Helical complexes of polyriboinosinic acid with copolymers of polyribocytidylic acid containing inosine, adenosine and uridine residues

J. 1MoZ.Biol. (1971) 62, 591-611

Helical Complexes of Polyriboinosinic Acid with Copolymers of Polyribocytidylic Acid containing Inosine, Adenosine and Uridine Residues AMY C. WANG AND NEVILLE R. KALLENBACH

Department of Biology, Leidy Laboratories University of Pennsylvania Philadelphia, Pa. 19104, U.X.A. (Received 21 October 1970, and in revised form 26 August 1971) The consequences of incorporating non-complementary residues into the poly (I). poly (C) helix have been investigated. Complexes of poly (I) and copolymers of C with different mole-ratios of I, A and U residues have been prepared and denatured in a variety of solvents. The results of both denaturation and analysis of the stoichiometry of the reactions suggest that in poly (I).poly (C, I,) complexes, the I residues are excluded from the helix matrix, whereas in the poly (I)*poly (G, U,) and poly (I)*poly (C, A,) systems the minor component bases are retained. Preliminaries to a quantitative analysis of the transition data are presented, permitting rough estimates of the difference in stability between poly (I) *poly (C) and poly (I). poly (U) or poly (I). poly (A) pairs in these complexesthe results being 1.7 kcal./mole and 1.3 kcal./mole, respectively. The differences in behavior of poly (I). poly (C, I) complexes are found to be most evident in the presence of 8 M-urea.

1. Introduction Since Watson & Crick (1953) first invoked hydrogen bonding between nucleotide bases as a source of stability and specificity in their structural model for DNA, it has become clear that interactions other than hydrogen bonds have a significant role in stabilizing ordered nucleic acids in solution. These include solvent interactions, internal base-base interactions parallel to the helix axis and electrostatic contributions due to effects of counterions on the negatively-charged phosphate groups (see the reviews by Zimm & Kallenbach, 1962; Felsenfeld & Miles, 1967). The relative contributions of the different interactions involved have proved to be difficult to assess quantitatively since the over-all stability of helices represents a balance of fairly small contributions. That hydrogen bonding is responsible for much of the specificity of base-pair interactions is shown by the selective affinity of adenine for uracil and guanine for cytosine at the monomer level. In non-aqueous solvents the association constants and enthalpies of dimerization for the complementary pairs are greater than for the corresponding homologous interactions (see Voet & Rich, 1970 for a recent review). In aqueous solution, Tuppy & Kuchler (1964) have shown that uracil and cytosine are retarded by columns bearing linked adenine and guanine derivatives, respectively. The effects of non-complementary bases on the stability of synthetic polynucleotide bihelices have been investigated extensively since the first experiments of Steiner 591

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(1959) on complexes of poly (A) with poly (U) strands containing A residues in various amounts. From the spectral mixing curves for these complexes, Fresco & Alberts (1960) concluded that the additional uracil residues preferentially loop out into the solvent and are apparently excluded from the helix. Several other complexes have been found to lead to “helix-with-loops” structures, including complexes of poly (U) with poly (A$) and poly (A,G) (Bautz & Bautz, 1964) and complexes of poly (I) with poly (C,U) (Tsuboi, Matsuo & Nakanishi, 1968). The biological implications of these structures have been discussed by Bautz (1965) as well as Fresco & Alberts (1960). However, the energetics of suoh models have not been estimated. The possibility that, under certain circumstances, bases which fail to hydrogen bond in the normal sense may nevertheless be retained within a helix must be considered. In formulating the wobble theory for relating degenerate codons to anticodons, Crick (1966) pointed out that the steric requirements for base-pair formation at the end of a helical segment may be less stringent than within a helix. Thus, the wobble pairs G-U and 1-A were postulated as feasible only at the end of a codon. Whether or not these pairs are forbidden sterically at an internal position is open to question, although the fidelity of genetic mechanisms argues that they must be rare. We have undertaken a detailed investigation of the behavior of complexes of poly (I)*poly (C) in which the pyrimidine strands contain different concentrations of I, U and A residues. We report here the thermal denaturation of these complexes in three different solvent systems at neutral pH : (1) O-1 M-Na+, (2) 0.5 M-N&+, and (3) loNa+, 8 M-urea. The poly (I)*poly(C,I,) series of complexes exhibits a progressive loss of hyperchromism and co-operativity as the mole fraction x increases, in contrast to the behavior of the poly (I)*poly (C,U) and poly (I)*poly (CA) series. A statistical analysis of the spectral mixing curves for these interactions leads to the conclusion that the break in the mixing curve of poly (I) + poly (CJ,.,,) occurs very nearly at 42 moles y0 poly (I), and not at 50 moles y0 as in the cases of poly (I) + poly (C) and the other complexes studied. Finally, the progressive shift in density with increasing x is most severe in the poly (I)*poly (CJ) system, suggesting availability of the excess I residues in the equimolar complexes for multistrand interactions. These results indicate that the I residues in the C strand are excluded from the helix with formation of a helix-with-loops structure, in agreement with results of Tsuboi et al, (1968). However, the behavior of U and A residues in the C strand fails to reveal a comparable tendency of these residues to loop out, and we propose that they remain completely included within the helix structure, at least for the range of mole fractions investigated.

