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 phos- phate. 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 spec- trum 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. I 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 's '~a 2' °, 2A R^ 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. C2~~~~~~~~~ o 2 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.

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