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Conformational stability of dinucleotides in solution
J. Afol. Biol. (1967) 25, 481-495
Conformational
J. BR&Mst,
J. C.
Stability of Dinucleotides in Solution MmmzoTt
AND
A. M.
MICHELSON
Centre de Recherches sur les MacromoE6cules 6, rue Boussingault, ii’trasbourg Institut de Biologic Physicochimipue 13, rue Pierre Curie, Paris, France (Received17 Octobw 1966) A variety of 3’ + 5’ and 2’ -+ 5’ dinucleoside phosphates (at neutral pH) have been studied using circular dichroic and absorption spectra in a range of temperature of - 20 to + 80°C. Two kinds of circular dichroic spectra can be observed represented by ~3,pair of bands of at low temperature : one “conservative” opposite sign, the other ‘Lnon-eonservative” composed of one main positive band, characteristic of guanine and cytosine compounds. Both spectra are consistent with a model of dissymmetrical, single-stranded helical conformation. Thermodynamic parameters for the process order +G disorder in. dinucleotides have been estimated by following thermally induced changes in circular dichroic spectra. Values are similar for all the 3’--+5’ derivatives and are of the order of 6 to 7.5 k&./mole for dHo, about 20 to 25 e.u./mole for AS0 ad about 0.5 kcal./mole at 0°C for APO. This absence of major differences in th.ermodynamic parameters does not permit one to divide dinucleotides into groups characterized by a stacked or unstacked conformation; for example, 3’ -+ 6’ uridylyl-uridine (UpU) is appreciably stacked at 20°C. Examination of corresponding 2’ -+ 5’ dinucleoside phosphates gave quite different results in the same range of temperature, indicating that stacking is much less marked compared with the 3’-+ 5” compounds. An intramolecular hydrogen bond involving the 2’-hydroxyl group of the ribose is postulated to explain these differences.
1. Introduction In our previous studies using circular dichroism, we have shown that oligo and po$nucleotides of adenylic acid (Brahms, Michelson & Van Nolde, 1966) and cytidylie rahms, Maurizot & Michelson, 1967) assumea single-strand, helical conformation under appropriate conditions. The stability of such structures is determined by forces other than intermolecular base interactions, but does involve stacking interactions between parallel bases. The thermal denaturation was shown to be essentially a non-co-operative process. Similar results and conclusions have been obt&ned using optical rotatory dispersion (Poland; Vournakis & Scheraga, 1966) and rdtraviolet absorption (Leng & Felsenfield, 1966). The single-strand, helical polynzrcleotide chain can be represented as a collection of stacked dimers which melt independently. T Present address: Institut
de Biophysique
MolBculaire,
481
45 OrlBans-La
Source.
482
J. BRAHMS,
J. C. MAURIZOT
AND
A. M. MICHEESOPU’
Studies of the conformation and conformationalstability of simple dimers of various base composition can provide data for a better understanding of the conformation of nucleic acids in solution. One can consider that RNA’s may have a fraction or the entire molecule in a single-strand conformation in the absenceof complementary base pairing. The formation and stability of such stacked basestructures will depend upon the different thermodynamic values of interaction between residues of different base composition. At present, such values have been experimentally determined only for adenylates and cytidylates; other available values are the theoretical predictions of De Voe $ Tinoco (1962), Pullman, Claverie & Caillet (1966), Claverie, Pullman & Caillet (1966), Pollack & Rein (ZYKJInt. Biophya. Congr. Abstr. p, 139, 1966). This paper presents studies using oircular dichroism which indicate that various 3’ -+ 5’ dinucleoside phosphates assumeat neutral pH a dissymmetrical conformation with stacked basesin the absence of intermolecular hydrogen-bonded base pairing. This is in general agreement with the earlier work of Michelson (1959) on hypochromism in simple dimers, and the results of optical rotatory dispersion studies of Warshaw & Tinoco (1965) and Massoulie & Michelson (1964). The thermodynamic parameters for the thermal denaturation presented here are very similar for all 3’ -+ 6’ dinucleoside phosphates and are in contrast with the behaviour of 2’ +- 5’ derivatives. It is proposed that an internal hydrogen bond between the 2’-hydroxyl group and a phosphate oxygen atom contributes to the oonformation of singlestranded helical polyribonucleotides as well as the stacking interactions previously considered.
2. Experimental
Methods
(a) Materials The 3’ + 6’ and 2’ -+ 5’ dinucleosidephosphateswere synthesized by chemical teohniques (Michelson, 1959). A2’p5’C was purchased from Waldhof Co. The solvent used was 4.7 M-KF-O-01 M-Tris at pM 7.5. This solvent was chosen in ord.er to extend the spectral measurements to relatively low temperatures. The freezing point of the solvent is below - 20%. The use of low temperatures serves two purposes in that the resolution of circular dichroic spectra is improved and also gives the low-temperature parameters for the thermodynamic analysis. It should be emphasized that RF is transparent in the ultraviolet spectral region under investigation. There was no detectable effect of salt concentration on circular dichroic spectra of dinucleoside phosphates at neutral pH, indicating that the ionic strength has no important influence on singlestranded helical conformation. (b) Meusurernent of circular diclwoim and absorption spectra These spectra of dinucleoside phosphates were made in a concentration range of low4 to 10w3u (per nucleotide residue). In general, there was no detectable influence of concentretion on oircular dichroic spectra. However, study of 3’ + 5’ GpC showed that at lower temperatures circular dichroism was dependent on concentration between 10d4 and 10-3nix. We have chosen to perform the measurements at high dilution, in conditions excluding all irttermolecular association processes. Cells of 0.1, 0.2, 0.5 cm path length were employed. The experiments were carried out at controlled temperatures using a Lauda cryostat and a Haake thermostat. The temperature was monitored by a thermocouple in contact with the cell and connected to a Sefram recorder. In order to prevent evaporation, a closed cell was used. The circular dichroism and the rotational strength are given per nucleotide residue and were calculated were determined by as previously indicated (Brahms, 1963). E values for the dinucleotides alkaline hydrolysis to the monomer.
DINTJCLEOTIDES
IN
SOLUTION
48
3. Results and ~i§~~$~i~n (a) Ci’rcular
dichoism and absorption spectra
We haw measured the circular dichroism and absor@ion speotra of ten 3’ --> 5’ ribodinucleoside phosphates at neutral pII (in 1.7 M-HF--@O1 M-!&S, pII 7.5) at various temperatures between -20 and 80°C. Figure 1 showsthe results of circular diohroism measurementsof three dimers at the lowest temperature used. Also shown are the absorption spectra of the dimers and the circular dichroism curves of the monomer constituents. From the comparison of the results of Fig. 1, one can clearly seethat the eirdac dichroism speotrum of each 3’ -+ 5’ dinucleoside phosphate is qualitatively and quaut~tatively very different from that of its monomer components. T&s is reflected in differences in general shape of the circular dichroic spectra, in the position and even in the sign of the bands (seealso Fig. 2). In contrast, the absorption spectrum of each dimer is very similar to that of its corresponding components, apart from slight hypoehromie effects near h mBx. The general pattern of circular dichroism curves iti
PIG. 1. Circular dichroic spectra of three 3’+ 5’ dinucleoside phosphates at neutral pEI in 4.7 - 16 to -20% (-); the absorption spectra (-- -) m-KF-OGl-x-T& and at low temperature, we measured at 25% The circular diohroism of some of the monomer oonstituents (. . ~) is shown for comparison.
different for various dimers. In Fig. 1 are shown sometypical examples. It is apparen; that there are two categories of circular dichroic speotra,. The first category of circular dichroic spectra is represented essentially by a pa.k of jacent positive and negative bands with an intersection point situated at a wavelength very near that of the absorption maximum. The su.mof the roMiona1 strengths is approximatively equal to zero in the spectral region under investigation. The shape of these spectra is in agreement with the prediction of the exciton theory of optical rotatory power of polynucleotides (Tinoco, 1964), according to which the ~~te~a~tiol~among identical chromophores in a dimer will give rise to the splitting of the monomer band into two bands. It was previously shown that the exciton model tith single-strand helical geometry when applied to Apia (Van IIolde, Brahms & XEeheSson,1965; ‘Warshaw, Bush $ Tinoco, 1965) and to oligoadenylates (Brahms et al., 1966) allows satisfactory prediction of the shape of the observed circular dichroism curve, including the position of the bands and their rotational strength v&es. It is to be noticed that the predictions for non-degenerate interactions in a dimer
484
J.
BRAHMS,
J.
C. MAURIZOT
AND
A.
M.