2. Materials and Methods (a) Chem;cazs B’diphosphates The synthetic polynucleotides poly (I) and poly (C) and nucleotide used in copolymer syntheses were obtained from Calbiochem. Polynucleotide phosphorylase was obtained as a nuclease-free preparation from P. L. Biochemical% (b) Polymer Three series x: denotes the general, input the synthesis

synthesis

of copolymers, poly (C,I,), poly (C,U,) and poly (C,A,), where the subscript mole fraction of IDP, UDP and ADP, respectively, were synthesized. In values of z were 0.1, OS, and 0.3 for each series. The reaction mixture for contained the following components: 0.015 M-MgCl,, 0.1 M-Tris buffer (pH

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9.0), 0.002 m6-EDTA, O-03 M total nucleotide diphosphates, and 9 units of enzyme in a total volume of 3 ml. (Howard, Frazier & Miles, 1969). The reactions were carried out in a capillary viscometer maintained at 37” C, and the course of reaction was followed by the increase of outflow time of the mixture in the viscometer. When the outflow time attained its maximum value, the reaction was stopped by chilling, the solution was extracted once in phenol, precipitated with ethanol and dialyzed successively against ,O*OOl M-EDTA + 0.1 M-NaCl solution, 0.001 M-EDTA, and distilled water.

(c) Churacteri2ation

of polymers

was measured in O-1 M-Nat, The sedimentation coefficient (8 2,,, w) of each polymer O-005 M-sodium cacodylate (pH 7*0), at polymer concentrations of about 10s4 M, using a Spinco model E ultracentrifuge equipped with U.V. absorbtion optics. Molar extinction coefficients and base ratios of all copolymers were determined from the u.v. spectra of alkaline hydrolysates of the polymers (Loring, 1955). A sample of each copolymer was hydrolysed at room temperature in 0.3 N-KOH for at least 18 hr. The hydrolysate was then adjusted to pH 7 (pH 2 for the poly (C, U) hydrolysates) by addition of HCl and the U.V. spectrum measured on a Cary no. 16 spectrophotometer. In certain cases extinction coefficients were also measured by phosphate analysis (Ames, 1966), and the base ratios cheoked by fractionating the alkaline hydrolysates by chromatography on a Dowex formate column (Hayashi & Spiegelman, 1961). The results were in very good agreement with the values obtained spectroscopically. (d) Mixing

cwves

Ultraviolet spectroscopic mixing curves were performed by the method of continuous variation (Felsenfeld & Rich, 1957). For each copolymer, a series of mixtures was made with varying ratios of poly (I) to copolymer and constant total nucleotide concentration. After equilibrating the series of mixtures overnight, the absorbance of each mixture was measured at several wavelengths with a Gary no. 16 spectrophotometer. In certain cases, measurements were repeated after a further interval of 24 hr to ensure attainment of equilibrium.

(e) Thermal denaturation

po$les

The equimolar complexes of poly (I) with the copolymers in each series were melted in Teflon-stoppered cuvettes held in a thermostatically controlled cell holder in a Gilford model 2000 recording spectrophotometer. The profile of absorbance at 250 nm as a function of time was recorded while the temperature of the solutions was increased linearly with time at a rate of approximately 0.5 deg. C/ mm, using a New England Scientific temperature programmer and bath. The temperature of the block holding the cuvettes was measured by a thermistor calibrated with a thermocouple.

3. Results (a) Xingle-stranded

polymers

Ultraviolet absorption spectra of the copolymers in 0.1 M-Na+, pH 7, and at room temperature are shown in Figure 1. In the series poly (CJ), a single isosbestic point at 262 nm is observed; in poly (C, A), the isosbestic point occurs at 268 nm. The series poly (C,U) shows two isosbestics, at 243 mn and 274 nm. In each case, a regular change of the spectral curves with composition is seen. A summary of sedimentation coe&ients and molar extinction ooeikients is given in Table 1. For the series of copolymers poly (CA), the base composition of the polymers and the input mole ratio of CDP to ADP were found to be the same over the range of values indicated. In the poly (CJ) series, the base composition differs from the input mole ratio CDP:IDP by a constant factor of 0.8, while in the poly (C,U) series this factor decreases to 0.7. Nearest neighbor or sequence analysis of the copolymers has not been performed, so that deviations from random sequence of the two bases may exist. Earlier studies by

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IO 9 8 7 "? 9 ",

6 5 4 3 2 I 240

260

280

300

240

260

280

300

240

260

260

300

Wavelength (nm)

FIG. 1. Extinction ooefficients of polymers in 0.005 M-sodium cacodylate, 0.1 M-Na+, pH 7, at room temperature. (a) curves 1 to 5, poly (I) ; copolymers : poly (C ,I o.24), ~01~ (W o.15)1~01~ (C&.OS), and ~01~ (C). (b) Curves 1 to 5, poly (A); copolymers: poly (C,&.& poly (C,&.& poly (C,&.1), and poly (C), respectively. (c) Curves 1 to 6, poly (U); copolymers: poly (C,U,.s,), poly (C,U,.,,), poly (C,U,.,,), poly (C,U0.07), and poly (C), respectively.

