BIOCHEMISTRY
The Secondary Structure of Polyadenylic Acid. Inferences from Its Reaction with Formaldehyde" Charles L. Stevens and Alita Rosenfeld
We have measured the kinetics of the pseudo-first-order reaction of formaldehyde with polyadenylic acid (poly A) and with adenosine between 30 and 45 ". The reaction, followed spectrophotometrically, does not appear to be cooperative. The forward and reverse rate constants are somewhat larger for adenosine than for poly A. The apparent equilibrium constants for the two compounds are between 3 and 10 (1. mole-') at all experimental temperatures; the values at each temperature, however, are not significantly different. Formaldehyde does not destroy the
ABSTRACT:
I
t has been suggested that the sugar-phosphate backbone of (single-stranded) ribonucleic acid (RNA)' in solution folds back on itself permitting the formation of limited regions of hydrogen-bonded base pairs. Compared to deoxyribonucleic acid (DNA), these regions are presumed to be less extensive and the bases imperfectly paired in some similar but less stable manner (Fresco et al., 1960). Like some species of RNA, polyadenylic acid (poly A) in solution of neutral pH has a broad thermal-denaturation profile and upon heating gradually loses optical rotatory power. Because of these and other properties, it was proposed that poly A has a structure like that suggested for RNA (Fresco and Klemperer, 1959). On the basis of optical rotatory dispersion and ultraviolet absorption studies of polycytidylic acid (poly C) in aqueous formaldehyde (pH above 6) and other organic solvents, however, Fasman et al. (1964) have reported that this polymer possesses helical structure, but amino-group hydrogen bonds have a negligible influence on the
2714
* From the Department of Biophysics, University of Pittsburgh, Pittsburgh, Pennsylvania. Receioed January 31, 1966; reaised May 18, 1966. This is publication No. 123 of the Department of Biophysics, University of Pittsburgh. Work was supported by U. S . Public Health Service Grant G M 10403 and further by an Andrew Mellon Predoctoral Fellowship and a U. S. Public Service Predoctoral Fellowship to Miss Rosenfeld. The data presented in this paper were taken in part from a dissertation presented by Miss Rosenfeld to the University of Pittsburgh in partial fulfillment of the requirements for the degree of Master of Science, Dec 21, 1965. A preliminary report of this work was presented before the Biophysical Society at its 9th Annual Meeting, San Francisco, Calif., Feb 1965. 1 Abbreviations used in this work are: AMP, adenosine monophosphate; ApA, adenylyl-(3',5')-adenosine; poly (A+U), the two-stranded complex of poly A and poly U ; poly (I+C), the two-stranded complex of poly I and poly C ; A , , relative absorbance.
C H A R L E S L.
STEVENS A N D
ALlTA
ROSENFELD
heat-susceptible secondary structure of poly A usually associated with the hypochromic effect. Also, there are fewer than 10% of the adenosine monophosphate (AMP) residues in poly A which could be masked from formaldehyde, even at 20". When compared to the AMP residue, the chemical environment of the formaldehyde-reactive group (probably the C-6 amino group) is largely unaltered in the polymer. As already suggested by others, there appears to be no deoxyribonucleic-like base pairing in poly A in aqueous solution near neutral pH.