MICHELSON
composed of different bases like ApU (Bush, 1965) are in good agreement with observed circular dichroic spectra (Fig. 1). This type of circular dichroic spectra is also observed for ApC, CpA (Fig. 2) and CpU (Fig. 3). In these cases the agreement may be fortuitous, since the presence of more than one band in cytosine may cause overlapping and cancellation. The second category of circular dichroic spectra consists of one or two positive bands as shown for GpA, GpC (Fig. 1) and also CpC. One can clearly see that the sum of rotational strengths is not equal to zero in the spectral region under investigation. This type of spectra is not predicted by the exciton theory of optical rotatory power. The differences between these two categories are not so clearly seen from the optical rotatory dispersion curves. It must be emphasized that these circular dichroic spectra are exhibited by dimers with guanine and/or cytosine. It is well known that the absorption spectra of guanine and cytosine at neutral pH are complex and have at least two overlapping bands in the spectral region of 210 to 320 rnp. Hence calculation of the rotational strengths for all dimers containing guanine or cytosine should include the contribution arising from non-degenerate interaction of these two near ultraviolet bands (Bush, 1965). The predicted rotational strengths will still sum to zero. The predominant positive rotational strength in the near ultraviolet can be calculated if one includes the interaction with the far ultraviolet transitions. This contribution is of non-conservative character and depends on the direction of the electronic transition dipole moment (Bush & Brahms, 1967). The orientation of the transition moment of guanine and cytosine must be such that the contribution of far ultraviolet terms is large when in a single-strand structure, whereas for adenine and uracil it is small. In general, the calculated values for a model of stacked bases with the geometry of a single-strand DNA are consistent with the experimental results (Van Holde et al., 1965; Warshaw et aZ., 1965; Bush & Brahms, 1967). It will be noted that the circular dichroio spectra were obtained with highly dilute solutions (10w4~), in conditions which rule out the possibility of intermolecular hydrogen bonding or other associative processes as an explanation of the optical activity (see Experimental Methods). Further support for the stacked-model conformation can be found in the X-ray data on crystals of 2’ -+ 5’ ApU (Shefter, Barlow, Sparks & Trueblood, 1964).
I”
-44 220
”
230
240
250
260
270
280
290
Wavelength,
220
”
”
”
I
230
h(rnp)
FIG. 2. Circular clichroic spectraof 3’+ 5’dinucleoside phosphatesisomers (-) The absorption tures (-18 to -20°C) in 4.7 M-KF-0.01 M-Tris at neutral pH. corresponding 3’ +6’ compounds are: left: (---) UpG and (-.-.-) GpU; right (-.-.-) ApC. Circular dichroic spectra (. . .) of the monomer constituents.
at lowtemperaspectra of the (---) CpA and
DINUCLEOTIDES
IN
SOLUTION
485
Figure 2 shows the circular dichroic and absorption spectra of four 3’ + 5’ sequenee isomers taken as an example. It can be clearly seen that the circular dichroic spectrum is very sensitive to the base sequence. Thus 3’ -+ 5’ GpU at about - 20°C exhibits four well-resolved bands in a spectrum quite different from that of 3’ -+ 5’ UpG (see Table 1). The absorption spectra show one broad band which is very similar for both isomers. Circular diehroism measurements thus allow detection of the contdbution of several
TABLE
1
Circular dichroism and absorption spectral data Substance
3’ 3 2’ 3 3’4.5’ 3’ -+ 2’ -+ 3’ -+ 3” -+ 3’ -+ 3’ 3 3’+ 3’ --f 3’ + 2’ -a
)Imax (mp)
Absorption cx 10-3 (cm-lmol-ll.)
spectrum A1 (mp)
Circular dichroism R2X 104” h3 (w-4
-
5’ cpc 5’ cpc UpU 5’ ApC 5’ ApC 5’ CpA 5’ UpG 5’ GpU 5’ cpu 5’ GpC 5’ GpA 5’ ApU 5’ ApA
269 269.5 260 261 262 261.5 255.5 258 264.5 266 258 260 258.5
7.9 7.8 9.1 10.0 10.0 IO.0 IO..% 10.0 8.1 8.9 13.1 11.4 12.9
280 275 272 274.5 277.5 274.5 278.5 281.5 276 284 284.5 268.5 270.5
+ 25.9 +13 + 14.5 i 14.5 + 6.7 t 12.7 -t 3.5 + 0.6 +20.5 + 3.3 - 0.6 + 7.8 + 9.7
2465 255.5 260.5 265 257.5 266.5 275.6 255 252.5 257.5 258.5
CNIP UMP AXP GXP
271 262 259.5 252
9.0 10.0 15.4 13.7
272 265
+12 + 9.3
242 244
246
+ 0.9
245 236 251-5 235.5 258 267 237 254 266 249 249
- 8.9 -14.7 - 8.5 - 7.5 - 1.4 - 1‘3 - 8 + 4.0 t 4.7 - 3.2 - 4.9
R, x 1OklJ
232
218
+
1.2
228 249
+
4.5
229 228
-
1.5 4.2
Symbols are as follows: AmaX is the maximum wavelength in mp; E is the extinction coefficient at this wavelength. For the circular dichroic spectrum, hl, Xs, & are the wavelengths of the extremnm, and R1, Rz, Rs are the rotational strengths of the corresponding circular dichroic bands and & the orossing point. The circular dichroic spectra of dinucleoside phosphates were measured at relatively low temperatures of about -20°C (for details see Experimental Methods).
transitions not resolved in the absorption spectrum. The sequence dependence of the circular dichroism is also shown in spectra of 3’ --t 5’ ApC and CpA (Fig. 29. In general, our circular dichroic results are in good agreement with absorption spectra! data of Michelson (1963) and optical rotatory dispersion data of Viiarshaw & Tinoco <1965j, showing clearly that optical methods and more particularly circular dichroism and optical rotatory dispersion can be useful tools for the determination of base sequence. In Table 1 are summarized circular dichroic and absorption spectra characteristic of various 3’ -+ 5’ dinucleoside phosphates and some 2’ + 5’ derivatives. In contrast to the circular dichroism, the absorption spectrum of each dimer is very similar to that of its corresponding monomer components. The hypochromicity neaLI x Ill&Xis relatively small and is not greater than some 15%. We notice that almost al! 3’ -+ 5” dimers exhibit a positive longer wavelength circular dichroic band which can be correlated with the right-handed helical senseat least for dimers containing bases
486
J. BRAHMS,
J.
C. MAURIZOT
AND
A.
I%. MiPICHELSON
possessing a single absorption band in the near ultraviolet; namely, ad&no, uraeil. However, in one case, that of 3’ -+ 5’ GpA, we observe that the longer wavelength band is of negative sign and of relatively weak intensity. The detailed circular dichroic spectra described above were detectable only by recording at relatively low temperatures of about -20°C. We have not attempted to predict the circular dichroism characteristics for various dimers, since the exact parameters, such as the orientation of electric dipole transition moments, are not determined for guanine and cytosine. However, it has been shown that using reasonable approximations for a single-strand, stacked model of CpC, a positive near-ultraviolet circular dichroic band in agreement with the observed curve can be predieted (Bush & Brahms, 1967). (b) ThermaZ denaturtiion Since the intensities of the circular dichroio bands of various dinwleoside phosphates are very different, it is diEcult to derive a yrmntitative understanding of the stacked helical conformation from the results of measurements made at one temperature only. For this reason and also in order to determine the thermodynamia parameters, the circular dichroic spectra were measured at various temperatures ranging from -20 to 80°C.
FIG. 3. Circular dic’hroic spectra of 3’+5’ dinucleoside phosphates at various Left: 3’-+-5’ CpU at: (1) -20°C; (2) 3%; (3) 28’C; (4) 60°C; (5) 72°C. Centre: 3’+5’UpU at: (1) -18’C; (2) 2°C; (3) 26OC; (4) 41%; (6) 67%. Right: 3’+5’ CpA at: (1) -20°C; (2) 0°C (3) 23°C (4) 81°C. Conditions are as described in Figs 1 and 2. Dotted line: the circular dichroio monomers.
temperatures.
spectra
of the
Figure 3 shows the results for circular dichroism at various temperatures of three 3’ -+ 5’ dinucleosides phosphates as examples. It can be readily seen that the intensity of circular dicbroic bands changes markedly in the range of temperature used. At higher temperatures the intensity of the longer wavelength positive band decreases and approaches that of the monomer oomponents. Since there is no spectral shift of the position of the maximum of circular dicbroic bands at ah. temperatures used, it is possible to follow quantitatively the thermal denaturation process either by measuring the circular dichroism at the wavelength of the maximum or the rotational strength of the positive band as a function of temperature. We have chosen to
DINUCLEOTIDES
FIG.
IN SOLUTI0.N
487
4. Temperature dependence of circular dichroism of 3’ + 5’ and 2’ + 5’ dinucleoaide in 4-7 M-KF-0.01 ix-Tris at neutral p’II. The magnitude of circular diehroism is taken maxim,m of the main, longer wavelength positive band (il~),~,. The symbols are ae
phosphates at the
+5’cpU;(@)3’-+5’ApC; 2’+
5’AipA;
(A)
2’+
5’CpC;
(0)
2’+
5’ApC;
(0) 3’+5‘UpU; (A)
3’+
( ) 3’+ 5’ ApU; (TJ)
5’GpA.
determine the changes in (Q,--Ed) at various temperatures, since calculation of the rotational strengths is difficult for dimers showing complex circular dichroic spectra, especially at higher temperatures. Figure 4 shows the circular dichroism thermal proiiles for a series of 3’ -+ 5’ dinucleoside phosphates. Despite the difference in intensity of circular dichroism (hIl,X) for dinucleoside phosphates of different base composition at a given t,emperatnre, it can be seen that the “melting” curves are of similar, sigmoidal shape and of gradual character, At higher temperatures, the values of de,,, approach those of the monomer components (Table l), These residual values depend on the nature of thp? monomer, and as a result the various melting curves of the 3’ -+ 5’ dinucEeoside
488
J.
BRAHMS,
J.
C.
MAURIZOT
AND
A.
M.
MICHELSON
phosphates are displaced on the intensity scale. The 2’ -+ 5’ dimers studied (ApC, ApA, CpC) show relatively very small changes of AC,,, over the entire range of temperatures. This will be analysed later. It is di&ult to make a quantitative comparison of the thermal denaturation curves of the various 3’ -+ 5’ dinucleoside phosphates shown in Fig. 4. However, the data can be used to construct a graph of the fraction of stacked bases as a function of temperature. For this we use the fact that at high temperatures the dimers have almost reached a completely disordered conformation. One can obtain the limiting value of AC,,, at low temperature, characteristic of a stacked conformation, by two
I
I
I
I
I
I
cpc
2
ApC
3
CpA
4
ApU
5
cpu
0
FIG. 5. Fraction Fig. 4).
