Binding of Polycyclic Aromatic Hydrocarbons to Polyadenylic Acid A

Proceeding8 of the National Academy of Science8 Vol. 67, No. 3, pp: 1337-1344, November 1970

Binding of Polycyclic Aromatic Hydrocarbons to Polyadenylic Acid A. Morrie Craig and I. Isenberg

DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS, OREGON STATE UNIVERSITY, CORVALLIS, OREGON 97331 Communicated by Norman Davidson, August 13, 1970 Abstract. A number of polycyclic aromatic hydrocarbons bind. to the double- stranded, acid form of polyadenylic acid (poly A). Model building shows that these hydrocarbons may intercalate in the helix, and be well protected from contact with the aqueous medium. Hydrocarbons that are too large to be so protected are found not to bind. A size criterion for the binding of hydrocarbons to poly A therefore exists. This criterion differs from one that was previously found for DNA. The size criteria for DNA and poly A, together, serve as strong evidence for the intercalation model for hydrocarbon complexes. Model-building experiments show that only a small portion of the hydrocarbon need extend into the medium to prevent binding. This finding implies that in two cases (1,2,5,6-dibenzanthracene - poly A and 3,4-benzpyrene DNA) the structure of the complex is almost completely determined by the size criterion alone. It is now established'-" that a group of polycyclic aromatic hydrocarbons will complex to DNA. An intercalation model of these complexes, proposed by Boyland and Green' and Liquori et al.,2 has been central to all subsequent dis- cussions of hydrocarbon-DNA interaction. Although an intercalation model has appeared reasonable, compelling evidence in its behalf has been lacking. In an intercalation model, the hydrocarbon slips between the base pairs of a distorted Watson-Crick helix. If only intercalation is postulated, however, unspecified degrees of freedom still remain. The hydrocarbon may stick out more or less from the helix, and it may rotate about an axis parallel to the helical axis. Intercalation, therefore, properly refers to a class of models rather than to any particular one. It has recently been- proposed7" 0 that a size criterion might exist for the com- plexing of aromatic hydrocarbons with DNA. The size criterion severely restricts the degrees of freedom in an intercalation model. It states that only those hydrocarbons will bind to DNA that can intercalate and orient themselves so that they are well protected from contact with the medium. A hydrocarbon that cannot so orient will be found not to bind, or to bind only slightly. The size criterion is based on the extremely low solubility of hydrocarbons in water. However, its validity cannot rest on a priori considerations alone. It presupposes that hydrophobic interactions not only exist, but play a dominant role in complex formation, at least in first approximation. Contrary possibili- 1337 Downloaded by guest on September 25, 2021 1338 CHEMISTRY: CRAIG AND ISENBERG PROC. N. A. S. ties may easily be imagined. If, for example, specific orientation-dependent forces between the nucleotides and hydrocarbon, rather than hydrophobic interactions, dominated binding, the size criterion would be found to be invalid. We have recently examinedcP the predictive value of the size criterion, and reported that all of the predictions tested were verified. Thus, for example, in the series anthracene, 9-methylanthracene, and 9-phenylanthracene, the first two hydrocarbons, but not the last, were found to complex with DNA. Models show that the phenyl group must extend into the medium. Further- more, in all three complexes, the anthracene moiety, itself, may intercalate. We have noted11 that, despite its predictive success, the size criterion could not be considered as rigorously established. Larger hydrocarbons, which do not satisfy the criterion, also tend, in general, to have higher crystal binding energies than smaller ones. Since the hydrocarbon-DNA binding experiments measure an equilibrium between crystal #nd complex, it was possible that the apparent verification of the size criterion merely reflected an accidental agreement with a crystal energy progression. In the present paper we rule out this possibility. In this paper we will describe the testing of a size criterion for the biiding of hydrocarbons to the double-stranded, acid form of polyadenylic acid (holy A). Fig. 1 compares the cross sections of DNA and of double-stranded poly A. The different shapes imply different size criteria. We report that the bipding of hydrocarbons to poly A follows the size criterion appropriate to it. Flow dichroism studies are also consistent with an intercalation model. 9Y~~~~~~~~~~~~~~~~~~~~~~~~~,0<

FIG. 1. Cross sections of double-stranded poly A12 and G-C base pair of DNA.18 The distance between riboses is shorter in poly A than in DNA, but the dimension- perpendicular to the ribose-ribose axis is longer. Downloaded by guest on September 25, 2021 VOL. 67, 1970 HYDROCARBON COMPLEXES WITH POLY A 1339