Ochoa and his co-workers (Heppel, Ortiz & Ochoa, 1957 ; Ortiz & Ochoa, 1959) indicated that random base sequence was maintained over a variety of input base ratios in the poly (A,U) series of copolymers. All the copolymers were found to exhibit hyperchromism at 250 nm in the temperature range from 0°C to 80°C in the presence of O-005 M-sodium cacodylate, 0.1 %I-Na + at pH 7. The hyperchromism is lowest for the poly (C,U) series, and decreases with increasing x value-12% for x = 0.21 and 15% for x = 0.07. In the poly (C,I) series, the hyperchromism is independent of x-all exhibit roughly 18%. In conjunction with the observed isosbestic point for the poly (C,I) series (Pig. l), it is unlikely that extensive formation of intramolecular C.1 helices occurs under these conditions. The hyperchromism of the poly (CA) series is greater than that exhibited by the other series-35% for x = 0.30 and 33% for x = O-10. Since the single-strand structure responsible for the hypochromism of neutral poly (A) melts non-co-operatively (Leng & Felsenfeld, 1966; Brahms, Michelson & Van Holde, 1966; Poland, Vournakis & Scheraga, 1966) and is present in the dimer ApA, the hyperchromism of these copolymers should simply reflect the sum of the contribution of all possible nearest-neighbor pairs weighted by the frequency of occurrence of these pairs in each case. If the bases are inserted randomly, these frequencies are predictable from the base ratio of each copolymer. With appropriate optical data for the component dinucleoside phosphate pairs (Warshaw & Tinoco, 1966) the single-strand hyperchromicities we observe could in principle be used to verify the random sequence hypothesis. We have so far observed only qualitative agreement in the case of copolymers for which the requisite data are available. Thus the relatively large hyperchromism of the poly (CA) series is consistent with the fact that all the possible component dinucleotide pairs (CPA, ApC, ApA and CpC) are strongly stacked (seeTable 1 of Warshaw & Tinoco,

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TABLE 1

Characteristics of polynucleotides used in complex jormation Polymer

Input mole ratios for synthesis

I C

-

(C,I) series cLJ.08 CJO.lS wl.,4

s

20.w

12.4 3.4

248 268

10.26 6.30

0.20 0.30

3.8 7.3 7.3

268 267 266

6.18 6.00 6.81

(C,U) series GUO.07 c,uo.14 GUO.,, GUo.35

0.10 0.20 0.30 0.50

9.7 5.4 10.9 3.9

267 267 266 264

6.47 6.64 6.80 7.15

(C,A) series GA, .I CA,., GGJ.3

0.10 0.20 0.30

5.8 7.1 5.0

267 265 263

6.35 6.50 6.60

0.10

1966). In the poly (C,U) series, two of the constituent pairs (UpU and UPC) are only weakly stacked. Quantitation of these correlations requires that the low temperature limit of the absorption profiles be established, which proves to be difficult to attain experimentally. The solvent reported by Cox & Kanagalingam (1967) to unstack single-stranded nucleic acids, 1 M-Na+ plus 8 M-urea, did lead to diminished values of the hyperchromism for all the copolymers, the decrease being to a level about one-half those observed in 0.1 M-Na+. However, the absorbance changes evident in the presence of urea indicate that a substantial residual structure remains. While the absorbance-temperature profile of poly (C) in 0.1 M-Na+ increases roughly linearly over the temperature range observed, above 20°C poly (I) in this solvent exhibits no further change in absorbance at 250 am. In the presence of O-5Mthe multistranded self-structure of poly (I), presumably poly(I.I.I), becomes Iva+, evident, as described by Rich (1958). The broad profile recorded in Figure 5 may reflect the presence of some low molecular weight material. (b) Thermal transition studies of copolymer complexes with polyinosinic acid Melting curves of equimolar mixtures of poly (I) with poly (C) and the copolymers of each series were performed for three different solvents, all buffered at pH 7 by 0.005 N-sodium cacodylate: (1) 0.1 M-Na+, (2) 1 M-Na+ plus 8 M-urea and (3) O-5 MNa + . Solvents (1) and (3) were chosen to determine the effect of a simple change in ionic strength on the stability of the complexes with non-complementary bases as opposed to the parent helix, poly (I)*poly (C). Solvent (2) represent a different balance of stabilizing interactions from the aqueous systems-increasing the stability by high sodium-ion concentration while decreasing it to a roughly similar extent with 8 M-urea (Cox & Kanagalingam, 1967). As noted above, this solvent leads to a decrease in the apparent structure of the single-stranded copolymers.

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The profiles for the poly (I)*poly (CJ,) series of complexes are shown in Figure 2, where it is evident that both the melting temperature (T,) and the hyperchromism decrease progressively with the value of x, the mole per cent I in the copolymer.

60 -

/”

0 I M-Na+

TemperoWe PIG. 2. Absorbance-temperature profiles at 250 nm for equimolar mixtures of poly (I) and poly (CJ,) in three solvents indicated. All solutions were buffered at pH 7 by 0.005 x-sodium cacodylate. From left to right: poly (I) *poly (C,I,.,,), poly (I) .poly (C,Io.ls), poly (I)*poly (G10.d and ply (I)*poly (C).