-20
0
of stacked
bases
20 Temperature, (x) as a function
40 f (“C)
60
of temperature
80
(conditions
as described
in
procedures: by the extrapolation of the sigmoidal melting curve to a limiting value of de (see Experimental Methods and the accompanying paper, Brahms et ccl., 1967), and as twice the value of AE at the mid-point of the transition. The value of AE at the mid-point of transition was obtained from the shapeof the available part of the curve for which the high-temperature limit can be quite well approximate. As shown in Fig. 5, all the 3’ --f 5’ dinucleoside phosphates yield curves of very similar shape, almost parallel but somewhat displaced on the temperature scale. The same gradual profile and similar steepnessof a,11the curves suggest that the same basic process, essentially non-co-operative, characterizes the melting of all 3’ --f 5’ dinucleoside phosphates. From Fig. 5 it can be seenthat dimers containing pyrimidine residuesonly, such as CpC, have almost the samefraction of stacked basesat various temperatures as dimers containing purine and pyrimidine residues, such as ApC or CpA. Dinucleoside ‘phosphates containing uracil, such as UpU, ApU, CPU, have at 20°C an appreciable fraction of the basesin a stacked conformat;on, contrary to the widespread opinion that the presenceof uridine in a polynucleotide chain causesunstacking (Richards, Flessel & Fresco, 1963; Warshaw & Tinoco, 1965). Furthermore,
DINUCLEOTIDE
S IN
SOLUTION
489
the conformational stability of dimers such as ApU and CpU is not too dissimilar to that of CpA, in which both bases are purines. However, dimers containing uracil doi have a rather smaller fraction of stacked bases at a given temperature than CpC or ApC. Nevertheless, we believe that classifioation into groups of sequences which will be in a stacked or unstacked conformation is unfortunate. From these (thermal denaturation) data, it is possible to determine the apparent equilibrium constant for the denaturation process of various 3’ --+ 5’ dinucleoside phosphates. This can be written as a simple reaction DC,) +Dcd). The apparent equihbrium constant oan be expressed:
Cd
K=Co=_l
IO - I, a t
where Id and I,, represent the limiting values of dernax at high and low temperatures where all the dimers are in a completely unstacked or stacked form, respectiveby; I, is the magnitude of At-,,, at a given temperature. Having obtained values of the equilibrium constant, we calculated standard state enthalpy change and standard state entropy change for the thermal denaturation process. Figure 6 shows the plot
I cpu 2 CpA 3 ApU 4 cpc 5 /\pC
I
28
3.0
I
32
F’ra. 8. A van t’Hoff plot of the thermal (The da&a are taken from Figs 3 and 4.)
I
I
3.4 (I/TX IO? denaturation
CJ * n El A
3.6
3.8
of some 3’ + 5’ dinucleoside
phosphates,
of log K veraus l/T for some dimers taken as examples. The lines of the van t’Hoff plot are straight with similar slopes, indicating that AH0 must be similar for each case. The thermodynamic parameters of 3’ --+ 5’ dinucleoside phosphates are summarized in Table 2. The standard state enthalpy changes are about + 6 k~a~./rn~~e
490
J. BRAHMS,
J.
C, MAURIZOT
AND
A.
M.
MVIICHELSON
to + 7-5 kc&/mole, and the entropy change about + 22 to 25 e.u./mole. The uncertainty of our procedure arisesfrom the estimate of the limiting values of circular dichroism at low temperatures. As indicated previously (Van Holde et al., 1965; Brahms et al., 1966), extreme variations in the extrapolated I,, value do not lead to more than & 10% difference in the calculated value of ALP and AL!?. The technique usedin the present work, applying temperatures of about - 20°C reduces the possible error in the estimates of the low-temperature limit of 1,. Since other errors are relatively small, the over-all limit of uncertainty is not greater than f- 15%. The values of AH0 listed in Table 2 are in good agreement with those obtained for oligo A by Poland et al. (1966), and lower but of similar magnitude when compared with the data of Leng & Felsenfeld (1966). The values of AH” and AX” presented here for various dimers are slightly smaller than those given previously for ApA by Van lyolde et al. (1965) and Brahms et al. (1966). We think that this rather small difference is due mainly to our present use of lower temperatures. Nevertheless, despite different experimental conditions, the values of AH0 and AX” obtained are of the same magnitude (Table 2). TABLE 2 Thermodynamic parametersfor thermal denaturation af 3’4 5’ dinucleosidephosphates(pH 7.5, 4.7 ~-KB’-0~01 M-Tris) Substance
CPA APC CPU APU CPC GPA ApAt
7.0 6.1
PolyU c
6.0 5.3 7.9
P$y The values t The data t Van Holde,
(k0.i~ole)
6.8 6.7 7-5 6.1 8.0
At
(e.$zole)
(kcal.,%e)
(0%)
24 21 24 24 25 22 28
i-o.4 +0.6 $0.2 +0*3 +0*7 +0*2 +0.4
21 16
-I-o.3 -to.9 +1.1
25
are expressed per mole of nucleotide residue. are taken from Van Holde, Brahms & Michelson, 1965 and from 1966; solvent used is 0.1 M-NaGI-0.01 M-Tris (pH 7.4).
18 25 6
11 24 9 25 15 59 40
Brahms,
Michelson
It is clear that fundamental differences in AH0 and dXO values for the 3’ -+ 5’ dinucleoside phosphates cannot be observed. The free energy change at 0°C is of the order of half a kcal./mole in favour of stacking. However, small differences in AF” are present (Table 2). It appears that the stability of a stacked conformation will be favoured by the presenceof cytosine and adenine rather than of uracil or gaanine at neutral pH. From Table 2 it can be seen that small differences in thermodynamic parameters have an effect on the value of T,, the temperature at the mid-point of the thermal “denaturation”. The results of thermal denaturation for single-strand helical polymers, and data found previously for ApA and poly A (Van Holde et al., 1905; Brahms et al., 1966) are also included, It can be seenthat the AH” data for the
DINUCLEOTIDES
IN
SOLUTION
491
are very similar to those of the polymers, confirming previous conchrsions that the melting of such structures has the characteristics of a largely non-co-operative process(Leng & Felsenfeld, 1966; Poland, Vournakis & Scheraga, 1.966;Brahms et al., 1966). Thus regardless of the chemical nature of the residues, the single-strand belices melt in similar fashion. The value of dP” for a polymer is somewhat larger than for a dimer. Thus our results on 3’ -+ 5’ dimers of various base composition can be applied to a ribopolynucleotide chain when in a single-strand conformation, has t,o bear in mind that a pair of stacked residuesis slightly more stable when in a polymeric array. dimers
(c) The 2’ -+ 5’ dinucleosidephosphates Figures 4 and 8 show the thermal denaturation of 2’ --f 5’ isomers compared w&h that of 3’ -+ 5’ compounds, and Fig. 7 showsthe circular diehroism spectra of 2’ -+ 5”
ApC at various temperatures. Examination of these curves leads to the fo1lowin.g observations. (1) The 2’ + 5’ isomers studied (ApC, ApA, CpC) yield circular dichroic spectra different from those of the constituent monomers. The general shape of the circular dichroic curves of 2’ --f 5’ derivatives is similar to that of the 3’ + 5’ compounds;
2 3 ‘4 2 0 -2
IL_-__I280 Wovelength, FIG. 7. Circular dichroism spectra tsmperaturos: (1) -17%; (2) 25’C;
of 2’-+ 5’ ApC (3) 79%.
290
301)
A(qr) in 4.7 M-KP-0.01
N-T&
(pH
7,5),
at ~a,riou~
however, the intensity of the circular dichroic band is relatively small (Figs 7 and S), also the band position is slightly displaced. (2) The changes in intensity of the circular dichroic bands of the 2’ + 5’ isomers are some 10 to 40% over the temperature range of about lOO”C, namely from + 86°C to - 2O”C, wherea.sfor similar 3’ -+ 5’ dimers the intensity can change by a factor of three. (3) The thermal denaturation curves of 2’ -+ 5’ derivatives (Figs 4 and 8) are very different from those of the corresponding 3’ -f- 5’ dinucleoside phosphates. Figures 4 and 8 show that the dependence of circular dichroism at the wavelength of the maximum (dam,,) is almost linear for 2’ + 5’ isomers, in contrast to the sigmoidal melting of 3’ + 5’ compounds. 33
492
J.
BKAHMS,
J.
