
( 3 (3
25. C (3 (3
(I
(3
Fig. 11. Itaman spectra of 2c0dGMP in II2O a t 25" and 84°C. The top spectrum was taken after being a t 84°C for 4 hr.
and Clark43using tritium labeled water concluded that only the purine nucleosides exchange and t h a t the half-time for exchange a t 100°C was about 1 hr. Doppler-Bernardi and F e l ~ e n f e l dhave ~ ~ described an exchange method for producing tritium labeled native DNA in vitro with minimal physical damage to the DNA. At temperatures above 70"C, we have observed a large shift to lower frequency of a base band a t 1484 cm-lin DKA (Figure 5 ) , which is assigned to adenine and guanine ring vibrations. The spectra in Figure 5 a t 75", 80", and 90°C were equilibrated for 2 hr a t the corresponding temperatures to show the changes in the 14S4-cm-l band. At 84°C this band shifts from 1484 t o 1457 em-' in about 4 hr. A study of the same band in d G N P (Figure 11) and dAMP (Figure 12) in D,O a t S4"C produced a similar shift in 4 hr.45 I n d G N P (Figure 11) the 1480 cm-' band shifts t o 1457 cm-' while in d A 3 P (Figure 12) the band shifts from 1481 to 1465 cm-'. YO changes were found for DNA, dGMP, or d A N P in aqueous solutions under the same conditions. These changes are not reversible upon lowering the temperature to 23°C. From these observations, we have assigned this frequency shift from 1484 t o 1457 cm-I in DNA to the slow druteration of the C-8 protons of adenine and guanine. Quantitative measurements of the rate process have been made and will be reported elsewhere.
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ERFURTH AND PETICOLAS
Fig. 12. Rama.n spectra of 2 7 , dAMP in 1 1 2 0 at 25" and 84°C. The top spectrum was taken after being at 84°C for 4 hr.
CONCLUSIONS Certain Raman bands arising from each of the four bases, adenine, thymine, guanine, and cytosine undergo a gradual increase in intensity prior to the melting point. These changes indicate that disruption of vertical base-base stacking interactions occurs beginning a t about 50"C and increasing up until the melting point. However, observation of the various deoxyribose-phosphate backbone vibrations between 828 and 1060 em-' shows that the DNA backbone. does not undergo any significant conformational changc. prior to the melting point. This indicates that the backbone conformation of the helix is not disordered by the premeltirig phenomenon. As long as the temperature remains below the T , the changes arc completely reversible upon rapid cooling. Temperatures above the T , permit breaking of hydrogen bonds and unstacking of t h r bases rtlsulting in a t least partial separation of the double helix and formation of a random-coil form, which is irreversible in terms of reformation of the original orderrd duplex. However, rven aftw melting out, the deoxyribose.-phosphatc backbont. in DNA found a t low temperature is a very stable potential energy minimum,
MELTING AND PREMELTING I N DNA
263
which seems to be fully recovered after melting and then partial renaturation of the helices. Beginning a t about 50°C the carbonyl stretching band of thymine, involved in the interbase hydrogen bonding, was found to undergo a large gradual shift of 14 em-' to a lower frequency, with a concurrent increast. in intensity. Similar shifts to lower frequency with concurrent intensity changes have been found in the ir and Raman spectra of polyribonucleotidcs containing uracil.20 33-36 I'rintz and von Hippel' have shown that DNA is subject t o local structural fluctuations in the prcmelting region, u hich results in frequent opening and closing of the structure a t the level of the nucleotide pair. Thus both interbase hydrogen-bond breaking and bas0 unstacking appear to be occurring in the premelting region. At this time, however, we cannot distinguish between hydrogen-bond breaking and base unstacking in the carbonyl region. By comparing the spectra of DNA a t various temperatures to those of previously published spectra of known backbone conformation, i.e., the A and B forms,22it is possible to sec that DNA does not go into the A conformation either in the premelting or melting region. Rather the DNA remains in the B-form geometry up to the melting point, a t which time it goes into a disordered random-coil form. Finally, a large shift from 1484 to 14.57 cm-I has been observed in an inplane ring vibration assigned to guanine and adenine at temperatures above 70°C. At 84°C this band in deuterated DNA, dGJIP, and dAJI1' was found to shift in about 4 hr. This frequency shift has been tcntatively assigned to the slow deuteration of the C-S protons of adenine and guanine. This same shift has recently been observed by Thomas.46