This effect is particularly pronounced in the urea solvent. The decrease in absorbance prior to the transition of poly (I)*poly (C) in 05 M-salt is due to the self-structure of poly (I) at this salt concentration. If mixing is performed at O-1 M-Salt and the salt concentration adjusted to O-5 M, no absorbance decrease prior to melting is seen and the transitions are fully reversible. Absorption spectra of these complexes in O-1 Msalt, pH 7, at room temperature and at 90°C are shown in Figure 3. The hyperchromism is most pronounced in the wavelength region between 240 nm and 260 nm for all cases. Profiles of the thermal transitions of equimolar complexes of the poly (I)*poly (C,U) series at 250 nm are shown in Figure 4. In each solvent used, the dependence of T, and the hyperchromism on the z-value are less than in the poly (I)*poly (CJ) case. This difference is most obvious in the urea solvent data (Fig. 2). The curves shown in Figure 4, corresponding to the salt case, were obtained by mixing the polymers in low salt first. However, it is likely that the decrease in hyperchromism seen for poly (I)*poly (C,U,.,,) in high salt reflects residual poly (I) self-structure. If the polymers were mixed in high salt, and allowed to equilibrate overnight, the biphasic profiles shown in Figure 5 result. The transition profile of poly (I) is included to show that there is self-structure in poly (I) above the T, for poly (I)*poly (C,U,.,,) and within the

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0.6 -

0.6 0.4 OZ220 240

260

200 300

240

260 280 300

Wavelength(nm) FIG. 3. Absorption spectra of 1: 1 mixtures of polymers in 0*005Imn-sodium cacodylate, O-1 anNa+ (pH ‘7) before and after melting. In each case the upper curve represents the spectrum in coil form (90’ C) and the lower curve represents the spectrum in helix form (room temperature). The total polymer concentration in each mixture is 03 x 10e4 M. (a) Poly (I) .poly (C); (b) poly (I) .poly (C,I,.os); (01 POSY(1) .poly (Wo.is); (d) ~01~ (I) .poly GL,.,,).

1 ure 6 shows the absorption spectra of the melting range of poly (I)*poly (C,U,.,,). F’g 1: 1 complexes of this series in 0.1 M-Na+ at low and high temperatures. Maximum hyperchromism is again found between 240 mn and 260 nm in all the complexes. The general features of the transitions of 1: 1 complexes between poly (I) and poly (CA) resemble those of poly (I) and poly (C,U) (Fig. 7). Spectra of these complexes at low and at high temperatures are shown in Figure 8. The stability of the poly (I)*poly (CJ) system was also investigated in the presence of tetramethyl ammonium bromide, to see whether specific ions influence structures in which bases appear to be looped out from the helix. The tetramethylammonium ion has been shown to bind far more weakly than Na + to DNA (Ross & Soruggs, 1964). The complexes were dialyzed exhaustively against tetramethylammonium bromide in the cold (7 days) and the pH adjusted to 7.0 with tetramethylammoniun hydroxide just before the melting experiment. The results in Figure 9 show that the stability is substantially decreased when the counterion is the tetramethylammonium ion. There is a slight differential effect on the relative stabilities of the complexes as a function of z in this solvent. The T, values of the poly (I)-poly (C,I,.,s) and poly (I)-poly (CJ, .J complexes are closer to that ofpoly (I)-poly (C) inthis case by about l-4 deg.C, but the result suggests that the looping-out behavior of this system is insensitive to the nature of the monovalent oounterion present.

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0I

t+Na+

60 .: ; !

60

IO

20

30

40

50

60

70

Temperature PC)

FIG. 4. Absorbance-temperature profiles at 250 nm for equimolar mixtures of poly (I) and poly (C,U,) in three solvents indicated. All solutions were buffered at pH 7 by 0.005 M-sodium caeodylatte. From left to right: poly (I)*poly (C,Ue.ar), poly (I)*poly (C,Uo.ln) and poly (I).poly (C,-fJ,.o7).

0.5 hi-Nat 0.8 -

w* .x0

0.7 -

,.-

/.L.-.

-.-*-.

,/’

o-504/--

0

10

20

30

40

50

60

70

Temperatwo PC)

FIG. 5. Transition profiles of poly (I) .poly (C,U,) in 0.005 M-sodium cacodylate, 0.5 M-Na+, pH 7. The 1: 1 oomplexes were mixed after adjusting each polymer to 0.5 M in salt concentration. Top profile: poly (I); bottom profiles, from left to right: poly (I) .poly (C,U,.,,), poly (I).poly G,Uo.21), poly (1).poly (C,UO.IA &ml POSY (I)*poly (CUa.0,).

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I

I

1

(b) z x & x a

599

I

(cl

060.40.2 220

240

260

200 300 Wavelength (nm)

FIG. 6. Absorption spectra of 1: 1 mixtures of polymers in 0.005 M-sodium cacodylste, 0.1 MNa+ (pH 7) before and after melting. In each case the upper curve represents the spectrum in coil form (90’ C) and the lower curve represents the spectrum in helix form (room temperature). The total polymer concentration in each mixture w&s 0.8 x 100~ M. (a) Poly (I)*poly (C,Uo.O,); b) poly (1) *poly (GUo.4; (0) poly (I) poly (GUo.,,).

I

60 -

O-IM-Na*

40 20 -

I

I 40 -

I

I

I

I

I

8 M-‘&o

5 5 F

a z

20-

O60 -

I

!Ul

I

O-5 M-Nat

4020-

O-

. IO

20

30

40

50

I 70I

J

60

Temperature (“Cl

FIG. 7. Absorbance-temperature profiles at 250 nm for equimolar mixtures of poly (I) and poly (C,A,) in the three solvents indicated. All solutions were buffered at pH 7 by 0.005 M-sodium cacodylate. From left to right: poly (I) .poly (C,A,.,), poly (I).poly (C,A,.,) and poly (I) .poly vu,.,).

