BIOCHEMISTRY
Polyriboadenylic and Polydeoxyriboadenylic Acids. Optical Rotatory Studies of pH-Dependent Conformations and Their Relative Stability* Alice J. Adler, Lawrence Grossman, and Gerald D. Fasman
ABSTRACT: The ultraviolet optical rotatory dispersion, circulm dichroism, and absorption properties of polyriboadenylic acid, polydeoxyriboadenylic acid, and the corresponding dinucleoside phosphates were examined as a function of pH and dioxane content. The objective of the study was to examine similarities and differences among the structural forms assumed by the ribose and by the 2’-deoxyribosecontaining polymers under various conditions. Circular dichroism at neutral pH, conditions under which the polymers form single-strand helices, confirms the optical rotatory dispersion finding of low-magnitude Cotton effects for polydeoxyriboadenylic acid, especially at high wavelength. The lack of dependence upon ionic strength of this ellipticity band implies that phosphate repulsion is not responsible for this reduced rotation. However, the relative stability of neutral polydeoxyriboadenylic acid toward dioxane may indicate a
T
he ultraviolet rotatory properties of DNA differ from those of RNA, probably reflecting different geometries for the asymmetric macromolecular structures. These differences affect the related phenomena of optical rotatory dispersion (Samejima and Yang, 1965) and circular dichroism (Brahms and Mommaerts, 1964) of these nucleic acids. Previous work in this laboratory has utilized these methods to study the effect of the pentose configuration upon the structure of cytidine compounds (Adler et al., 1967,1968). The present work compares poly rA with poly dA as conformational models for the ribo- and deoxyribonucleic acids. Poly rA, the corresponding dinucleoside monophosphate (rApA), and larger rA oligomers have previously been studied by several optical methods. These are ultraviolet spectroscopy (Fresco and Klemperer, 1959; Leng and Felsenfeld, 1966; Applequist and Damle, 1965, 1966), optical rotatory dispersion (Holcomb and Tinoco, 1965; Warshaw et al., 1965; Sarkar and Yang, 1965; Poland et al., 1966; Michelson et al., 1966), and circular dichroism (Van Holde et al., 1965; Brahms et al., 1966; Hashizume and Imahori, 1967; Bush and Scheraga, 1969). X-Ray diffraction in the solid state (Rich et al., 1961) and in solution (Luzzati et al., 1964), calorimetry (Epand and
* Publication No. 654 from the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154. Received April 2, 1969. This work was supported by research grants from the National Science Foundation (GB 6208 to L. G., GB-8642 to G. D. F.), the National Institutes of Health (AM-05852 to G. D. F.), and the Atomic Energy Commission (AT30-1-3449 to L. G.). L. G. is a Research Career Development awardee (Award No. I 310 nm (Le., scattered light). The optical equipment (Cary Model 14 spectrophotometer, and Cary Model 60 spectropolarimeter with 6001 circular dichroism accessory) was operated in a manner similar to that reported by Adler et a/. (1968). The spectrophotometer was flushed
with nitrogen for use below 220 nm. All experiments were performed at 22”, unless another temperature is indicated. Large pen periods and slow scanning speeds were required tor optical rotatory dispersion and circular dichroism runs at X < 225 nm. Optical rotatory dispersion results are reported in terms of [ ~ n ‘(residue ] rotation per mole of adenine residue, corrected for refractive index), circular dichroism in terms of [e] (residue ellipticity). Both parameters have units of (deg cm2); dmole. Formulas used for calculation are [m’] = (1OaObsd/ /c)(3/(n2 2)) and [e] = IO Oobsd//c, where a o h s d = rotation (degrees), e o b s d = ellipticity (degrees), / = path length (decimeters), c = residue concentration (M), and n = refractive index. The optical rotatory dispersion and circular dichroism curves could be measured to ~t0.0005deg in the wavelength range above 225 nm, and to iO.001 deg below 225 nm. Signal values at peaks and troughs were greater than 0.01 deg. except in the case of mononucleotides. A set of experiments was performed in which complex formation between poly r U and the adenyl polymers was monitored by ultraviolet absorption. Solutions for these studies M poly rA and poly dA, and 6 x were made of 3 X M poly rU, in acetate or formate buffers. The pH was adjusted independently for each solution with 1 M HCI or NaOH. For each pH the adenylate (A) and poly U solutions, at the same pH, were placed in separate 1-cm path-length compartments of a tandem cell, and the ultraviolet absorption spectrum was scanned in the 260-nm region. Then the A and U solutions were removed and quickly mixed, and the mixture was placed in both cell compartments. Spectra were taken at frequent intervals, starting at 2 min after mixing. The kinetics of complex formation were monitored by spectral changes, particularly by a decrease in absorbance at.,,X, A Corning Model 12 pH meter with expanded scale was used for titrations, as was a Manostat mercury-filled 0.1-ml digital readout micrometer buret, read to 0.0005 ml. The volume for titrations was 3.0 ml; the solvent was 0.1 M NaCl; the temperature was 23 i 1 ”. Titrations were stopped when opalescence occurred (pH N 2 for poly dA, and pH E 3 for poly rA). Spectrophotometric titrations were conducted in 1-cm square, stoppered cuvets containing magnetic stirring bars. The titrant was 0.1 M HCI, and volume increases were kept below 1%. Absorbance curves were scanned from 250 to 270 nm at each pH. Potentiometric hydrogen ion titrations were conducted separately; solvent blanks were measured and subtracted, and 0.01 M HCl was used. Paper electrophoresis was carried out in pH 3.5 sodium formate buffer, on Whatman No. 3MM paper, using 27,000 V. Samples and markers (adenine, deoxyadenosine, dAMP5’, d(pA)z, poly dA, purine, and deoxyinosine) were located under ultraviolet light.
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Results and Discussion The work will be presented in two parts: (A) data taken at neutral pH, where poly rA is a single-strand helix; (B) experiments performed under acidic conditions, where poly rA is a double-strand helix. A . Single-Strand Forms OpticalProperties.Optical rotatory dispersion (Holcomb and Tinoco, 1965) and circular dichroism (Brahms et al., 1966)have
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BIOCHEMISTRY
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1 : Circular dichroism of adenylate compounds at pH 8.5 in unbuffered 0.1 h.1 NaF at 22“. (a) rA compounds: poly rA. -; ,’ d(pA)t. dAMP-5‘, rA-2’-O-methyl-prAp, -.-.; rAMP-5’, . . . . . . (b) dA compounds: poly dA, rA (3’p5’)rA, FIGURE
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established that poly rA and the corresponding oligomers exist partially as single-strand helices, with a fraction of the bases stacked, when the adenine bases carry no charge. These rA compounds exhibit two high-wavelength Cotton effects, of opposite sign and nearly equal intensity. The optical rotatory dispersion pattern of single-strand poly dA shows a great reduction of the highest wavelength, positive Cotton effect (Ts’o et al., 1966; Vournakis ef al., 1967), although the lower wavelength optical rotatory dispersion data are similar for the two polymers. Circular dichroism data for poly rA, poly dA, and for several dinucleoside phosphates and mononucleotides are presented in Figure 1 and in Table I. These data, which extend t o 190 nm, confirm the loss in intensity of the first, positive