stability of the structure. They suggest that the molecule is a single-stranded helical structure stabilized by interactions between stacked bases. Holcomb and Tinoco (1965) concluded that poly A has a similar structure, and on the basis of small-angle X-ray scattering, Luzzati et af. (1964) and Witz and Luzzati (1965) concluded that poly A is rodlike and helical with stacked base planes perpendicular to the helix axis with no hydrogen bonds linking the bases. Although the thermal denaturation behavior of poly A appears to be characteristic of single-stranded RNA, when data for poly A and oligomers of AMP are analyzed according to the van't Hoff equation, it becomes apparent that the secondary structure of poly A is not cooperatively formed. The derived value of the apparent A H (Stevens and Felsenfeld, 1964) is about the same as that measured calorimetrically (Rawitscher et a[., 1963). Leng and Felsenfeld (1966) refined these measurements and, further, showed that the apparent A H was almost independent of chain length over the range of dimer, ApA, to poly A. They concluded that the temperature-dependent hypochromism of poly A arises from a pair-wise stacking interaction between adjacent bases. Virtually the same conclusions were expressed by Van Holde et al. (1965) on the basis of the optical properties of poly-N6hydroxyethyladenylic acid and ApA ; by Vournakis et a/. (1966) on the basis of the optical properties of ApApCp; and by Warshaw and Tinoco (1965) and Cantor and Tinoco (1965) based on experiments with various dinucleoside phosphates and trinucleoside diphosphates. We have completed a systematic study of the kinetics of the reaction of poly A with formaldehyde which upholds this conclusion. The reaction with nucleotides does not go to completion at ordinary concentrations
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of formaldehyde (about 1%); thus one would expect that an appreciable alteration of the chemical environment of the corresponding base (such as polymerization) would result in a shift in the chemical affinity of the reaction. By measuring the forward and reverse rate constants spectrophotometrically for both poly A and adenosine we have estimated this affinity and find that the two materials give nearly the same values. The results also show that at 20°, there can be no more than 10% of the residues of poly A participating in DNA-like base pairing, even though the material retains about one-half or more of its maximum hypochrornism. Our results are consistent with the proposal that poly A is a single-stranded helix with stacked base planes enabling the C-6 amino group to react freely with formaldehyde. Materials and Methods Poly A was supplied by the Miles Laboratories, Elkhart, Ind. The method of preparation of stock solutions and the absorptivity of the polymer have been reported (Stevens and Felsenfeld, 1964). The solutions were extracted against freshly distilled, water-saturated phenol and the residual phenol was removed by ether extraction. Dissolved ether was removed either by dialysis or by bubbling carbon dioxide free nitrogen through the solution until no odor of ether remained. The solutions were stored frozen until used. The poly A had a sedimentation coefficient of 5.1 S when dissolved in 0.08 ionic strength phosphate buffer, at pH 7.5, and 19.6”. All reactions were carried out in this buffer solution. Reagent-grade aqueous formaldehyde was used. The material was distilled and stored in a light-tight glass vessel in the refrigerator. Concentration was determined by titration of the base formed upon the reaction of formaldehyde with sodium sulfite (Walker, 1953). Low concentrations of formaldehyde were determined by an adaptation of the method of Bricker and Johnson (1945) using chromotropic acid. Under the conditions of the test, formaldehyde and chromotropic acid form a relatively stable complex with a molar absorptivity at 580 mp of 18,400. To 1 ml of solution containing 10 pg or less of formaldehyde, 0.3 ml of chromotropic acid solution was added. This solution consisted of 2.5 g of 4,5-dihydroxy-2,7napthalenedisulfonic acid disodium salt (Eastman) and 50 mg of stannous chloride in 25 ml of water (Walker, 1953, p 369). Concentrated sulfuric acid (3 ml) was added slowly to the test sample and mixed thoroughly. The tubes were covered, heated for 15 min in a boiling water bath, and cooled, and 1.0 ml of water was added. The absorbance was measured at 580 mp within 0.5 hr after cooling. For the determination of formaldehyde in the presence of poly A, blanks containing poly A in various concentrations but without formaldehyde were used. The absorbance due to poly A in the test samples was subtracted from the measured value to obtain the corrected value; these corrections were never larger than 6 %.
THE
Absorbance measurements were made on a Cary Model 14M recording spectrophotometer. Cuvets were placed in a cored cell block for temperature control. Water was circulated from a Haake Model F thermostat to the cell block. Temperature was measured with a thermistor (Victoreen Engineering Co. No. 51A35) inserted in a dummy cuvet; resistance was measured with a Wheatstone bridge circuit with the thermistor making up one arm of the bridge. Selfheating of the thermistor was negligible. For the kinetic experiments, solutions of poly A and formaldehyde at the appropriate concentration were preequilibrated at the desired temperature and mixed. Maximum absorbance values at zero time varied between 0.4 and 1.2 but rate constants were independent of concentration of nucleotide between these limits. The first measurements could be obtained about 30 sec after mixing. Usually, the spectrum between 220 and 320 mp was recorded. The absorbance “at infinite time” was taken after there was no further change of absorbance, usually after 24-48 hr. The concentration of formaldehyde was about 2 % (g/IOO ml of solution) or less in all experiments; it has been reported that formaldehyde is in the hydrated, monomeric state under these conditions (Walker, 1953, Chapter 3). In some instances, the poly A-formaldehyde complex was removed from unreacted formaldehyde by gel filtration with Sephadex G-25 (Pharmacia Company, Upsala, Sweden). A column about 1 cm in diameter was filled to the 10- or 20-cm mark with gel equilibrated with buffer, and the poly A and formaldehyde mixture (about 1 ml) was placed on the column. The resulting 1-ml effluent fractions were assayed for poly A by ultraviolet absorption. In one instance, an effluent fraction was placed in the spectrophotometer and the rate of release of formaldehyde was measured directly. In others, the fractions were assayed for formaldehyde with chromotropic acid as well as for poly A. The thermal dependence of the hypochromism of poly A in 2.2% formaldehyde was measured; the poly A-formaldehyde mixtures were heated or cooled in steps and the spectrum was recorded after temperature equilibration and after absorbance no longer changed. Absorbances from these nonisothermal experiments were corrected for thermal expansion of solvent (water). Results and Discussion The reaction of formaldehyde with nucleotides and nucleosides follows pseudo-first-order kinetics and does not go to completion for formaldehyde concentrations of about 1 or 2%. If the reaction with adenosine is represented by the following formula
the differential equation for the reaction is
SECONDARY
S T R U C T U R E OF
POLYADENYLIC
A C
B IO C H EM I S T K Y
45
Adanorin.