600

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1

I

I

(a)

; :: ::0’

I

1

I

I (cl

(b)

06-

0,4-

a 0.2 i 220

1 240

I 260

t 280

! 240

300

I 260

1 280

, 240

300

I 260

I 260

300

Wavelength (ml

FIG. 8. Absorption spectra of 1: 1 mixtures of polymers in 0.005 M-sodium oacodylate, 0.1 M-&S+ (pH 7) before and after melting. In each case the upper curve represents the spectrum in coil form (90°C) and the lower curve represents the spectrum in helix form (room temperature). (a) Poly (I) .poly (GA,.,); lb) poly (I) *poly (C.Ao.,) : (0) poly (I) *poly (‘&A,.,).

(c) Mixing

curves of polyinosinic

acid and copolymers

Since the thermal transition data revealed differences between the complexes of poly (I) with poly (CJ) on the one hand and poly (C,U) and poly (CA) on the other, the stoicbiometry of the reactions of these polymers was analyzed by ultraviolet mixing experiments. The mixing curves at 250 nm and 233 nm for the reaction of poly (CJ,.,,), poly (C,UO,& and poly (CA,.,,) with poly (I) are shown in Figure 10. The corresponding curves of poly (I) and poly (C) are very close to those of poly (I) plus poly (C,U0.21), exhibiting a single discontinuity at 50 moles y0 poly (I) (Chamberlin & Patterson, 1965).

05M 60

/a-.

-

I

I

I

I

I

I

10

20

30

40

50

60

Temperature PC)

FIG. 9. Melting profiles in 1 M- and 0.5 xl-tetramethylammonium bromide, right: polv (I).poly (C,Io,Is), holy (I).poly (CJ,.,,) and poly (I).poly (C).

pH 7. From left to

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Mole % poly I

FIG. 10. Ultraviolet mixing curties for poly (I) and the copolymerspoly and poly (C,A,.,), respectively. Absorbances at 250 nm and 233 mn were plex. The experiments were carried out at room temperature (23°C) and in late, 0.1 ~a-Na+ (pH 7). (a) Poly (I) + poly (GA,.,); (b) poly (I) + poly POlY (C,U,.2d.

(C,IO.,,), poly (C,U,.,,), recorded for each com0.005 w-sodium cacody(CJ,.,,): (c) poly (I) +

While the results suggest that the minimum in the poly (CJ, .24)mixing experiment is reached below 50 moles o/o poly (I), it is difficult to conclude that there is a real difference without further analysis. A detailed statistical analysis of the mixing curve data is presented in the Appendix, along with a general method for performing the analysis for any continuous variation experimental data. The results provide statistical evidence that the minimum of the poly (I) + poly (CJ,.,,) mixtures occurs at 42 moles y. poly (I); only in this case can the hypothesis that the minimum occurs at 50 moles y. poly (I) be rejected. This is very near the value expected if all 24 moles o/oI residues in the copolymer strand are completely excluded from the helix, i.e. 43 moles o/o poly (I). Because the mixing curves for poly (C,U,.& and poly (GA,.,,) fail to indicate a minimum other than at 50 moles y. poly (I), both the U and A residues participate in the helix structure to the extent of occupying sites adjacent to an opposite I residue. Similar mixing data with poly (C,U) copolymers with higher mole fraction of U led Tsuboi et al. (1968) to conclude that the U residues loop out, as we have found for the poly (CJ) complexes. A possible explanation for the apparent discrepancy is given below. Complete spectra of a continuous variation experiment with poly (I) + poly (CJ,.,,) revealed two isosbestic points for the mixtures : one at 248 nm for the range 0 to 50 moles y. poly (I) and the second at 262 nm for the range 50 to 106 moles y. POlY (I)39

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sedimentation in cesium chloride-cesium sulfate gradients

The buoyant density of equimolar complexes of poly (I) with the copolymers of each series was investigated as a possible means of distinguishing looping-out structures from these in which the non-complementary bases are retained within the helix. Since poly (I) bands at a heavy position in Cs,SO,, due to formation of multistrand aggregates with high charge density, it is expected that the additional I residues present in the equimolar poly (I).poly (C,I) can interact to form similar aggregates and thus produce more drastic density changes with increasing x than in the case of equimolar complexes of poly (I) with poly (C,U) or poly (C,A). If the excess I residues in the looping-out case condense at the end or ends of a helix, both longer regions and higher concentration of I strands are available for aggregation as x increases. In order to minimize the problem of insolubility of RNA in Cs,SO, solution, we used the 8 : 1 CsCl-Cs,SO, mixtures recommended by Lozeron t Szybalski (1966). The results are shown in Figure 11. In each case a &bromouracil-containing T4 DNA was included as a reference. This DNA was isolated from bacteriophage provided

*

t

1

lb)

(b)

(cl

(c)

[d)