Materials and Methods. 25 Highly polymerized poly A, 2 K salt, Lot 157B-1840, from Sigma Chemical Co., was further purified by phenol ex- 20 traction. It was then dialyzed against 0.001 M NaCl in 0.01 M cacodylate buffer, 15 pH 7.1 for 6 days. Fractions were taken according to the / E 1o method of Eisenberg and Fels- enfeld.'4 Molecular weights 10 were determined by sedimenta- tion equilibrium (meniscus de- pletion method of Yphantis'5). 5 V = 0.55 was used.'4 The fraction used to study hydrocarbon binding had a weight- 0 average degree of polymeriza- 40 50 60 70 80 tion of 6100. The poly A solu- pH tions were dialyzed against 0.001 M NaCl-0.01 M acetate FIG. 2. Ae' (eL minus ER) versus pH for the poly A fraction buffer, pH 5.0. A small used in the binding studies. amount of gelatinous material formed during dialysis. This was removed by centrifugation at 1000 X g for 20 min. The preparations were concentrated by flash evaporation to 0.0145 M in phosphate. They were then dialyzed against 0.001 M NaCl-0.01 M acetate buffer, pH 5.0 for 2 days. pH titrations, using circular dichroism measurements16 to monitor helix formation, showed our stock solution to be double-stranded (Fig. 2). These measurements were taken on a Durrum-Jasco circular dichroism spectrometer. The flow dichroism assembly was essentially that of Callis and Davidson.17 All hydrocarbons were commercial products and were purified as previously described. Approximately 3 mg of crystalline hydrocarbon was added to 12 ml of stock poly A. Samples were shaken at 4VC for at least 2 weeks. To remove remaining crystalline hydrocarbon, the samples were then centrifuged at 34,800 X g for 1 hr. Absorbance spectra of the supernatant were obtained in a Cary model 14 spectrophotometer, with 10-cm sample cells. Flow dichroism studies were made when the hydrocarbon absorbance spectrum was sufficiently removed from that of poly A to make such work feasible. 7-ml aliquots of the supernate were then pipetted into 100-ml beakers and diluted with water to 40 ml. After these solutions had been filtered through Whatman no. 1 paper, the filtrate was extracted three times with 6-ml portions of cyclohexane. The extracts were combined and evaporated to 7 ml. Absorbance spectra were then recorded. As with DNA,7 we found that cyclohexane extracts of poly A yielded uv-absorbing material. When necessary, our measurements were corrected for this absorbance. Model-building experiments: To establish a size criterion for poly A, model- building experiments were undertaken for all hydrocarbons to be tested with space- filling Corey-Pauling-Koltun models. Figs. 3-6 show both stick models and space- filling models. Stick models are useful for illustrative purposes, but space-filling models are necessary to examine the steric hindrances that are involved in the size criterion. Fig. 3 illustrates the attempted model of 1,2,3,4-dibenzanthracene -poly A. The hydrocarbon has been intercalated and inserted as far as possible into the helix. Further insertion is prevented by steric hindrance between the hydrogens of the hydrocarbon and the ribose C2' hydrogen on one chain and the C3' hydrogen of the other. As a result, the hydrocarbon extends into the medium. The existence of this protrusion predicts that 1,2,3,4-dibenzanthracene will not complex to poly A. Downloaded by guest on September 25, 2021 1340 CHEMISTRY: CRAIG AND ISENBERG PROC. N. A. S.

FIG. 3. Presumed model of 1,2,3,4-dibenzanthracene poly A. The hydrocarbon has been inserted as far as possible into the helix. Hindrance between the hydrocarbon hydrogens and the C2' and C3' hydrogens prevent further insertion. Arrows point to hydrocarbon hydrogens.

,° 2A

FIG. 4. Model of 1,2,5,6-dibenzanthracene * poly A in which hydrocarbon is maximally shielded from contact with the medium. Arrows point to hydrocarbon hydrogens. Downloaded by guest on September 25, 2021 VOL. 67, 1970 HYDROCARBON COMPLEXES WITH POLY A 1341

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FIG. 5. Presumed model of tetracene DNA. Hindrance between hydrocarbon hydrogens and C2' hydrogens of the sugars prevents further insertion into the helix. Arrows point to hydrocarbon hydrogens.

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FIG. 6. Model of 3,4-benzpyrene DNA showing hydrocarbon maximally shielded from contact with the medium. Arrows point to hydrocarbon hydrogens. Downloaded by guest on September 25, 2021 1342 CHEMISTRY: CRAIG AND ISENBERG PROC. N. A. S.