40
35
-1
.-
30
E.
/
0
/
0 x
25
Y
20
15
10
5
' I I
.1
.2
.3
1--
~
.4
30
- r
Ad
I
-7 E
Y
-
1_1
20
0
x Y
/ /
15
40 '
/ /
/ / 10
/
35O
5 /
/
/
3 0'
/
-,---
2716
I
I
.1
.2
I
I
.3
.4
[HC H 01 [ m o I i/ I i 1. r ) apparent rate constants ( k ' ) for the reaction of adenosine with formaldehyde (a) and poly A with formaldehyde (b). The graphs show the dependence of k' on formaldehyde concentration and on temperature.
FIGURE 1: The
C H A R L E S L.
STEVENS
A N D
ALlTA
KOSENFELD
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1966
AUGUSI'
The Second- ( k z )and First- ( k l )Order Rate Constants for the Reaction of Adenosine and Poly A with Formaldehyde.
TABLE I:
0
_____
Adenosine Temp -
a
~-
-
___
~
Poly A
( "0
kz X lo3 (1. mole-lmin-l)
kl X l o 3 (min- l )
k2 X l o 3
30 35 40 45
14.83 f 1 . 5 5 3 0 . 9 1 1 2.48 45.32 f 1 . 7 3 6 8 . 5 0 & 4.20
2.41 f 0.39 3.48 + 0 . 6 3 8 . 4 6 f 0.44 20.03 f 1.07
5.94 f 0.29 17.90f 0.56 25.50 f 0 . 4 8 58.42 f 1 . 4 2
___ kl X lo3 ~.
1.49 f 0.07 1.81 f 0 . 1 4 5.49 f 0 . 1 2 9.13 f0.36
The solvent was 0.08 ionic strength sodium phosphate buffer solution, pH 7.5.
A is the concentration of unreacted adenosine at time t, A . is the initial concentration of adenosine, Ar is the concentration of adenosine reacted with formaldehyde at time t , and y is the initial concentration of formaldehyde. The forward rate constant is k2 and the reverse constant is kl. If, as t + m , A A,, and because y remains virtually constant, the solution of the differential equation can be written
-
analogous to that given by Penniston and Doty (1963). Figure 1 shows a plot of k' 6s. formaldehyde concentration for poly A as well as for adenosine. Linear plots have been obtained with both materials for all temperatures tested (30-45 "). Straight lines were fitted to the data by the method of least squares, and the slopes and intercepts of these lines are listed in Table I. The limits on the real rate constants were calculated from the root-mean-square deviation of the measured k' about the least-square line, S,, and expressed as
If the stoichiometric coefficient for formaldehyde in (1) were not 1, say n,(3) would be ki
A - A, -= Ao - A ,
+ kp))t
exp -(kl
(In these experiments, however, we find that the value of n is 1.) The left side of (3) is a fraction which varies from 1 at zero time to 0 at equilibrium. In the spectrophotometric method used, this parameter is represented by E,
-
em
- eo
E
(4)
if Beer's law is valid for formalized and unformalized adenosine. Here E is given by E = fie1
+ he2
where fi and A are the mole fractions of adenosine and formalized adenosine at time t and el and e2 are the respective molar absorptivities. A plot of the natural logarithm of (4) with time, then, should yield a straight line with slope of - k' where k'
=
ki
+
k2y
(5)
and k' is the apparent rate constant. The values of k' for adenosine and for poly A are independent of wavelength between 253 and 280 mp. Equation 4 is
f
-)+
S,( y2
)'l
Y2
- Yl
where yt and yl are the highest and lowest concentrations of formaldehyde used in a particular series. The point on the ordinate of the graph for poly A in Figure 1 was obtained by the direct spectrophotometric measurement of the dissociation of formaldehyde from poly A at 40 " after separating the unreacted formaldehyde by gel filtration. The point is within 1.5% of the intercept by least-squares analysis of the 40 O data. Formaldehyde is known to react irreversibly with nucleotides forming an intermolecular methylene bridge (Fel'dman, 1962). It appears, however, that this crosslinking proceeds far too slowly to be significant under the conditions of our experiments. On the basis of the observed rate of cross-linking of DNA by high concentrations of formaldehyde (12 %) (Freifelder and Davison, 1963), Penniston and Doty (1963) estimated that perhaps 0.3 % of the molecules of soluble ribonucleic acid (sRNA) might become cross-linked in 320 hr in 0.3 % formaldehyde, neutral pH, at 25 '. Haselkorn and Doty (1961) found that, if they removed the reacted formaldehyde by dialysis, the regenerated poly A was able to form a complex again with poly U and the temperature of the midpoint of the thermal transition was unaltered. In addition, these authors concluded that 2.76% formaldehyde does not induce a chain scission of single-stranded poly I after 15 min at 65"