21
FIG. somer
8. Temperature (0). Conditions
dependence as described
C. MAURIZOT
I -20
I 0
AND
I 20
40
Temperature,
t?Cl
of circular in Fig. 4.
diohroism
A. 111. MZCHELSON
I 60
of
I 00
3’ + 5’ ApC
(a)
and
the
Y-t
5’
(4) In agreement with earlier observations (Michelson, 1963) the 2’ --t 5’ isomers are hypochromic. A proposed structure for 2’ -+ 5’ dinucleoside phosphates must be consistent with all these observations. The existence of circular diohroic spectra of relatively weak intensity indicates the presence of a dissymmetrical structure with weakly interacting bases. Since the changes in circular dichroism with temperature are very small, it is logical to think that the structural transition to an ordered, stacked form must occur at much lower temperature. Thus in the region of temperatures used, the predominant conformatiom of 2’ + 5’ isomers is close to random coil. Consequently we may assume that, at extremely low temperatures, both 2’ -+ 5’ and 3’ -+ 5’ compounds will have similar conformations, with stacked bases rotated as in a DNA single-stranded helix. The following evidence indicates that there is no reason to suppose that the 2’ -+ 5’ linked isomers cannot form a stacked base conformation, aa a result of steric restrictions. (1) The circular dichroio spectra (Figs 7 and 2) of 2’ --f 5’ derivatives are of similar shape when compared with 3’ -+ 5’ linked dimers. The displacement in the position of the circular dichroic bands and in the intersection point (Table 1) may reflect the changes in the position of the internucleotide linkage. (2) It is possible to construct a Courtauld model of 2’ -+ 5’ dinucleoside phosphates with stacked bases. (3) X-Ray results on a crystal of 2’ + 5’ ApU show that the planes of the bases are almost parallel and the two bases have a certain overlap (Shefter et al., 1964). (4) At very low temperature (77°K) the excitation fluorescence spectra and electron spin resonance data on 2’ -+ 5’ ApC are different from that of the monomer components, but they do not differ significantly from the 3’-+ 5’ compou.nds (Glueron, Shulman & Eisinger, personal communication, 1966; llelenr;, Donzou & Michelson, 1966).
DINUCLEOTIDES
IN
SOLUTION
493
Thus we are led to conclude that in. appropriate conditions at low temperature the 2’ .+ 5” isomers can adopt a stacked base conformation. The differences in thermal denaturation behaviour between 2’ --t 5’ and 3’ -+ 5’ linked dinucleoside phosphates may be explained by the importance of the 2’-hydroxyl group of ribose for the formation of a stacked conformation. It is possible that the 2’hydroxyl hydrogen allows formation of an internal hydrogen bond with an oxygen atom. This is also confirmed by our investigations of deoxy GpG, which yields exceedingly weak circula:~ dichroie spectra. It was found that the changes in circular dichroism of d GpG over the same range of temperatures, i.e. down to - 2O”C, were small. However, since the 3’ -+ 5’ riboguanylyl guanosine was not available the absolute comparison is difficult.
4. Conclusions The results obtained by circular dichroism investigations are consistent with the idea that, at low temperatures and neutral pH, 3’ --t 5’ dinucleoside phosphates adopt a d&symmetrical conformation similar to the beginning of a single-stranded helix. Evidence for the stacked conformation comes from both optical and thermodynamic properties, Essentially two categories of circular dichroic spectra were observed in the spectral region of base absorption: one, conservative, composed of two adjacent circular dichroic bands of opposite sign and almost equal rotational strength, and the second “non-conservative” represented by one (CpC) or two (GpC) positive circular dichroic ba,nds. Calculations based on an exciton model of stacked ApA predict very satisfactorily the first type of conservative circular dichroic spectra, (Van Holde et al,, 1965; Warshaw et al., 1965; Brahms et al., 1966). The second non-conservative type of spectra agreeswith predictions for a stacked CpC model if other contributions than the exciton effect are added: i.e., the contribution arising from interactions between near ultraviolet and far-ultraviolet transitions (Bush & Brahms, 1967). The higher resolution of circular diehroism allows detection of spectral details which cannot be readily seen from optical rotatory dispersion studies. The dissymmetrical stacked base structure is consistent with previous studies of hypochromicity (Michelson, 1963) and other optical properties (MassouliB & Michelson, 1964), and the optical rotatory spersion studies ofvarious 3’--+ 5’ dinucleotide phosphates (Warshaw & Tinoco, 1965). At about 2O”C, 3’ -+ 5’ dinucleotides still have an appreciable fraction of bases in a stacked conformation. The analysis of the thermal denaturation process shows that the thermodynamic parameters are essentially similar for all 3’ -+ 5’ compounds. The standard state enthalpy change is about 6 to 7.5 kcal./mole and -the standar entropy change 20 to 25 e.u./mole. Thus we cannot observe any fundamental ences in thermodynamic parameters which allow a division into categories. However, there are small differences in AF” which indicate that the Ecee energy changes for dimers containing uracil or guanine at neutral pH are slightly less in favour of stacking thftan for dimers with cytosine and adenine. In general, the AF” .values at 0°C i~re rather small in favour of stacking. However, from Table 2 it can be seen that a pair of stacked residues is slightly more stable when in a polynucleotide single-stranded helix. Thus it is perfectly possible that certain sequences in RNA possess a single* strand helical conformation. In contrast to the similarity of the thermodynamic parameters of all 3’ -+ 5’ dinucleoside phosphates indicating stacking, the 2’ --t 5’ derivatives ApC, ApA, GpC (and also deoxy GpG) are essentially in a disordered conformation in the range of temperature used, i.e. from - 20 to + 80°C.
494
J. BRAHMS,
J. C. MAURIZOT
AND
8. M. MICHELSON
Thus important differences in conformational stability appear to depend on the presenceor absenceof a 2’-hydroxyl group of the ribose. It is possible that a hydrogen bond between the 2’-hydroxyl group and another group is contributing to the formation of a favourable configuration for basestacking in 3’ --t 5’ ribo-oligonucleotides. One can consider, for example, that this internal hydrogen bondis formed between 3’Jinked ribose 2’-hydroxyl and the phosphate oxygen. Although we prefer the phosphate oxygen since this bond definitely restricts rotational freedom of the phosphodiester linkage, another possibility is a bond between the 2’-hydroxyl group and the 2-keto group in pyrimidines or N(3) in purines as postulated by (Ts’o, Rapaport & Bollum, 1966). However, this type of hydrogen bond will only restrict rotation about the glycosyl linkage, without affecting the flexibility of the chain. It could well be present in monomers (Ts’o, 1966, personal communication) yet absent in polymers, if binding to the phosphate oxygen causesan over-all increase in conformational stability. We feel that a third possibility, hydrogen bonding between the 2’-hydroxyl group and the ether oxygen of the sugar is sterioally unlikely. Also other possibleexplanations, such as some small differences in basebaseinteractions or other steric effects, cannot satisfactorily account for the observed striking differences between compounds with or without the 2’-hydroxyl group. The internal hydrogen-bond energy probably contributes little to the enthalpy change of the order-disorder process in 3’ -+ 5’ dinucleoside phosphates, but is important in that it facilitates base stacking interactions. In 2’ -+ 5’ dinucleoside phosphates (and in deoxy GpG), the absenceof the 2’-hydroxyl group doesnot allow the formation of this hydrogen bond. Thus the freedom of rotation about the covalent bonds of the backbone chain is not restricted. Consequently one can expect that the stacking in a helical single-strand conformation will occur at much lower temperature. The same arguments can be applied to chains of poly 2’-deoxynucleotides. In conclusion, the present investigation suggests that the stability of a helical single-strand polyribonucleotide chain is due not only to the stacking interactions but also to the effect of an internal hydrogen bond. One might expect that in a RhTA chain the “hot spot” of disordered conformation will be situated in the region where the ribose moiety has the 2’-hydroxyl group substituted by a methoxyl group. We thank Professor C. Sadron for his interest and encouragement of this work. Mrs M. L. Welter and Miss C. Monny provided excellent technical assistance. This work was presented at the Second Jnternational Biophysics Congress held in Vienna, September S-9,1966. REFERENCES Brahms, J. (1963). J. Amer. Chem. Sot. 85, 3298. Brahms, J., Maurizot, J. C. & Michelson, A. M. (1967). J. Mol. Biol. 25, 465. Brahms, J., Michelson, A. M. & Van Holde, K. E. (1966). J. Mol. Biol. 15, 1467. Bush, A. C. (1965). Thesis, University of California. Bush, A. C. & Brahms, J. (1967). J. Chem. Phys. 46, 79. Claverie, P., Pullman, B. & Caillet, J. (1966). J. Theoret. Biol. 112, 419. De Voe, H. & Tinoco, I., Jr. (1962). J. Mol. Biol. 4, 500. Helene, C., Douzou, P. & Michelson, A. M. (1966). Proc. Nat. Acad. Sci., Wash. 55, 376. Leng, M. & Felsenfeld, G. (1966). J. Mol. Biol. 15, 455. Massoulie, J. & Michelson, A. M. (1964). C.R. Acad. Sci. Paris, 259, 2923. Michelson, A. M. (1959). J. Chem. Sot. 3655. Michelson, A. M. (1963). The Chemistry of Nucleosides and Nucleotides. New York: Academic Press.
DINUCLEOTIDES
IN SOLUTION
495
Poland, z)., Vournakis, J. N. & Soheraga, H. A. (1966). Bio~oZymers, 4, 223. Pullman, B., Claverie, P. & Caillet, J. (1966). Proc. Nat. Acad. Sci., Wash,, 55, 904. IZichards, E. G., Flessel, C. R. & Fresco, J. R. (1963). Biopolymers, 1, 431. Sheftar, E., Barlow, M., Sparks, R. & Trueblood, K. (1964). J. Amer. C%em. Sot. 86, 1872. Tincoo, I., Jr. (1964). J. Amer. Chem. Sot. 86, 297. Ts’o, P.O.P., Rapaport, S. A. & Bollum, F. J. (1966). Biochemistry, 5, 4151. Van Holde, K. E., Brahms, J. & Michelson, A. M. (1965). J. Mol. Biol. 12, 726. Warshaw, M. M., Bush, C. A. & Tinoco, I., Jr. (1965). Biochem. Biophys. Res. cbmm. 1 633.
Wajrshaw,
M. M. & Tinoco,
I., Jr. (1965). J. Mol. Biol. 13, 54.