Fig. 4 shows a model of 1,2,5,6-dibenzanthracene-poly A. For this hydrocarbon, it is possible to build a model and have good shielding of the hydrocarbon by the poly A. We predict, then, that 1,2,5,6-dibenzanthracene will complex to poly A. Two DNA models are shown in Figs. 5 and 6. Tetracene (Fig. 5) cannot be shielded by DNA. The hindrance preventing such shielding is between the hydrocarbon hydrogens and the C2' hydrogens of the sugars. 3,4-Benzpyrene may be shielded by DNA, as shown in Fig. 6. The prediction is that tetracene will not bind to DNA and 3,4-benzpyrene will. These predictions, along with a number of others, have been shown to be verified" experimentally. Results. Table 1 summarizes our results on binding. In this table, the < sign means that no hydrocarbon binding was detected. The value after the sign is an estimated upper limit of experimental error. TABLE 1. Saturation binding values for hydrocarbon -poly A complexes. Amount hydrocarbon (Mmol) per Flow dichroism mol poly A phosphate measured by at 21,250 sec-' Direct Extraction average absorption by cyclohexane Group A Class 1 Anthracene N.F. 140 200 1,2-Benzanthracene AAIA = 0.07 590 390 1,2,5,6-Dibenzanthracene N.F. 24.0 39.0 Tetracene N.F. 32.0 57.0 Class 2 1,2,3,4-Dibenzanthracene N.F. <7.4 <7.4 Pentacene N.F. <0.1 <0.1 Group B Class 1 Anthracene N.F. 140 200 9-Methylanthracene AA 1A = 0.07 1070 990 Class 2 9-Phenylanthracene N.F. <5.8 X 10-3 <5.8 X 10-3 Group C Class 1 Pyrene AA/A = 0.07 750 840 3,4-Benzpyrene AA/.A = 0.07 450 680 Class 2 1,2,5,6-Dibenzpyrene N.F. <5.4 X 10-3 <5.4 X 10-3 All values are averages of two or more determinations. A less than sign means that no conmplexed hydrocarbon was found. The value after the less than sign is an estimated upper limit of experimental error. N.F. means that flow dichroism measurements were not feasible. Group A contains benzyl derivatives of anthracene of various sizes. Group B contains anthracene derivatives with substitutions in the 9 position. Group C consists of three pyrene derivatives. Classes 1 and 2 are hydrocarbons that do and do not bind respectively. It may be noted that the two methods of determining the amount of bound hydrocarbon agree to within a factor of two. Such agreement rules out techni- cal artifacts of measurement, as discussed in a previous paper."I All of the results satisfy the qualitative predictions of a size criterion, based on the model-building experiments described above. Several cases call for particular comment. As predicted by the size criterion for DNA," 1,2,5,6- dibenzanthracene and tetracene do not bind to DNA. The size criterion for poly A predicts binding, however, and this is what we have found. On the other hand, 1,2,3,4-dibenzanthracene binds to DNA but not to poly A, and this accords with the poly A size criterion. Downloaded by guest on September 25, 2021 VOL. 67, 1970 HYDROCARBON COMPLEXES WITH POLY A 1343

Work on the size criterion, reported in this or in previous papers,7"10"11 is sum- marized in qualitative form in Table 2. In addition, we note that, in all cases tested,7 no binding of hydrocarbons to single-stranded poly A has been found.

TABLE 2. Predictions of binding based on size criterion. DNA Poly A Group A Anthracene Yes Yes 1,2-Benzanthracene Yes Yes 1,2,3,4-Dibenzanthracene Yes No 1,2,5,6-Dibenzanthracene No Yes Tetracene No Yes Pentacene No No Group B Anthracene Yes Yes 9-Methylanthracene Yes Yes 9-Phenylanthracene No No Group C Pyrene Yes Yes 3,4-Benzpyrene Yes Yes 1,2,3,4Dibenzpyrene No No 1,2,5,6-Dibenzpyrene No No

We have observed that the absorbance spectra of poly A-hydrocarbon complexes are red-shifted 12 nm with respect to the spectrum in cyclohexane. This red shift is similar to that found for DNA complexes.7"0 Flow dichroism measurements were made on double-stranded poly A alone and poly A hydrocarbon complexes. Absorbances were measured at 257 nm for poly A, 351 nm for 1,2-benzanthracene, 395 nm for 9-methylanthracene, 346 nm for pyrene, and 395 nm for 3,4-benzpyrene. AA/A at 21,250 sec-' was 0.07 in all of these cases. AA/A, for a given molecular weight, is a function of the distribution of the orientation of the chromophores. In our flow dichroism assembly, a positive value of AA/A implies an orientation in which a 7r-7r* tran- sition moment is perpendicular to the helical axis. The values obtained are consistent, therefore, with an intercalation model for hydrocarbon complexes (Table 1). Discussion. The division of hydrocarbons into two classes-those that complex and those that do not-depends on the polynucleotide, and it appears as if the primary role of the polynucleotide is to serve as a protective agent to shield the hydrocarbon from contact with the solvent. The fact that the size criterion is found to be valid for double-stranded poly A and DNA has a number of consequences: (a) The size criterion appears valid for complex formation, and does not merely reflect differences in hydrocarbon crystal energy. Were the latter the case, the hydrocarbons that form complexes would be the same for poly A and DNA. (b) There can be little remaining doubt that intercalcation is the mode of binding. (c) Hydrophobic interactions, at least to first approximation, dominate the binding. If other interactions were of the same order of magnitude, one might expect to find exceptions to the size criterion in at least a few instances. (d) The water around DNA and poly A behaves as liquid water, at least with respect to hydrophobic interactions. Downloaded by guest on September 25, 2021 1344 CHEMISTRY: CRAIG AND ISENBERG PROC. N. A. S.