THE SECONDARY S T R U C T U R E O F
271 7
P O L Y A D E N Y L I C A C I D
BIOCHEMISTRY
c I
-. ,
1
poly A
2.0
I
*
1.6
0 X
c
.-
1.2
E 8 6
-8
0.0
-0.4
-
1
I
1
I
1
1
I
I
I
i I I
l 1 I
I
I
I
1
I I I I I
I 1 1 I 1
I I I I I I
I
'
I I 1 1 I I I
I I
-
-
I
formaldehyde
I -
I
I
I
I
I
I
I
0 7
8
9
10
11
12
13
14
15
16
fraction no.
FIGURE 2: Separation of unreacted formaldehyde from a poly A-formaldehyde complex with Sephadex G-25. Poly A was incubated for 48 hr in 2% formaldehyde at 20"; the separation was carried out also at 20" and completed within 15 min after placing the mixture on the column. Except for the use of a longer column for the second separation, the experiments are duplicates.
2718
in 0.12M phosphate buffer solution of pH 6.8. There are, however, two important questions to answer before a comparison of the behavior of poly A and adenosine toward formaldehyde can be made. The affinity of the formaldehyde reaction with nucleotides is small, and the possibility exists that there is a class of primary amino groups of poly A which remains unavailable to reaction with formaldehyde (e.g., groups participating in amino-group hydrogen bonds). Indeed, this is generally believed to be the case for RNA in solution and has been suggested for poly A (Fresco and Klemperer, 1959). The kinetic method cannot detect this. A sample of poly A was incubated in 2% formaldehyde at 20" for 48 hr. Unreacted formaldehyde was removed from aliquots of the reaction mixture by gel filtration. The conditions of formalization were chosen such that, if all AMP residues of poly A were free, at least 90% of them would have reacted by the end of the incubation period. On the other hand, if the hypochromism of poly A under these conditions results from a DNA-like base pairing in which the AMP residues are masked from formaldehyde, more than 50% of them would not react. Figure 2 shows that at least 90 of the residues were complexed with formaldehyde.
C H A R L E S L.
STEVENS
A N D
ALITA
ROSENFELD
The other question involves the possibility that formaldehyde induces a structural alteration of poly A which unmasks the AMP residues. Haselkorn and Doty (1961) have concluded that this is, indeed, the situation with the system poly (A+U) in formaldehyde. While it is likely that the kinetic method used here would detect this, the absorption spectra of poly A in formaldehyde show that it does not occur. Figure 3 is a tracing of superimposed spectra obtained at successive times during the reaction with formaldehyde. They reveal an isosbestic point at 255.0 (*OS) mp for adenosine and one at 252.5 ( * O S ) mp for poly A. Molar absorptivity at 252.5 mp for poly A, however, is very sensitive to the extent of thermal denaturation. Formaldehyde, therefore, does not induce this type of structural transition in poly A. Virtually all the AMP residues of poly A, then, are available for the reaction with formaldehyde, and the complex forms without appreciable alteration of the short-range order of the residues. Recent viscosity studies by T. E. Cartwright. and L . L. Larcom (private communication) have shown that the reduced viscosity of poly A increases only about 4 % in 4 % formaldehyde at 30", while that of RNA from tobacco mosaic virus increases almost three fold under the same conditions. This suggests that the long-
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0.9 \
/'
0.8
1%
HCHO
0.7 0.6
0.5
EC
0.4
0
.a
z 3
L
2so
0.7
1
260
170
0
280
Adenor in.