Cd)

i.Ocm

I

FIG. I. 1. Buoyant density of equimolar complexes of poly (I) and copolymers of different species and composition. Eaoh experiment represents & tracing of a u.v. absorption photograph after 22 hr at 44,770 rev./min in a Spinco model E centrifuge. The eel1 contained 0.4 ml. CsCl solution (37.14 g- optical grade CsCl in 20 ml. 0.01 M-Tris, pH S), O-06 ml. CsaSOc solution (8.35 g optical &de CQSO, in b ml. 0.01 lur-Tris, pH 8, with 2 x 1oe4 MLEDTA) and 0.06 ml. sample. The simple consisted of about 0.03 AQnn T4 DNA, about 0.03 A,,,-__ unit of --- unit of 5-bromourakl-substituted the complex, diluted to 0.05 ml. in O-01 M-T& buffer, pH 8. Left hand panel: (a) poly (I) *poly (C); (b) ~01~ (I)*poly (C,%,.0,); (0) poly (I)*poly NAUo.14);(d) poly (I)*Po~Y (CXk,,). Center Pnel: (a) poly (I).poly (C); (b) poly (I)*poly (C,A,.,); (c) poly (I)*poly (C!,A,.,) (reduaed in scale to fit); (d) poly (I)*poly (C,A&. Right hand panel: (a) poly (I).poly (C); (b) poly (I)*poly (C,I+&; (o) poly (I)*poly (C,Io.16); (d) poly (I).poly (C,I,.,,). The arrow at the left in each panel ma& the position in the gradient of the reference DNA (bromoumoil-substituted T4 phage DNA).

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by Dr A. Kozinski. Absolute density values were not determined, since the requisite parameters for mixed salt solutions are not available. However, experiments of Lozeron (personal communication) indicate that the relative separation in density between normal and 5-fluorouracil-containing tobacco mosaic virus RNA is similar over a range of CsCl to Cs,SO, mixtures to that observed in Cs,S04 alone. Within each series of complexes, the buoyant density is observed to increase with increasing x. The density increment is clearly greatest for the poly (I)*poly (CJ) series, In the case of poly (I)*poly (C,I,,.& a very broad peak near the density of pure poly (I) alone is observed, suggesting marked aggregation by association of extrahelical I residues, possibly according to the scheme:

in which the I strands are represented by straight lines and the copoly (CJ) strands by Dhe pleated lines. In no other case is this observed, even though poly (I)*poly (C,A,.,,) was used, which contained 6 moles o/0 more of the minor component than in the I complex. These results therefore support the contention that only in the poly (CJ) complexes are the non-complementary residues physically excluded from the helix structure.

4. Discussion We have attempted to define the consequences of incorporating classically noncomplementary bases into the poly (I)*poly (C) helical matrix. From the results of thermal denaturation profiles, spectral mixing curves, and buoyant density measurements, we conclude that A and U residues incorporated into the poly (C) strand are preferentially retained in the helix in all the solvents studied, while I residues are excluded. We emphasize that this conclusion rests primarily on analysis of spectral mixing curves, which requires several stringent assumptions. We therefore make no claim to have proved this with the evidence so far presented. However, the poly (I).poly (CJ) series of polymers can be considered to be analogous to the case investigated by Fresco & Alberts (1960), who proposed the “helix-with-loops” model to describe the exclusion of defective residues in the poly (A) + poly (U) system. It is worth remarking that the hyperchromicities observed within the series of poly (I)*poly (CJ,) complexes do not increase linearly with 5, but fall off considerably more sharply (Fig. 2). To an extent this is a reflection of the spectral shifts seen in Figure 3. However, the assumption that the change in molar absorbance on denaturation of the complexes, 4POlY

WPOlY

G&N = u-4

WPOlY

(I)-POlY

(C)l,

which neglects single-strand corrections, may fail because of the increase with x of the probability of configurations in which one or two C residues enclosed by I residues are excluded from the helix. This alone predicts a greater dependence of the hyper39*

604

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chromicities on x, according to the expression: ds[poly (I)*poly (CJ,)] w [l-x

- 2x2 + 3x3 - x4]ds[poly (I)*poly (C)l,

in which the contribution of such residues is subtracted. According to this equation, for example, the ratio of the hyperchromism of poly (I)*poly (CJ,.,,) to that of poly (I).poly (C) should be about 0.68, while the linear expression gives 0.76, and the actual observed value is 0.48. From Crick’s (1966) discussion of the wobble codon-anticodon pairs, it is clear that hydrogen-bonded pairs of I with C, U and A are feasible, although not neoessarily compatible with each other in the context of a long helical structure. Available fiber diffraction data on the poly (I)*poly (C) helical duplex (Davies, 1960) suggest that this system resembles double-stranded reovirus RNA (Langridge & Gomatos, 1963), with the base-pair planes tilted with respect to the helix axis. If the regular 3.4 A spacing between adjacent base-pair planes is maintained, the question of how the compatible residues (1,U) and (LA) fit into a common matrix with the standard Watson-Crick type IC base pair is of some concern. In terms of Crick’s base-pairing models (1966), the C;---C; distance for the hypothetical 1-U pair lies very close to the value for the standard pairs, i.e. about 11 A. That for the corresponding 1.A pair on the other hand is appreciably longer (over 13 A). Thus if Crick’s hydrogen-bonding scheme is involved in stabilizing the (LA) residues, a fairly severe distortion of the unsubstituted poly (I)*poly (C) helical structure must be possible, implying that there is some flexibility in the structure. Alternatively, we can suppose that this pairing is not involved in our complexes, and that the (IA) residues do not disrupt stacking interactions,whereas (II) residues do. No such obvious incompatibility between the 1-U pairing scheme and IC base pairs exists, except that the angles of the glycosidic bonds may be wrong, and there is thus no reason not to assume pairing, although it must be emphasized that we have no direct evidence establishing this. The situation of the poly (I)*poly (C,U) and poly (I)*poly (CA) complexes can be considered as an extreme of the random base-paired DNA model: that is, two species of “base pairs” IC and 1-U (or I-A), are present, one with a stability substantially in excess of the other. The thermal denaturation for this case has been analyzed (see, for example, Crothers & Kallenbach, 1966) in terms of the following simple model. Define so as the stability constant for formation of an IC pair at the end of a helix of IC pairs, and similarly let sy be the corresponding constant for either the 1-U or I-A helices. Neither species is known to be stable as double-stranded complexes, although poly (A). 2 poly (I) is stable (Gabbay, 1967). Then if we assume that the heats of formation of the different species are not very different, the ratio of these constants, k = sy/sc is fixed : ln~=~(~~--