References 1. Printz, M. P. & von Hippel, P. H. (1965) Proc. Nut. Acad. Sci. U.S. 53,363-370. 2. von Hippel, P. H. & Printz, M. P. (1965) Fed. Proc. 24, 14.18-1463. 3 . McConnell, B. & von Hippel, P. H. (1970) J . Mol. Biol. 50, 297-316. 4. McConnell, B. & von Hippel, P. 13. (1970) J . Mol. Riot. 50, 317-332. 5 . Utiyama, H. & I h t y , P. (1971) Riochemistry 10, 1254-1264. 6. von Hippel, P. H. & Wong, K. Y . (1971) J . d f o l . Biol. 61,587-613. 7. Ts'o, P. 0. P. & Helmkamp, G. (1961) Tetrahedron 13, 198-207. 8. Fresco, J. R. (1961) Tetrahedro,L 13, 183-197. 9. Brahms, J. & Mommaerfs, W. F. H. M. (1964) J . Mol. Biol. 10.73-88. 10. Ts'o, P. 0. P., Itappaport, S. A. & Bollrim, F. J . (1966)Riochemistry5,41.?S-4170. 11. Gennis, It. B. & Cantor, C. It. (1972) J . Mol. 13iot. 65, 381-399. 12. Freund, A. & Bernardi, G. (1963) i\'ature 200, 1318-1319. 13. Tomlinson, B. L. & Peticolas, W. L. (1970) J . Chem. Phys. 52,2154-2136. 14. Peticolas, W. L., Nafie, L., Stein, P. & Fanconi, B. (1970) J . Chem. Phys. 52, 1576-1384. 15. Thomas, Jr., G. J., Mederios, G. I>. & Hartman, K. A. (1971) Biochem. Biophys. Res. Cornmu?,.44, 587-592. 16. Aglward, N. N. & Koenig, J. I,. (1970) Macromolecules 3 , 570-596. 17. Thomas, Jr., G. J. & Hartman, K. A. (1973) Riochim. B i o p h y s . Acta 312,311-322. 18. Thomas, Jr., G. J., Chen, M. C. & Hartman, K. A. (1973) Riochim. Biophys. Acla 324,3749.
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19. Small, E . W. & Peticolas, W. L. (1971) Biopolymers 10, 69-88. 20. Small, E. W. and Peticolas, W. L. (1971) Biopolymers 10, 1377-1416. 21. Thomas, Jr., G. J. (1970) Biochim. Riophys. Acta 213,417-423. 22. Erfurth, S. C., Kiser, E. J . & Peticolas, W. L. (1972) Proc. -\at. Acad. Sci. U.S. 69,938-941. 23. Brown, K. G., Kiser, E. J . & Peticolas, W. L. (1972) Hiopolymers 11, 1855-1869. 24. Lafleur, L., Rice, J. & Thomas, Jr., G. J. (1972) Biopolymers 11, 2423-2437. 25. Small, E. W., Brown, K. G. & Peticolas, W. L. (1972) Biopolymers 11, 1209-1215. 26. Hartman, K. A., Clayton, N. & Thomas, Jr., G. J. (1973) Biochem. Biophys. Res. Commuii. 50,942-949. 27. Pezolet, M. & Peticolas, W. L. (in preparation). 28. Lord, R . C. & Thomas, Jr., G. J. (1967) Speclrochim. Acta25A, 2551-2591. 29. Sober, H. A,, Ed. (1970) Haudbook of Biochemistry, 2nd ed., Chemical Rubber Co., Cleveland, Ohio. 30. Mandel, M. & Marmur, J. (1968) dtethods Euzymol. 12B, 195-206. 31. Schimanouchi, T., Tsuboi, M. & Kyogoku, Y. (1964) Advan. Chem. Phys. 7,435498. 32. Tsuboi, M. (1957) J . Amer. Chem. SOC.79,1351-1354. 33. Bode, I)., Heinecke, M. & Schernau, U. (1973) Biochem. Biophys. Res. Commun. 52,1234-1240. 34. Miles, H. T . & Frazier, J. (1964) Biochem. Biophys. Res. Commun. 14,21-28. 35. Morikawa, K., Kyogoku, Y . & Tsuboi, M. (1969) cited in Tsuboi, M., Appl. Spectrosc. Rev. 3 , 4 5 9 0 . 36. Morikawa, K. Tsuboi, M., Takahashi, S., Kyogokn, Y., Mitsui, Y . , Iitaka, Y. & Thomas, Jr., G. J. (1973) Biopolymers 12,799-816. 37. Erfurth, S. C. & Peticolas, W. L., unpublished results. 38. Davies, 1). It. & Baldwin, It. L. (1963) J. Mol. Biol. 6,251-235. 39. Brown, E. & Peticolas, W. L., unpublished results. 40. Tsuboi, M. (1969) Appl. Spectros. Rev. 3 , 4 5 9 0 . 41. Bullock, F. J. & Jardetzky, I). (1964) J. Org. Chsm. 29, 1988-1970. 42. Schweizer, M. P., Chan, S. I., Helmkamp, G. K. & Ts’o, P. 0. P. (1964) J. Amer. Chem. SOC. 86,696-700. 43. Shelton, K. It. & Clark, J. M. (1967) Biochemistry 6,2735-2739. 44. Doppler-Bernardi, F. & Felsenfeld, G. (1969) Biopolymers 8,733-741. 4.5. Kiser, E. J., Yu. T . J. & Peticolas, W. L. (in preparation). 46. Thomas, G. T., private communication.
Received August 13, 1974 Accepted September 25, 1974