0.6
0.8% HCHO
30'
0.5 0.4
0.3 0.2
.
1
A
I
250
260
270
. 280
290
w a v o l o n g r h in m y
3: Superimposed spectra from the reaction of poly A and of adenosine with formaldehyde. The spectra were respecrecorded at successive times after adding formaldehyde. Formaldehyde concentrations were 1 and 0.8 tively, and the temperature was 30".
FIGURE
z,
range order of poly A also remains virtually unaltered by formaldehyde. We have also measured the thermal denaturation of formalized poly A. It is evident from Figure 4 that the formalized polynucleotide still possesses hypochromism. Poly A is similar to poly C in this respect (Fasman et al., 1964). When the loss of hypochromism is measured at the isosbestic point, as we have done, one observes that formaldehyde perhaps even stabilizes the structure rather than destabilizes it as previously supposed. (In the absence of data for the complete thermal transition of formalized poly A, however, this conclusion cannot be stated for certain.) Others have observed that Ne-substituted poly A still retains temperaturedependent hypochromism. Solutions of poly Nehydroxyethylriboadenylic acid of neutral pH show an increase of absorbance on heating. However, the extent
of the increase is not as large as that for poly A, at least between 20 and 90" (Van Holde et al., 1965). Also, poly-N6-methylriboadenylic acid and poly-Nedimethylriboadenylic acid display gradual hyperchromicity at pH 7.5 with increasing temperature (Griffin et al., 1964). The plot of k' with formaldehyde concentration for adenosine and for poly A yield good straight lines in accord with eq 5, suggesting that the reaction is not cooperative for poly A as well as for adenosine. Thus, the calculation of an apparent equilibrium constant from the real rate constants for poly A seems justified. These values appear in Table 11. The values for adenosine agree fairly well with those calculated for AMP by Grossman er al. (1961) by a slightly different method; this agreement shows that the simple kinetic model of the reaction of formaldehyde with nucleotides is ade-
THE SECONDARY
STRUCTURE
OF
POLYADENYLIC
2719
A C I D
I3 1 0 C H E M 1 Y T R Y
1.20
-
POLY A IN FORMALDEHYDE dependence of absorbance on temp, 1 5 3 mp
1.16
without HCHO
"
[I--cl 2.2% HCHO
Ar 1.12
-
1.08
-
1.04
..
1.00
IO
20
30
40
50
60
70
1EMP.
FIGURE 4: Thermal denaturation of poly A and of poly A in the presence of formaldehyde. The measurements were taken at 253 mp, the wavelength of the isosbestic point for the formaldehyde reaction. Formaldehyde concentration was 2 . 2 z .
The Apparent Equilibrium Constants for the Reaction of Adenosine and Poly A with Formaldehyde Calculated as the Ratio of the Second- and First-Order Rate Constants.
TABLE 11:
1. mole-L
Temp
2720
("C)
Adenosine
Poly A
30 35 40 45
6.15 f 1.98 8.88 f 2 . 8 2 5.36 f 0 . 4 8 3.42 =k 0.39
3.99 i 0 . 4 3 9.89 1.10 4.64 i 0 . 1 9 6.40 f 0.40
*
quate. While the real rate constants for adenosine and for poly A are somewhat different, the apparent equilibrium constants are within 50% of one another. There is sufficient scatter in the data to prevent an estimation of the enthalpy of the reaction from a van't Hoff analysis; we cannot tell, then, if the similarity of equilibrium constants reflects a compensating shift in the enthalpy and entropy of reaction for poly A com-
C H A R L E S L.