;J,

(1)

where T, is the T, of poly (I).poly (Y) and Tc that of poly (I)*poly (C), and dH is the heat of formation of the two species. The equilibrium constant for a molecule of mole fraction x of the 1-Y species can be taken as s;sk-” provided gross effects of sequence on stability are absent. We find then: (21

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In Figure 12, such plots are shown for the poly (I)-poly (C,U) and poly (I).poly (C,A) complexes. Extrapolation to 2 = 1 gives - 17°C for the T, of poly (I)*poly (U) and -3°C for poly (I)*poly (A) in 0.1 M-Na+, pH 7. Although such an extrapolation entails considerable uncertainty, the resulting numbers should represent upper bounds in each case. As noted before, the data on the thermal transitions of equimolar poly (1) plus ~01~ (A) mixtures (Steiner & Beers, 1961) correspond most probably to the

n(CU)

xm1

x(CA)

Fro. 12. The dependence of the reciprocal of the melting temperature, T,, on the mole fraction (2) of I, U, or A in the copolymer strand for the equimolar mixtures of poly (I) and the copolymers. -o-o----, 0.5 M-NZ%+ ; -.--.--, 0.1 M-NE%+ ; - n-A---, lm6-N~+plusS~-urea;-x-x-, 0.5 M-tetra~methylammonium bromide, all 1.0 M-tetramethyl&mmonium bromide; -a--•---, at pH 7.

three-stranded complex (see Gabbay, 1967). With the value of -56 kcal./mole base pair for the heat of formation of poly (I)*poly (C) (ROSS& Scruggs, 1969), assuming the other species have the same heats, we find k = O-077 for the (C,U) series and O-135 for the (C,A) series. The difference in free energy from the 1-C system in each case, taken as AC’ = -RT Ini?, is then l-70 kcal./mole for the 1-U system and l-3 kca1.j mole for the I*A. These estimates are obviously subject to the assumptions of ideality which are made (see Crothers & Kallenbach, 1966). In particular these calculations assume that stacking interactions are not dependent on species. According to this model also, the increase in transition breadth observed with increasing x is strictly a consequence of heterogeneity of the two species, and should be maximal for x = l/2. Consider the apparent “helix-with-loop” complexes now. While we have not yet performed a detailed analysis of the helix-coil transitions in this case, some insight into the behavior of these molecules can be obtained by regarding the process of helix formation as analogous to crystallization of poly (CJ) onto a fixed lattice of poly (I). Following Flory (1953) the melting point depression occurring in a random copolymer consisting of A units capable of crystallization and B units which are not is given by 1 ---= Tm

1 T:

(3)

606

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C. WANG

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N.

R.

KALLENBACH

where T& denotes the melting temperature of the pure crystallizing component, AH, is the heat of fusion per A monomer unit and N, is the mole fraction of this component. Identifying the A species with the C residues, the B with I, and putting NA = 1 - x, we obtain 1 -=$+-&ln(l-x)FCi$--

T mw

c

0

(

-&z

1

for small x, where AH is taken as -AH,. The resulting form of the equation is the same as equation (2), but the slope has a different interpretation. As noted by Flory (1953), the form of equation (3) and thus of equation (4) is dictated by the GibbsDuhem equation, and is general, provided the activity of the B component is proportional to its concentration. Evaluation of the slope of the data, for the poly (I)*poly (CJ) complexes plotted according to this equation yields a value of AH = - 1.8 kcal./mole C residues for the enthalpy of formation of an IC pair, a value about onethird the apparent calorimetric heat (Ross & Scruggs, 1969). Since the details of the denaturation process have not been taken into account, this discrepancy is not surprising. The difference between the results we have obtained with the system poly (I)*poly (C,U) and those of Tsuboi et al. (1968) remains to be accounted for. According to the estimates given above, the intrinsic stabilities of the “pairs” 1.U and I-A are very low, the extrapolated T, values being below 0°C in O-1 M-Nit+ solution, pH 7. At room temperature therefore these “pairs” can be maintained in the helical matrix only by residual interactions from runs of neighboring 1-C pairs (“stacking” interactions). If the x values of the U or A components in the copolymers are increased to sufficiently high values, it is reasonable to expect that a point will be reached where the runs of U or A residues become sufficient in length and frequency to lead to disruption of the stacking interactions responsible for maintaining these bases in register. Such an effect has in fact been reported by Uhlenbeck, Harrison & Doty (1968) in the series of complexes poly (U).poly (A,G)-below x = O-43, the complexes melt co-operatively with similar hyperchromicities, while at x = 0.43 both the hyperchromism and cooperativity of the transition decrease. This behavior may, therefore, be characteristic of all random complexes in which a very weak “pair” is admixed with a stable onethe value of x at which the change is observed should itself provide a measure of the relative stabilities of the species involved. In the absence of actual data, it is difficult to predict whether these statistical effects should produce relatively sharp or gradual changes from complete inclusion to exclusion of the non-complementary bases. The data of Tsuboi et al. (1968), based on two (C,U) copolymers, containing 0.33 and 0.57 mole o/o U, respectively, can conceivably be viewed as representing the situation beyond the critical x-value for this system. Investigation of the complexes of poly (A)*poly (U,A,) at very low s-values may reveal a value of z below which the excluded components are retained. It may prove difficult to detect this behavior experimentally, since, for example, the mixing curves lose resolution at low x. The detailed chemical basis for the behavior of these residues as defects in a double helical structure remains unknown. Specific monovalent-ion effects on the stability of helix-with-loop structures are absent since the relative stability of poly (I)*poly (CJ,) complexes is similar in the presence of tetramethylammonium ions or Na +. In view of the known effects of Mg2 + on RNA structure, it is possible that divalent cations may be capable of differentially affecting the looping-out of residues for