It is of interest to see how much of the hydrocarbon need project into the medium to prevent complex formation. It may be seen (Fig. 3) that only a small slice of 1,2,3,4-dibenzanthracene sticks out into the medium; yet this is enough to prevent complex formation. In Fig. 5 we see that a tetracene-DNA model may be built in which only a small fraction of tetracene protrudes. In addition to these, we find that models of two other presumed complexes-1,2,5,6- dibenzanthracene * DNA and 1,2,5,6-dibenzpyrene - poly A-also show only small pieces of hydrocarbon extending from the helix. Experimentally, we find that none of these complexes form (Tables 1 and 2). The model-building experiments on the 1,2,5,6-dibenzanthracene -poly A complex, and that of 3,4-benzpyrene -DNA complex, show that if one demands that the hydrocarbon not jut into the medium, the configuration is determined to within the accuracy of the Corey-Pauling-Koltun models. The hydrocarbons have no remaining degree of freedom. It may be noted that, in poly A complexes, the helix distorts by a simple extension to accommodate the hydrocarbon. For DNA, the helix not only extends, but the angle between the base pairs adjacent to the hydrocarbon de- creases. For the 3,4-benzyprene complex, the angle reduces from 360 without the hydrocarbon to 150 with the hydrocarbon. In summary, we find that the binding of hydrocarbons to poly A follows a size criterion appropriate to poly A. These findings, together with earlier size- criterion studies of DNA complexes, leave little doubt that the binding is by intercalation and that hydrophobic inter actions are dominant. We thank Drs. Callis and Davidson for help and advice in the construction of our flow dichroism assembly. Abbreviation: poly A, polyadenylic acid. This work was supported by U.S. Public Health Service, National Cancer Institute grant CA-10679. M. C. was supported by the National Institutes of Health Training grant GM 00253. 1 Boyland, E., and B. Green, Brit. J. Cancer, 16, 507 (1962). 2 Liquori, A. M., B. DeLerma, F. Ascoli, C. Botre, and M. Trasciatti, J. Mol. Biol., 5, 521 (1962). 3 Ts'o, P. 0. P., and P. Lu, Proc. Nat. Acad. Sci. USA, 51, 17 (1964). 4Lerman, L. S., 5th Nat. Cancer Conf. Proc. (Philadelphia: Lippincott, 1964), p. 39. 5 Ball, J. K., J. A. McCarter, and M. F. Smith, Biochim. Biophys. Acta, 103, 275 (1965). 6Nagata, C., M. Kodama, Y. Tagashira, and A. Imamura, Biopolymers, 4, 409 (1966). 7Isenberg, I., S. L. Baird, Jr., and R. Bersohn, Biopolymers, 5, 477 (1967). 8Green; B., and J. A. McCarter, J. Mol. Biol., 29, 447 (1967). 9 Lesko, S. A., Jr., A. Smith, P. 0. P. Ts'o, and R. S. Umans, Biochemistry, 7, 434 (1968). 10 Isenberg, I., S. L. Baird, Jr., and R. Bersohn, Ann. N.Y. Acad. Sci., 153, 780 (1969). 1 Craig, M., and I. Isenberg, Biopolymers, 9, 689 (1970). 12 Rich, A., D. R. Davies, F. H. C. Crick, and J. D. Watson, J. Mol. Biol., 3, 71 (1961). 13 Langridge, R., D. A. Marvin, W. E. Seeds, H. R. Wilson, C. W. Hooper, M. H. F. Wilkins, and L. D. Hamilton, J. Mol. Biol., 2, 38 (1960). 4 Eisenberg, H., and G. Felsenfeld, J. Mol. Biol., 30, 17 (1967). 6Yphantis, D. A., Biochemistry, 3, 297 (1964). 6 Brahms, J., A. M. Michelson, and K. E. Van Holde, J. Mol. Biol., 15, 467 (1966). Callis, P., and N. Davidson, Biopolymers, 7, 335 (1969). Downloaded by guest on September 25, 2021