ellipticity peak of poly dA even in comparison with d(pA)z. While this work was in progress, Bush and Scheraga (1969) collected some similar circular dichroism data for these compounds; the two sets of measurements are in substantial agreement. Poly dA is unique among synthetic polynucleotides for the small magnitude of its first circular dichroism band (the other bands being relatively normal); only DNA in ethylene glycol exhibits similar behavior (Green and Mahler, 1968). Results identical with those in Figure 1 were obtained in pH 8.5 Tris buffer. (See Experimental Section for buffer compositions.) For purposes of comparison with other spectra, the small poly dA positive ellipticity band at 264 nm should probably be considered, rather than the peak at 282 nm. The latter peak is comparable with the shoulder at the same position in poly rA, and may be due to an n-rY transition (Bush and Scheraga, 1969). The 264-nm poly dA peak is much smaller than the 264-272-nm bands of poly rA, rA(3’pS’)rA, and even d(pA)z. (These circular dichroism bands are attributable to the T-T* absorption peaks at 257 nm; exciton interactions split these peaks into positive circular dichroism bands at about 270 nm and negative ones at about 250 nm.) The shorter wavelength part of the poly dA circular dichroism spectrum (below
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260 nm) is very similar to that of d(pA)z; this finding can bc inferred also from the optical rotatory dispersion results of Vournakis et al. (1967), when they are calculated on a molar residue basis. This situation is very different from that of cytidine compounds (Adler et al., 1967), where poly rC, poly dC, and the corresponding dinucleoside phosphates all have similarly shaped optical rotatory dispersion curves, and the differences in Cotton effect magnitudes can be explained by varying amounts of base stacking. In the rA series, only the difference between poly rA and rA(3 ’p5 ‘)rA can be explained on this basis, and only if the poly rA 282-nm circular dichroism shoulder is ignored. Ultraviolet absorption spectra were obtained for all the adenyl compounds in 0.1 M N a F from 190 to 320 nm. These spectra are all similar. The highest wavelength peaks are listed in Table I. In addition, poly rA and poly dA show shoulders at about 280 nm, and all the compounds have shoulders at about 205 nm (except for the monomers, which have well defined small maxima at this wavelength). The ultraviolet spectrum of poly dA is identical with that of poly rA, to within the experimental error. Therefore, the striking differences in circular dichroism cannot be caused by any differences in electric transition moments. The circular dichroism and absorption spectra of poly dA were analyzed by means of a DuPont 310 curve resolver. Eight gaussian bands were required for an adequate fit of the circular dichroism data above 190 nm. Of these, the bands af 262 and 251 nm both appear to derive from the absorption peak at 257 nm. Similarly, the 217- and 206-nm circular dichroism bands appear to be correlated with a single absorption maximum, thus giving evidence for exciton splitting. The circular dichroism peaks of rA(2’pS’)rA are only slightly smaller than those of rA(3’pS’)rA at 22” (Table I); this is a result of the choice of temperature. Brahms et ul. (1967) have shown that the temperature dependence of these two compounds is different. The methylated dinucleoside di-
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Ionic S t r e n g t h , FIGURE 2: Effect of NaF concentration upon circular dichroism peaks of single-strand adenine polymers. Conditions: pH 8.5, 22“. Poly rA peaks (nanometers): 264, 0 - 0 ; 248.5, A-A; 221, 7-7; 205, n-2. Poly dA peaks (nanometers): 281.5, 0-0; 250, A-A; 218, V-V; 204.5, M-m.