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example. The role of hydrogen bonding per se can be investigated by means of ehemitally modifying the minor components. Further experimentation with these and similar synthetic systems may clarify the particular requirements for inclusion or exclusion of non-complemetary residues from helical nucleic acids, which have possibly important biological consequences. This research was supported by National Science Foundation grant no. GB-11782. The authors wish to acknowledge helpful correspondence with Dr H. Lozeron concerning the mixed gradient system, and the capable technical assistance of Mrs S. Stefanovic in the equilibrium centrifugation experiments. REFERENCES Ames, B. (1966). In Methods of Enzymology, ed. by S. P. Colowiok & N. 0. Kaplan, vol. 8, p. 115. New York: Academic Press. Bautz, E. K. F. (1965). In Evolving Genes and Proteins, ed. by V. Bryson & H. J. Vogel, p. 419. New York: Academic Press. Bautz, E. K. F. & Bautz, F. A. (1964). Proc. Nat. Acad. Sk., Wash. 52, 1476. Brahms, J., Michelson, A. M. & Van Holde, K. E. (1966). J. Mol. Biol. 15, 467. Chamberlin, M. J. & Patterson, D. (1965). J. Mol. BioZ. 12, 410. Cox, R. A. & Kanagalingam, K. (1967). Bioclzem. J. 103,749. Crick, F. H. C. (1966). J. Mol. BioZ. 19,548. Crothers, D. M. & Kallenbach, N. R. (1966). J. Chem. Phys. 45, 91’7. Davies, D. R. (1960). Nature, 186, 1030. Felsenfeld, G. & Miles, H. T. (1967). Ann. Rev. Biochem. 36, 407. Felsenfeld, G. & Rich, A. (1957). Biochim. biophys. Acta, 26, 457. Flory, P. J. (1953). In Principles of Polymer Chemistry, pp. 456 to 571. Ithaca, New York: Cornell University Press. Fresco, J. R. & Alberts, B. M. (1960). Proc. Nat. Acad. Sci., Wash. 46, 311. Gabbay, E. (1967). Biopolymers, 5, 727. Hayashi, M. & Spiegehnan, S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1564. Heppel, L. S., Ortiz, P. J. & Ochoa, S. (1957). J. BioZ. Chem. 229, 695. Howard, F. B., Frazier, J. & Miles, H. T. (1969). J. BioZ. Chem. 244, 1291. Langridge, R. & Gomatos, P. 5. (1963). Science, 141, 694. Leng, M. & Felsenfeld, G. (1966). J. Mol. BioZ. 15, 455. Loring, H. S. (1955). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, pp. 191 to 199. New York: Academic Press. Lozeron, H. A. & Szybalski, W. (1966). Biochem. Biophys. Res. Comm. 23, 612. Ortiz, P. J. and Ochoa, S. (1959). J. BioZ. Chem. 234, 1208. Poland, D., Vournakis, J. N. & Scheraga, H. A. (1966). BiopoZymers, 4, 223. Rich, A. (1958). Biochim. biophys. Acta, 29, 502. Ross, P. D. & Scruggs, R. (1964). Biopolymers, 2, 79. Ross, P. D. & Scruggs, R. L. (1969). J. Mol. BioZ. 45, 567. Steiner, R. F. (1959). Ann. N. Y. Acad. Sci. 81, 742. Steiner, R. F. & Beers, R. F. (1961). In PoZynucZeotides, pp. 261 to 262. Amsterdam: Elsevier. Tsuboi, M., Matsuo, K. & Nakanishi, M. (1968). Biopolymers, 6, 123. Acta, 80, 669. Tuppy, H. & Kuchler, E. (1964). Biochim. biophys. Uhlenbeck, O., Harrison, R. & Doty, P. (1968). In Molecular Associations in Biology, ed. by B. Pullman, p. 107. New York: Academic Press. Voet, D. & Rich, A. (1970). Prog. Nucleic Acid Res. & Mol. BioZ. 10, 183. Warshaw, M. M. & Tinoco, I., Jr. (1966). J. Mol. BioZ. 20, 29. Watson, J. D. & Crick, F. H. C. (1953). Nature, 171, 737. Zimm, B. H. & Kallenbaeh, N. R. (1962). Ann. Rev. Phys. Chem. 13, 171.