Conformational
J. BR&Mst,
J. C.
Stability of Dinucleotides in Solution MmmzoTt
AND
A. M.
MICHELSON
Centre de Recherches sur les MacromoE6cules 6, rue Boussingault, ii’trasbourg Institut de Biologic Physicochimipue 13, rue Pierre Curie, Paris, France (Received17 Octobw 1966) A variety of 3’ + 5’ and 2’ -+ 5’ dinucleoside phosphates (at neutral pH) have been studied using circular dichroic and absorption spectra in a range of temperature of - 20 to + 80°C. Two kinds of circular dichroic spectra can be observed represented by ~3,pair of bands of at low temperature : one “conservative” opposite sign, the other ‘Lnon-eonservative” composed of one main positive band, characteristic of guanine and cytosine compounds. Both spectra are consistent with a model of dissymmetrical, single-stranded helical conformation. Thermodynamic parameters for the process order +G disorder in. dinucleotides have been estimated by following thermally induced changes in circular dichroic spectra. Values are similar for all the 3’--+5’ derivatives and are of the order of 6 to 7.5 k&./mole for dHo, about 20 to 25 e.u./mole for AS0 ad about 0.5 kcal./mole at 0°C for APO. This absence of major differences in th.ermodynamic parameters does not permit one to divide dinucleotides into groups characterized by a stacked or unstacked conformation; for example, 3’ -+ 6’ uridylyl-uridine (UpU) is appreciably stacked at 20°C. Examination of corresponding 2’ -+ 5’ dinucleoside phosphates gave quite different results in the same range of temperature, indicating that stacking is much less marked compared with the 3’-+ 5” compounds. An intramolecular hydrogen bond involving the 2’-hydroxyl group of the ribose is postulated to explain these differences.
1. Introduction In our previous studies using circular dichroism, we have shown that oligo and po$nucleotides of adenylic acid (Brahms, Michelson & Van Nolde, 1966) and cytidylie rahms, Maurizot & Michelson, 1967) assumea single-strand, helical conformation under appropriate conditions. The stability of such structures is determined by forces other than intermolecular base interactions, but does involve stacking interactions between parallel bases. The thermal denaturation was shown to be essentially a non-co-operative process. Similar results and conclusions have been obt&ned using optical rotatory dispersion (Poland; Vournakis & Scheraga, 1966) and rdtraviolet absorption (Leng & Felsenfield, 1966). The single-strand, helical polynzrcleotide chain can be represented as a collection of stacked dimers which melt independently. T Present address: Institut
de Biophysique
MolBculaire,
481
45 OrlBans-La
Source.
482
J. BRAHMS,
J. C. MAURIZOT
AND
A. M. MICHEESOPU’
Studies of the conformation and conformationalstability of simple dimers of various base composition can provide data for a better understanding of the conformation of nucleic acids in solution. One can consider that RNA’s may have a fraction or the entire molecule in a single-strand conformation in the absenceof complementary base pairing. The formation and stability of such stacked basestructures will depend upon the different thermodynamic values of interaction between residues of different base composition. At present, such values have been experimentally determined only for adenylates and cytidylates; other available values are the theoretical predictions of De Voe $ Tinoco (1962), Pullman, Claverie & Caillet (1966), Claverie, Pullman & Caillet (1966), Pollack & Rein (ZYKJInt. Biophya. Congr. Abstr. p, 139, 1966). This paper presents studies using oircular dichroism which indicate that various 3’ -+ 5’ dinucleoside phosphates assumeat neutral pH a dissymmetrical conformation with stacked basesin the absence of intermolecular hydrogen-bonded base pairing. This is in general agreement with the earlier work of Michelson (1959) on hypochromism in simple dimers, and the results of optical rotatory dispersion studies of Warshaw & Tinoco (1965) and Massoulie & Michelson (1964). The thermodynamic parameters for the thermal denaturation presented here are very similar for all 3’ -+ 6’ dinucleoside phosphates and are in contrast with the behaviour of 2’ +- 5’ derivatives. It is proposed that an internal hydrogen bond between the 2’-hydroxyl group and a phosphate oxygen atom contributes to the oonformation of singlestranded helical polyribonucleotides as well as the stacking interactions previously considered.
2. Experimental
Methods
(a) Materials The 3’ + 6’ and 2’ -+ 5’ dinucleosidephosphateswere synthesized by chemical teohniques (Michelson, 1959). A2’p5’C was purchased from Waldhof Co. The solvent used was 4.7 M-KF-O-01 M-Tris at pM 7.5. This solvent was chosen in ord.er to extend the spectral measurements to relatively low temperatures. The freezing point of the solvent is below - 20%. The use of low temperatures serves two purposes in that the resolution of circular dichroic spectra is improved and also gives the low-temperature parameters for the thermodynamic analysis. It should be emphasized that RF is transparent in the ultraviolet spectral region under investigation. There was no detectable effect of salt concentration on circular dichroic spectra of dinucleoside phosphates at neutral pH, indicating that the ionic strength has no important influence on singlestranded helical conformation. (b) Meusurernent of circular diclwoim and absorption spectra These spectra of dinucleoside phosphates were made in a concentration range of low4 to 10w3u (per nucleotide residue). In general, there was no detectable influence of concentretion on oircular dichroic spectra. However, study of 3’ + 5’ GpC showed that at lower temperatures circular dichroism was dependent on concentration between 10d4 and 10-3nix. We have chosen to perform the measurements at high dilution, in conditions excluding all irttermolecular association processes. Cells of 0.1, 0.2, 0.5 cm path length were employed. The experiments were carried out at controlled temperatures using a Lauda cryostat and a Haake thermostat. The temperature was monitored by a thermocouple in contact with the cell and connected to a Sefram recorder. In order to prevent evaporation, a closed cell was used. The circular dichroism and the rotational strength are given per nucleotide residue and were calculated were determined by as previously indicated (Brahms, 1963). E values for the dinucleotides alkaline hydrolysis to the monomer.
DINTJCLEOTIDES
IN
SOLUTION
48
3. Results and ~i§~~$~i~n (a) Ci’rcular
dichoism and absorption spectra
We haw measured the circular dichroism and absor@ion speotra of ten 3’ --> 5’ ribodinucleoside phosphates at neutral pII (in 1.7 M-HF--@O1 M-!&S, pII 7.5) at various temperatures between -20 and 80°C. Figure 1 showsthe results of circular diohroism measurementsof three dimers at the lowest temperature used. Also shown are the absorption spectra of the dimers and the circular dichroism curves of the monomer constituents. From the comparison of the results of Fig. 1, one can clearly seethat the eirdac dichroism speotrum of each 3’ -+ 5’ dinucleoside phosphate is qualitatively and quaut~tatively very different from that of its monomer components. T&s is reflected in differences in general shape of the circular dichroic spectra, in the position and even in the sign of the bands (seealso Fig. 2). In contrast, the absorption spectrum of each dimer is very similar to that of its corresponding components, apart from slight hypoehromie effects near h mBx. The general pattern of circular dichroism curves iti
PIG. 1. Circular dichroic spectra of three 3’+ 5’ dinucleoside phosphates at neutral pEI in 4.7 - 16 to -20% (-); the absorption spectra (-- -) m-KF-OGl-x-T& and at low temperature, we measured at 25% The circular diohroism of some of the monomer oonstituents (. . ~) is shown for comparison.
different for various dimers. In Fig. 1 are shown sometypical examples. It is apparen; that there are two categories of circular dichroic speotra,. The first category of circular dichroic spectra is represented essentially by a pa.k of jacent positive and negative bands with an intersection point situated at a wavelength very near that of the absorption maximum. The su.mof the roMiona1 strengths is approximatively equal to zero in the spectral region under investigation. The shape of these spectra is in agreement with the prediction of the exciton theory of optical rotatory power of polynucleotides (Tinoco, 1964), according to which the ~~te~a~tiol~among identical chromophores in a dimer will give rise to the splitting of the monomer band into two bands. It was previously shown that the exciton model tith single-strand helical geometry when applied to Apia (Van IIolde, Brahms & XEeheSson,1965; ‘Warshaw, Bush $ Tinoco, 1965) and to oligoadenylates (Brahms et al., 1966) allows satisfactory prediction of the shape of the observed circular dichroism curve, including the position of the bands and their rotational strength v&es. It is to be noticed that the predictions for non-degenerate interactions in a dimer
484
J.
BRAHMS,
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C. MAURIZOT
AND
A.
M.
MICHELSON
composed of different bases like ApU (Bush, 1965) are in good agreement with observed circular dichroic spectra (Fig. 1). This type of circular dichroic spectra is also observed for ApC, CpA (Fig. 2) and CpU (Fig. 3). In these cases the agreement may be fortuitous, since the presence of more than one band in cytosine may cause overlapping and cancellation. The second category of circular dichroic spectra consists of one or two positive bands as shown for GpA, GpC (Fig. 1) and also CpC. One can clearly see that the sum of rotational strengths is not equal to zero in the spectral region under investigation. This type of spectra is not predicted by the exciton theory of optical rotatory power. The differences between these two categories are not so clearly seen from the optical rotatory dispersion curves. It must be emphasized that these circular dichroic spectra are exhibited by dimers with guanine and/or cytosine. It is well known that the absorption spectra of guanine and cytosine at neutral pH are complex and have at least two overlapping bands in the spectral region of 210 to 320 rnp. Hence calculation of the rotational strengths for all dimers containing guanine or cytosine should include the contribution arising from non-degenerate interaction of these two near ultraviolet bands (Bush, 1965). The predicted rotational strengths will still sum to zero. The predominant positive rotational strength in the near ultraviolet can be calculated if one includes the interaction with the far ultraviolet transitions. This contribution is of non-conservative character and depends on the direction of the electronic transition dipole moment (Bush & Brahms, 1967). The orientation of the transition moment of guanine and cytosine must be such that the contribution of far ultraviolet terms is large when in a single-strand structure, whereas for adenine and uracil it is small. In general, the calculated values for a model of stacked bases with the geometry of a single-strand DNA are consistent with the experimental results (Van Holde et al., 1965; Warshaw et aZ., 1965; Bush & Brahms, 1967). It will be noted that the circular dichroio spectra were obtained with highly dilute solutions (10w4~), in conditions which rule out the possibility of intermolecular hydrogen bonding or other associative processes as an explanation of the optical activity (see Experimental Methods). Further support for the stacked-model conformation can be found in the X-ray data on crystals of 2’ -+ 5’ ApU (Shefter, Barlow, Sparks & Trueblood, 1964).