phosphate, rA-2’-O-methyl-prAp, displays greatly reduced ellipticity, even when compared with d(pA)z. A study of Courtauld models shows that the methyl group sterically interferes with base stacking. However, Bobst et al. (1969) have recently demonstrated that at neutral pH poly-2’-O-methyladenylic acid has a circular dichroism spectrum nearly identical with that of poly rA. In addition, it has been demonstrated that rApAp has a smaller circular dichroism band at 275 nm than does rApA, thus showing that the 3’-phosphate also causes destabilization (Bush and Scheraga, 1969). Effect of Salt Concentration. The influence of NaF concentration upon the major circular dichroism peaks of poly rA and poly dA was studied (Figure 2). N o change in peak wavelengths was observed, indicating that at pH 8.5 the polymers retain their single-strand conformation even at low salt concentration, where the adenine pK,’s rise. Vournakis et al. (1967) showed that lengthening the chain of dA oligomers beyond the dimer does not result in increased rotation. To account for this observation, they postulated a next-nearest neighbor repulsive potential, which might possibly involve the negative charges on phosphate groups. If phosphate repulsion were an important factor in structure destabilization, then the ellipticity of poly dA should increase at high ionic strength, where the charges are shielded. Figure 2 shows that salt has absolutely no effect on poly dA circular dichroism at any wavelength. This is true even at l o , where increased base stacking causes a 10% increase in circular dichroism peaks, and where destabilization should be revealed more readily. Therefore, phosphate repulsion is most probably not responsible for the weak rotatory properties of poly dA, or for the polymer’s apparent inability to form any but pairwise stacks. The ellipticity bands of poly rA decrease somewhat as the salt concentration is raised. This is opposite to the effect of ionic strcngth upon poly rC and poly dC (Adler et al., 1968),
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A N D
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and indicates that phosphate-phosphate repulsion is unimportant in poly rA. Furthermore, although the salt effect is small, perhaps the data may be used as evidence for possible hydrogen bonds in single-strand poly rA between the 2’-hydroxyl groups and phosphate oxygens. If such bonds exist, then a decrease in salt concentration would increase the effective charge on the phosphate oxygen, thus leading to stronger hydrogen bonds and a greater rigidity of helical structure. Effect of Dioxane upon Single-Strand Structure. Organic solvents are known to destroy the helical structure of nucleic acids (Geiduschek and Herskovits, 1961) by disrupting basestacking interactions. Dioxane is a particularly good denaturing solvent (Levine et al., 1963) and has no hydroxyl groups that could possibly hydrogen bond with the polynucleotide. The two ether oxygens of dioxane could serve as possible, weak, hydrogen-bond acceptors, but are not expected to do so in the presence of water. In the present work, dioxane in varying concentrations was used to investigate the base stacking properties of poly rA, poly dA, and the corresponding dinucleoside phosphates. In particular, a comparison of the circular dichroism and the optical rotatory dispersion (Vournakis et al., 1967; Poland et al., 1966) data for these compounds, especially at low wavelength, leads to the expectation that poly dA might be similar to d(pA), and to rA(3’pS’)rA in its structural properties (Le., stabilization factors); poly rA, with its larger optical rotatory dispersion and circular dichroism bands, would perhaps be expected to be different. Furthermore, single-strand poly rC (Fasman et al., 1964) was shown to be more stable than poly dC (Adler et al., 1967) toward ethylene glycol denaturation, presumably because of 2‘-OH hydrogen bonding. The optical rotatory dispersion data collected in dioxane solutions at neutral pH are presented in Figure 3. The optical rotatory dispersion trough at about 213 nm is utilized as well as the one at about 260 nm, since the latter is relatively small in poly dA. All compounds exhibited an increase in extinction coefficient as dioxane was added. Poly rA solutions became opalescent at 45 % dioxane; the other compounds were still soluble at 80% dioxane. Monomer rotations were unaffected by dioxane. Figure 3 shows that it is poly dA, and not poly rA, which differs from the other compounds, and which retains its secondary structure in moderate concentrations of dioxane. Poly rA is similar to the rA and the dA dinucleoside phosphates in that its rotation is sharply reduced by small concentrations of dioxane. Analogous behavior for poly rA was observed in its sensitivity toward ethylene glycol denaturation at neutral pH (Hanlon and Major, 1968). On the other hand, the rotation (throughout the optical rotatory dispersion spectrum) of poly dA is nearly constant until over 25 % dioxane has been added. (The possibility that the stability of neutral poly dA toward dioxane may be caused by double-strand helix formation can be excluded on the basis of temperature-dependence experiments (Ts’o et al., 1966; Riley et a[., 1966; Vournakis et al., 1967; Barszcz and Shugar, 1968). The noncooperative melting of poly dA, similar to that of poly rA, shows that poly dA is single strand at neutral pH.) The dioxane data indicate that there may be some unique structural property of singlestrand poly dA, not present even in d(pA)*,which allows it to maintain its asymmetric conformation. This uniqueness is suggested also by the high-wavelength, positive band in the circLiI:ir dichroism and optical rotatory tlispcrsioii s p c d r a
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