STEVENS A N D ALITA
KOSLNFELD
pared to adenosine. Barring this coincidence, it appears that there is no large alteration of the chemical environment of the formaldehyde-reactive group on poly A compared to the mononucleoside. It is clear that the structure of poly A in neutral solution does not consist of regions of DNA-like base pairing. The hypochromism as well as other properties indicates that the molecule, nevertheless, possesses a considerable degree of order. In light of the kinetic behavior of poly A, the group reactive to formaldehyde (probably the C-6 amino group) is not masked and its accessibility remains largely unaltered in the polymer. The conclusion that poly A is a single-stranded, probably helical molecule with no internucleotide hydrogen bonding seems to be established rather firmly. A number of investigators have reported that formaldehyde induces a structural alteration in different types of natural RNA and in some polynucleotide complexes. Haselkorn and Doty (1961) conclude that formaldehyde initially denatures polynucleotide complexes [uiz., poly (A+U) and poly (I+C)], then reacts with the susceptible residues thus unmasked. Using similar techniques, Penniston and Doty (1963) observe an analogous effect of formaldehyde on the structure
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of s-RNA. Stanley and Bock (1965) report that the sedimentation coefficient of the “23s” component of ribosomal RNA fell to about 18 S in 1 formaldehyde0.1 M potassium chloride-0.05 M potassium phosphate, pH 6.9, while that of the “16s” component fell from about 17 S to about 15 S. The structural transition induced by formaldehyde is demonstrated, perhaps most dramatically, by the viscosity study of RNA from tobacco mosaic virus by Cartwright and Larcom already mentioned. These transitions are probably analogous to that of DNA in formaldehyde observed by Luzzati et al. (1964). While it is apparent that poly A is not a good structural analog of RNA, it appears that it may be a structural analog of formalized RNA. Acknowledgments We thank Dr. Gary Felsenfeld for his helpful suggestions during the course of this investigation and Dr. Max A. Lauffer for supporting this work under a Program-Project Grant from the U. S. Public Health Service to the Department of Biophysics. Added in Proof After this manuscript was submitted, it was called to our attention that two additional papers demonstrating the noncooperative nature of the secondary structure of riboadenylates were in press. Poland et al. (1966) measured the optical rotatory dispersion of the oligonucleotides (PA),, (PA),, and PA)^ over the temperature range 5-85’ and concluded that the stacking interactions of successive residues could be treated as independent. Brahms et al. (1966), in general agreement with Leng and Felsenfeld (1966), concluded that the thermodynamic parameters associated with the thermal denaturation of oligomers of adenylic acid are essentially independent of chain length. References Brahms, J., Michelson, A. M . , and Van Holde, D. E. (1966), J . Mol. B i d . 15, 467.
Bricker, C . E., and Johnson, H. R. (1945), Ind. Eng. Chem. 17, 400. Cantor, C. R., and Tinoco, I., Jr. (1965), J . Mol. Bid. 13, 65. Fasman, G. D., Lindblow, C., and Grossman, L. (1964), Biochemistry 3, 1015. Fel‘dman, M. Ya. (1962), Biochemistry ( U S S R ) 27, 321. Freifelder, D., and Davison, P. (1963), Biophys. J. 3, 49. Fresco, J. R., Alberts, B. M., and Doty, P. (1960), Nature 188, 98. Fresco, J. R., and Klemperer, E. (1959), Ann. N . Y. Acad. Sci. 81, 730. Griffin, B. E., Haslam, W. J., and Reese, C. B. (1964), J. Mol. Biol. 10,353. Grossman, L., Levine, S. S., and Allison, W. S. (1961), J . Mol. Bioi. 3, 47. Haselkorn, R., and Doty, P. (1961), J . Bid. Chem. 236, 2738. Holcomb, D. N., and Tinoco, I., Jr. (1965), Biopolymers 3,121. Leng, M., and Felsenfeld, G . (1966), J . Mol. Biol. 15, 455. Luzzati, V., Mathis, A., Masson, F., and Witz, J. (1964), J. Mol. Bid. 10, 28. Penniston, J. T., and Doty, P. (1963), Biopolymers 1, 145. Poland, D., Vournakis, J. N., and Scheraga, H. A. (1966), Biopolymers, 4, 223. Rawitscher, M. A., Ross, P. D., and Sturtevant, J. M. (1963), J . Am. Chem. Soc. 85. 1915. Stanley, W. M., Jr., and Bock, R. M. (1965), Biochemistry 4, 1302. Stevens, C . L., and Felsenfeld, G. (1964), Biopolymers 2,293. Van Holde, K. E., Brahms, J., and Michelson, A. M. (1965), J . Mol. Biol. 12, 726. Vournakis, J. M., Scheraga, H. A., Rushizky, G. W., and Sober, H. A. (1966), Biopolymers 4, 33. Walker, J. R. (1953), Formaldehyde, 2nd ed, New York, N. Y., Reinhold, p 383. Warshaw, M. M., and Tinoco, I., Jr. (1965), J. Mol. Biol. 13, 54. Witz, J., and Luzzati, V. (1965), J . Mol. Biol. 11, 620.
272 1
1 ilk
YtCONDAKY
STKUCTUKE OF
POLYADENYLIC
A C I D