I”
-44 220
”
230
240
250
260
270
280
290
Wavelength,
220
”
”
”
I
230
h(rnp)
FIG. 2. Circular clichroic spectraof 3’+ 5’dinucleoside phosphatesisomers (-) The absorption tures (-18 to -20°C) in 4.7 M-KF-0.01 M-Tris at neutral pH. corresponding 3’ +6’ compounds are: left: (---) UpG and (-.-.-) GpU; right (-.-.-) ApC. Circular dichroic spectra (. . .) of the monomer constituents.
at lowtemperaspectra of the (---) CpA and
DINUCLEOTIDES
IN
SOLUTION
485
Figure 2 shows the circular dichroic and absorption spectra of four 3’ + 5’ sequenee isomers taken as an example. It can be clearly seen that the circular dichroic spectrum is very sensitive to the base sequence. Thus 3’ -+ 5’ GpU at about - 20°C exhibits four well-resolved bands in a spectrum quite different from that of 3’ -+ 5’ UpG (see Table 1). The absorption spectra show one broad band which is very similar for both isomers. Circular diehroism measurements thus allow detection of the contdbution of several
TABLE
1
Circular dichroism and absorption spectral data Substance
3’ 3 2’ 3 3’4.5’ 3’ -+ 2’ -+ 3’ -+ 3” -+ 3’ -+ 3’ 3 3’+ 3’ --f 3’ + 2’ -a
)Imax (mp)
Absorption cx 10-3 (cm-lmol-ll.)
spectrum A1 (mp)
Circular dichroism R2X 104” h3 (w-4
-
5’ cpc 5’ cpc UpU 5’ ApC 5’ ApC 5’ CpA 5’ UpG 5’ GpU 5’ cpu 5’ GpC 5’ GpA 5’ ApU 5’ ApA
269 269.5 260 261 262 261.5 255.5 258 264.5 266 258 260 258.5
7.9 7.8 9.1 10.0 10.0 IO.0 IO..% 10.0 8.1 8.9 13.1 11.4 12.9
280 275 272 274.5 277.5 274.5 278.5 281.5 276 284 284.5 268.5 270.5
+ 25.9 +13 + 14.5 i 14.5 + 6.7 t 12.7 -t 3.5 + 0.6 +20.5 + 3.3 - 0.6 + 7.8 + 9.7
2465 255.5 260.5 265 257.5 266.5 275.6 255 252.5 257.5 258.5
CNIP UMP AXP GXP
271 262 259.5 252
9.0 10.0 15.4 13.7
272 265
+12 + 9.3
242 244
246
+ 0.9
245 236 251-5 235.5 258 267 237 254 266 249 249
- 8.9 -14.7 - 8.5 - 7.5 - 1.4 - 1‘3 - 8 + 4.0 t 4.7 - 3.2 - 4.9
R, x 1OklJ
232
218
+
1.2
228 249
+
4.5
229 228
-
1.5 4.2
Symbols are as follows: AmaX is the maximum wavelength in mp; E is the extinction coefficient at this wavelength. For the circular dichroic spectrum, hl, Xs, & are the wavelengths of the extremnm, and R1, Rz, Rs are the rotational strengths of the corresponding circular dichroic bands and & the orossing point. The circular dichroic spectra of dinucleoside phosphates were measured at relatively low temperatures of about -20°C (for details see Experimental Methods).
transitions not resolved in the absorption spectrum. The sequence dependence of the circular dichroism is also shown in spectra of 3’ --t 5’ ApC and CpA (Fig. 29. In general, our circular dichroic results are in good agreement with absorption spectra! data of Michelson (1963) and optical rotatory dispersion data of Viiarshaw & Tinoco <1965j, showing clearly that optical methods and more particularly circular dichroism and optical rotatory dispersion can be useful tools for the determination of base sequence. In Table 1 are summarized circular dichroic and absorption spectra characteristic of various 3’ -+ 5’ dinucleoside phosphates and some 2’ + 5’ derivatives. In contrast to the circular dichroism, the absorption spectrum of each dimer is very similar to that of its corresponding monomer components. The hypochromicity neaLI x Ill&Xis relatively small and is not greater than some 15%. We notice that almost al! 3’ -+ 5” dimers exhibit a positive longer wavelength circular dichroic band which can be correlated with the right-handed helical senseat least for dimers containing bases
486
J. BRAHMS,
J.
C. MAURIZOT
AND
A.
I%. MiPICHELSON
possessing a single absorption band in the near ultraviolet; namely, ad&no, uraeil. However, in one case, that of 3’ -+ 5’ GpA, we observe that the longer wavelength band is of negative sign and of relatively weak intensity. The detailed circular dichroic spectra described above were detectable only by recording at relatively low temperatures of about -20°C. We have not attempted to predict the circular dichroism characteristics for various dimers, since the exact parameters, such as the orientation of electric dipole transition moments, are not determined for guanine and cytosine. However, it has been shown that using reasonable approximations for a single-strand, stacked model of CpC, a positive near-ultraviolet circular dichroic band in agreement with the observed curve can be predieted (Bush & Brahms, 1967). (b) ThermaZ denaturtiion Since the intensities of the circular dichroio bands of various dinwleoside phosphates are very different, it is diEcult to derive a yrmntitative understanding of the stacked helical conformation from the results of measurements made at one temperature only. For this reason and also in order to determine the thermodynamia parameters, the circular dichroic spectra were measured at various temperatures ranging from -20 to 80°C.
FIG. 3. Circular dic’hroic spectra of 3’+5’ dinucleoside phosphates at various Left: 3’-+-5’ CpU at: (1) -20°C; (2) 3%; (3) 28’C; (4) 60°C; (5) 72°C. Centre: 3’+5’UpU at: (1) -18’C; (2) 2°C; (3) 26OC; (4) 41%; (6) 67%. Right: 3’+5’ CpA at: (1) -20°C; (2) 0°C (3) 23°C (4) 81°C. Conditions are as described in Figs 1 and 2. Dotted line: the circular dichroio monomers.
temperatures.
spectra
of the
Figure 3 shows the results for circular dichroism at various temperatures of three 3’ -+ 5’ dinucleosides phosphates as examples. It can be readily seen that the intensity of circular dicbroic bands changes markedly in the range of temperature used. At higher temperatures the intensity of the longer wavelength positive band decreases and approaches that of the monomer oomponents. Since there is no spectral shift of the position of the maximum of circular dicbroic bands at ah. temperatures used, it is possible to follow quantitatively the thermal denaturation process either by measuring the circular dichroism at the wavelength of the maximum or the rotational strength of the positive band as a function of temperature. We have chosen to
DINUCLEOTIDES
FIG.
IN SOLUTI0.N
487
4. Temperature dependence of circular dichroism of 3’ + 5’ and 2’ + 5’ dinucleoaide in 4-7 M-KF-0.01 ix-Tris at neutral p’II. The magnitude of circular diehroism is taken maxim,m of the main, longer wavelength positive band (il~),~,. The symbols are ae
phosphates at the
+5’cpU;(@)3’-+5’ApC; 2’+
5’AipA;
(A)
2’+
5’CpC;
(0)
2’+
5’ApC;
(0) 3’+5‘UpU; (A)
3’+
( ) 3’+ 5’ ApU; (TJ)
5’GpA.
determine the changes in (Q,--Ed) at various temperatures, since calculation of the rotational strengths is difficult for dimers showing complex circular dichroic spectra, especially at higher temperatures. Figure 4 shows the circular dichroism thermal proiiles for a series of 3’ -+ 5’ dinucleoside phosphates. Despite the difference in intensity of circular dichroism (hIl,X) for dinucleoside phosphates of different base composition at a given t,emperatnre, it can be seen that the “melting” curves are of similar, sigmoidal shape and of gradual character, At higher temperatures, the values of de,,, approach those of the monomer components (Table l), These residual values depend on the nature of thp? monomer, and as a result the various melting curves of the 3’ -+ 5’ dinucEeoside
488
J.
BRAHMS,
J.
C.
MAURIZOT
AND
A.
M.
MICHELSON
phosphates are displaced on the intensity scale. The 2’ -+ 5’ dimers studied (ApC, ApA, CpC) show relatively very small changes of AC,,, over the entire range of temperatures. This will be analysed later. It is di&ult to make a quantitative comparison of the thermal denaturation curves of the various 3’ -+ 5’ dinucleoside phosphates shown in Fig. 4. However, the data can be used to construct a graph of the fraction of stacked bases as a function of temperature. For this we use the fact that at high temperatures the dimers have almost reached a completely disordered conformation. One can obtain the limiting value of AC,,, at low temperature, characteristic of a stacked conformation, by two
I
I
I
I
I
I
cpc
2
ApC
3
CpA
4
ApU
5
cpu
0
FIG. 5. Fraction Fig. 4).
