INTERACTION OF NUCLEIC ACIDS, I. PHYSICAL BINDING OF THYMINE, ADENINE, STEROIDS, AND AROMATIC HYDROCARBONS TO NUCLEIC ACIDS

In a previous communication,' it was shown that the helix-coil transition temperature, Tm, of thymus DNA and helical poly A is lowered by pyrimidines, purines, nucleosides, their analogues, and derivatives. The order of their effectiveness indicates that hydrophobic and stacking interactions are important. To investigate the mechanism by which the Tm is reduced is one principal purpose of this research. It has been proposed that these compounds may interact more strongly with the coil form of nucleic acids than with the helical form, thereby shifting the equilibrium in favor of the coil form.' The validity of this explanation has been investigated by equilibrium dialysis to measure the binding of these compounds to fiucleic acids. The coil form of nucleic acids was found to have a much higher affinity than the helical form for these compounds as predicted. The significance of the hydrophobic and stacking interaction of the bases in nucleic acids is now recognized,'-4 and has been quantitatively studied in our laboratory.5 This knowledge provides new insight into properties and functions of nucleic acids which were not previously understood or anticipated. Many hydrophobic compounds are biologically active but chemically inert. These include, for example, steroids and polycyclic carcinogens. This new concept suggests that such compounds may interact strongly with nucleic acids. The second principal purpose of this investigation is to measure the affinity of DNA for these compounds, again by equilibrium dialysis. Estradiol-,3-17, testosterone, and diethyl stilbesterol were chosen as representative of the sex hormones, naphthalene and phenanthrene as representative of aromatic hydrocarbons. The results indicate that the affinity of DNA for these compounds is interestingly high, especially the denatured form of DNA. It is noteworthy that the affinity of RNA, poly A, and poly U for these compounds is one or two orders of magnitude lower than that of denatured DNA. Materials and Methods.-Radiochemicals: Commercially available radiochemicals were used without further purification or carrier dilution. Thymine-methyl-H3 (6.64 c/mM), adenine-H3 (2.16 c/mM), estradiol-17,6-6, 7-H3 (150 mc/mM), and testosterone-i, 2-H3 (784 mc/mM) were obtained from New England Nuclear Corporation, Boston. Caffeine-i-methyl-C'4 (0.66 c/mM) was obtained from Tracerlab, Waltham, Mass. Diethyl stilbesterol-H3 (296 mc/mM) and phenanthrene-H3 (16.38 mc/mM) were obtained from Volk Radiochemical Company, Skokie, Illinois. Naphthalene-1-H3 (168 mc/mM) was obtained from International Chemical and Nuclear Corporation, City of Industry, Calif. Nucleic acids: Native DNA: Calf thymus DNA (highly polymerized) was purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio. A 10 mg/ml solution of this DNA in 0.0025 M Na2HPO4, 0.005 M NaH2PO4, and 0.001 II ethylene diamine tetraacetic acid, pH 7.1 (HMP) was sheared at 0-5°C in a VirTis "45" high-speed mixer (manufactured by the VirTis Corporation, Gardner, N. Y.) for 20 min at full speed. The solution was then centrifuged to remove a small amount of white residue. The final DNA solution was made up in 0.5 M in NaCl. This native DNA solution gave a 30% increase in optical density at 260 muA when heated above the Tm temperature (700 in HMP; 950 in HMP and 0.5 M NaCl).