-20
0
of stacked
bases
20 Temperature, (x) as a function
40 f (“C)
60
of temperature
80
(conditions
as described
in
procedures: by the extrapolation of the sigmoidal melting curve to a limiting value of de (see Experimental Methods and the accompanying paper, Brahms et ccl., 1967), and as twice the value of AE at the mid-point of the transition. The value of AE at the mid-point of transition was obtained from the shapeof the available part of the curve for which the high-temperature limit can be quite well approximate. As shown in Fig. 5, all the 3’ --f 5’ dinucleoside phosphates yield curves of very similar shape, almost parallel but somewhat displaced on the temperature scale. The same gradual profile and similar steepnessof a,11the curves suggest that the same basic process, essentially non-co-operative, characterizes the melting of all 3’ --f 5’ dinucleoside phosphates. From Fig. 5 it can be seenthat dimers containing pyrimidine residuesonly, such as CpC, have almost the samefraction of stacked basesat various temperatures as dimers containing purine and pyrimidine residues, such as ApC or CpA. Dinucleoside ‘phosphates containing uracil, such as UpU, ApU, CPU, have at 20°C an appreciable fraction of the basesin a stacked conformat;on, contrary to the widespread opinion that the presenceof uridine in a polynucleotide chain causesunstacking (Richards, Flessel & Fresco, 1963; Warshaw & Tinoco, 1965). Furthermore,
DINUCLEOTIDE
S IN
SOLUTION
489
the conformational stability of dimers such as ApU and CpU is not too dissimilar to that of CpA, in which both bases are purines. However, dimers containing uracil doi have a rather smaller fraction of stacked bases at a given temperature than CpC or ApC. Nevertheless, we believe that classifioation into groups of sequences which will be in a stacked or unstacked conformation is unfortunate. From these (thermal denaturation) data, it is possible to determine the apparent equilibrium constant for the denaturation process of various 3’ --+ 5’ dinucleoside phosphates. This can be written as a simple reaction DC,) +Dcd). The apparent equihbrium constant oan be expressed:
Cd
K=Co=_l
IO - I, a t
where Id and I,, represent the limiting values of dernax at high and low temperatures where all the dimers are in a completely unstacked or stacked form, respectiveby; I, is the magnitude of At-,,, at a given temperature. Having obtained values of the equilibrium constant, we calculated standard state enthalpy change and standard state entropy change for the thermal denaturation process. Figure 6 shows the plot
I cpu 2 CpA 3 ApU 4 cpc 5 /\pC
I
28
3.0
I
32
F’ra. 8. A van t’Hoff plot of the thermal (The da&a are taken from Figs 3 and 4.)
I
I
3.4 (I/TX IO? denaturation
CJ * n El A
3.6
3.8
of some 3’ + 5’ dinucleoside
phosphates,
of log K veraus l/T for some dimers taken as examples. The lines of the van t’Hoff plot are straight with similar slopes, indicating that AH0 must be similar for each case. The thermodynamic parameters of 3’ --+ 5’ dinucleoside phosphates are summarized in Table 2. The standard state enthalpy changes are about + 6 k~a~./rn~~e
490
J. BRAHMS,
J.
C, MAURIZOT
AND
A.
M.
MVIICHELSON
to + 7-5 kc&/mole, and the entropy change about + 22 to 25 e.u./mole. The uncertainty of our procedure arisesfrom the estimate of the limiting values of circular dichroism at low temperatures. As indicated previously (Van Holde et al., 1965; Brahms et al., 1966), extreme variations in the extrapolated I,, value do not lead to more than & 10% difference in the calculated value of ALP and AL!?. The technique usedin the present work, applying temperatures of about - 20°C reduces the possible error in the estimates of the low-temperature limit of 1,. Since other errors are relatively small, the over-all limit of uncertainty is not greater than f- 15%. The values of AH0 listed in Table 2 are in good agreement with those obtained for oligo A by Poland et al. (1966), and lower but of similar magnitude when compared with the data of Leng & Felsenfeld (1966). The values of AH” and AX” presented here for various dimers are slightly smaller than those given previously for ApA by Van lyolde et al. (1965) and Brahms et al. (1966). We think that this rather small difference is due mainly to our present use of lower temperatures. Nevertheless, despite different experimental conditions, the values of AH0 and AX” obtained are of the same magnitude (Table 2). TABLE 2 Thermodynamic parametersfor thermal denaturation af 3’4 5’ dinucleosidephosphates(pH 7.5, 4.7 ~-KB’-0~01 M-Tris) Substance
CPA APC CPU APU CPC GPA ApAt
7.0 6.1
PolyU c
6.0 5.3 7.9
P$y The values t The data t Van Holde,
(k0.i~ole)
6.8 6.7 7-5 6.1 8.0
At
(e.$zole)
(kcal.,%e)
(0%)
24 21 24 24 25 22 28
i-o.4 +0.6 $0.2 +0*3 +0*7 +0*2 +0.4
21 16
-I-o.3 -to.9 +1.1
25
are expressed per mole of nucleotide residue. are taken from Van Holde, Brahms & Michelson, 1965 and from 1966; solvent used is 0.1 M-NaGI-0.01 M-Tris (pH 7.4).
18 25 6
11 24 9 25 15 59 40
Brahms,
Michelson
It is clear that fundamental differences in AH0 and dXO values for the 3’ -+ 5’ dinucleoside phosphates cannot be observed. The free energy change at 0°C is of the order of half a kcal./mole in favour of stacking. However, small differences in AF” are present (Table 2). It appears that the stability of a stacked conformation will be favoured by the presenceof cytosine and adenine rather than of uracil or gaanine at neutral pH. From Table 2 it can be seen that small differences in thermodynamic parameters have an effect on the value of T,, the temperature at the mid-point of the thermal “denaturation”. The results of thermal denaturation for single-strand helical polymers, and data found previously for ApA and poly A (Van Holde et al., 1905; Brahms et al., 1966) are also included, It can be seenthat the AH” data for the
DINUCLEOTIDES
IN
SOLUTION
491
are very similar to those of the polymers, confirming previous conchrsions that the melting of such structures has the characteristics of a largely non-co-operative process(Leng & Felsenfeld, 1966; Poland, Vournakis & Scheraga, 1.966;Brahms et al., 1966). Thus regardless of the chemical nature of the residues, the single-strand belices melt in similar fashion. The value of dP” for a polymer is somewhat larger than for a dimer. Thus our results on 3’ -+ 5’ dimers of various base composition can be applied to a ribopolynucleotide chain when in a single-strand conformation, has t,o bear in mind that a pair of stacked residuesis slightly more stable when in a polymeric array. dimers
(c) The 2’ -+ 5’ dinucleosidephosphates Figures 4 and 8 show the thermal denaturation of 2’ --f 5’ isomers compared w&h that of 3’ -+ 5’ compounds, and Fig. 7 showsthe circular diehroism spectra of 2’ -+ 5”
ApC at various temperatures. Examination of these curves leads to the fo1lowin.g observations. (1) The 2’ + 5’ isomers studied (ApC, ApA, CpC) yield circular dichroic spectra different from those of the constituent monomers. The general shape of the circular dichroic curves of 2’ --f 5’ derivatives is similar to that of the 3’ + 5’ compounds;
2 3 ‘4 2 0 -2
IL_-__I280 Wovelength, FIG. 7. Circular dichroism spectra tsmperaturos: (1) -17%; (2) 25’C;
of 2’-+ 5’ ApC (3) 79%.
290
301)
A(qr) in 4.7 M-KP-0.01
N-T&
(pH
7,5),
at ~a,riou~
however, the intensity of the circular dichroic band is relatively small (Figs 7 and S), also the band position is slightly displaced. (2) The changes in intensity of the circular dichroic bands of the 2’ + 5’ isomers are some 10 to 40% over the temperature range of about lOO”C, namely from + 86°C to - 2O”C, wherea.sfor similar 3’ -+ 5’ dimers the intensity can change by a factor of three. (3) The thermal denaturation curves of 2’ -+ 5’ derivatives (Figs 4 and 8) are very different from those of the corresponding 3’ -f- 5’ dinucleoside phosphates. Figures 4 and 8 show that the dependence of circular dichroism at the wavelength of the maximum (dam,,) is almost linear for 2’ + 5’ isomers, in contrast to the sigmoidal melting of 3’ + 5’ compounds. 33
492
J.
BKAHMS,
J.
21
FIG. somer
8. Temperature (0). Conditions
dependence as described
C. MAURIZOT
I -20
I 0
AND
I 20
40
Temperature,
t?Cl
of circular in Fig. 4.
diohroism
A. 111. MZCHELSON
I 60
of
I 00
3’ + 5’ ApC
(a)
and
the
Y-t
5’
(4) In agreement with earlier observations (Michelson, 1963) the 2’ --t 5’ isomers are hypochromic. A proposed structure for 2’ -+ 5’ dinucleoside phosphates must be consistent with all these observations. The existence of circular diohroic spectra of relatively weak intensity indicates the presence of a dissymmetrical structure with weakly interacting bases. Since the changes in circular dichroism with temperature are very small, it is logical to think that the structural transition to an ordered, stacked form must occur at much lower temperature. Thus in the region of temperatures used, the predominant conformatiom of 2’ + 5’ isomers is close to random coil. Consequently we may assume that, at extremely low temperatures, both 2’ -+ 5’ and 3’ -+ 5’ compounds will have similar conformations, with stacked bases rotated as in a DNA single-stranded helix. The following evidence indicates that there is no reason to suppose that the 2’ -+ 5’ linked isomers cannot form a stacked base conformation, aa a result of steric restrictions. (1) The circular dichroio spectra (Figs 7 and 2) of 2’ --f 5’ derivatives are of similar shape when compared with 3’ -+ 5’ linked dimers. The displacement in the position of the circular dichroic bands and in the intersection point (Table 1) may reflect the changes in the position of the internucleotide linkage. (2) It is possible to construct a Courtauld model of 2’ -+ 5’ dinucleoside phosphates with stacked bases. (3) X-Ray results on a crystal of 2’ + 5’ ApU show that the planes of the bases are almost parallel and the two bases have a certain overlap (Shefter et al., 1964). (4) At very low temperature (77°K) the excitation fluorescence spectra and electron spin resonance data on 2’ -+ 5’ ApC are different from that of the monomer components, but they do not differ significantly from the 3’-+ 5’ compou.nds (Glueron, Shulman & Eisinger, personal communication, 1966; llelenr;, Donzou & Michelson, 1966).
DINUCLEOTIDES
IN
SOLUTION
493
Thus we are led to conclude that in. appropriate conditions at low temperature the 2’ .+ 5” isomers can adopt a stacked base conformation. The differences in thermal denaturation behaviour between 2’ --t 5’ and 3’ -+ 5’ linked dinucleoside phosphates may be explained by the importance of the 2’-hydroxyl group of ribose for the formation of a stacked conformation. It is possible that the 2’hydroxyl hydrogen allows formation of an internal hydrogen bond with an oxygen atom. This is also confirmed by our investigations of deoxy GpG, which yields exceedingly weak circula:~ dichroie spectra. It was found that the changes in circular dichroism of d GpG over the same range of temperatures, i.e. down to - 2O”C, were small. However, since the 3’ -+ 5’ riboguanylyl guanosine was not available the absolute comparison is difficult.
4. Conclusions The results obtained by circular dichroism investigations are consistent with the idea that, at low temperatures and neutral pH, 3’ --t 5’ dinucleoside phosphates adopt a d&symmetrical conformation similar to the beginning of a single-stranded helix. Evidence for the stacked conformation comes from both optical and thermodynamic properties, Essentially two categories of circular dichroic spectra were observed in the spectral region of base absorption: one, conservative, composed of two adjacent circular dichroic bands of opposite sign and almost equal rotational strength, and the second “non-conservative” represented by one (CpC) or two (GpC) positive circular dichroic ba,nds. Calculations based on an exciton model of stacked ApA predict very satisfactorily the first type of conservative circular dichroic spectra, (Van Holde et al,, 1965; Warshaw et al., 1965; Brahms et al., 1966). The second non-conservative type of spectra agreeswith predictions for a stacked CpC model if other contributions than the exciton effect are added: i.e., the contribution arising from interactions between near ultraviolet and far-ultraviolet transitions (Bush & Brahms, 1967). The higher resolution of circular diehroism allows detection of spectral details which cannot be readily seen from optical rotatory dispersion studies. The dissymmetrical stacked base structure is consistent with previous studies of hypochromicity (Michelson, 1963) and other optical properties (MassouliB & Michelson, 1964), and the optical rotatory spersion studies ofvarious 3’--+ 5’ dinucleotide phosphates (Warshaw & Tinoco, 1965). At about 2O”C, 3’ -+ 5’ dinucleotides still have an appreciable fraction of bases in a stacked conformation. The analysis of the thermal denaturation process shows that the thermodynamic parameters are essentially similar for all 3’ -+ 5’ compounds. The standard state enthalpy change is about 6 to 7.5 kcal./mole and -the standar entropy change 20 to 25 e.u./mole. Thus we cannot observe any fundamental ences in thermodynamic parameters which allow a division into categories. However, there are small differences in AF” which indicate that the Ecee energy changes for dimers containing uracil or guanine at neutral pH are slightly less in favour of stacking thftan for dimers with cytosine and adenine. In general, the AF” .values at 0°C i~re rather small in favour of stacking. However, from Table 2 it can be seen that a pair of stacked residues is slightly more stable when in a polynucleotide single-stranded helix. Thus it is perfectly possible that certain sequences in RNA possess a single* strand helical conformation. In contrast to the similarity of the thermodynamic parameters of all 3’ -+ 5’ dinucleoside phosphates indicating stacking, the 2’ --t 5’ derivatives ApC, ApA, GpC (and also deoxy GpG) are essentially in a disordered conformation in the range of temperature used, i.e. from - 20 to + 80°C.
494
J. BRAHMS,
J. C. MAURIZOT
AND
8. M. MICHELSON
Thus important differences in conformational stability appear to depend on the presenceor absenceof a 2’-hydroxyl group of the ribose. It is possible that a hydrogen bond between the 2’-hydroxyl group and another group is contributing to the formation of a favourable configuration for basestacking in 3’ --t 5’ ribo-oligonucleotides. One can consider, for example, that this internal hydrogen bondis formed between 3’Jinked ribose 2’-hydroxyl and the phosphate oxygen. Although we prefer the phosphate oxygen since this bond definitely restricts rotational freedom of the phosphodiester linkage, another possibility is a bond between the 2’-hydroxyl group and the 2-keto group in pyrimidines or N(3) in purines as postulated by (Ts’o, Rapaport & Bollum, 1966). However, this type of hydrogen bond will only restrict rotation about the glycosyl linkage, without affecting the flexibility of the chain. It could well be present in monomers (Ts’o, 1966, personal communication) yet absent in polymers, if binding to the phosphate oxygen causesan over-all increase in conformational stability. We feel that a third possibility, hydrogen bonding between the 2’-hydroxyl group and the ether oxygen of the sugar is sterioally unlikely. Also other possibleexplanations, such as some small differences in basebaseinteractions or other steric effects, cannot satisfactorily account for the observed striking differences between compounds with or without the 2’-hydroxyl group. The internal hydrogen-bond energy probably contributes little to the enthalpy change of the order-disorder process in 3’ -+ 5’ dinucleoside phosphates, but is important in that it facilitates base stacking interactions. In 2’ -+ 5’ dinucleoside phosphates (and in deoxy GpG), the absenceof the 2’-hydroxyl group doesnot allow the formation of this hydrogen bond. Thus the freedom of rotation about the covalent bonds of the backbone chain is not restricted. Consequently one can expect that the stacking in a helical single-strand conformation will occur at much lower temperature. The same arguments can be applied to chains of poly 2’-deoxynucleotides. In conclusion, the present investigation suggests that the stability of a helical single-strand polyribonucleotide chain is due not only to the stacking interactions but also to the effect of an internal hydrogen bond. One might expect that in a RhTA chain the “hot spot” of disordered conformation will be situated in the region where the ribose moiety has the 2’-hydroxyl group substituted by a methoxyl group. We thank Professor C. Sadron for his interest and encouragement of this work. Mrs M. L. Welter and Miss C. Monny provided excellent technical assistance. This work was presented at the Second Jnternational Biophysics Congress held in Vienna, September S-9,1966. REFERENCES Brahms, J. (1963). J. Amer. Chem. Sot. 85, 3298. Brahms, J., Maurizot, J. C. & Michelson, A. M. (1967). J. Mol. Biol. 25, 465. Brahms, J., Michelson, A. M. & Van Holde, K. E. (1966). J. Mol. Biol. 15, 1467. Bush, A. C. (1965). Thesis, University of California. Bush, A. C. & Brahms, J. (1967). J. Chem. Phys. 46, 79. Claverie, P., Pullman, B. & Caillet, J. (1966). J. Theoret. Biol. 112, 419. De Voe, H. & Tinoco, I., Jr. (1962). J. Mol. Biol. 4, 500. Helene, C., Douzou, P. & Michelson, A. M. (1966). Proc. Nat. Acad. Sci., Wash. 55, 376. Leng, M. & Felsenfeld, G. (1966). J. Mol. Biol. 15, 455. Massoulie, J. & Michelson, A. M. (1964). C.R. Acad. Sci. Paris, 259, 2923. Michelson, A. M. (1959). J. Chem. Sot. 3655. Michelson, A. M. (1963). The Chemistry of Nucleosides and Nucleotides. New York: Academic Press.
DINUCLEOTIDES
IN SOLUTION
495
Poland, z)., Vournakis, J. N. & Soheraga, H. A. (1966). Bio~oZymers, 4, 223. Pullman, B., Claverie, P. & Caillet, J. (1966). Proc. Nat. Acad. Sci., Wash,, 55, 904. IZichards, E. G., Flessel, C. R. & Fresco, J. R. (1963). Biopolymers, 1, 431. Sheftar, E., Barlow, M., Sparks, R. & Trueblood, K. (1964). J. Amer. C%em. Sot. 86, 1872. Tincoo, I., Jr. (1964). J. Amer. Chem. Sot. 86, 297. Ts’o, P.O.P., Rapaport, S. A. & Bollum, F. J. (1966). Biochemistry, 5, 4151. Van Holde, K. E., Brahms, J. & Michelson, A. M. (1965). J. Mol. Biol. 12, 726. Warshaw, M. M., Bush, C. A. & Tinoco, I., Jr. (1965). Biochem. Biophys. Res. cbmm. 1 633.
Wajrshaw,
M. M. & Tinoco,
I., Jr. (1965). J. Mol. Biol. 13, 54.