Ethidium bromide complexes with self-complementary deoxytetranucleotides. Demonstration and discussion of sequence...

The binding of ethidium bromide to the self-complementary deoxytetranucleotides containing guanine and cytosine bases has been studied by circular dichroism, visible absorption, and fluorescence spectroscopies. The circular dichroism spectrum of each of the four deoxytetranucleotides was measured and compared to the spectrum calculated by a nearest-neighbor approximation method which utilized the circular dichroism spectra of the deoxydinucleotides as a basis set. Reasonable agreement was obtained between the calculated and experimental spectra for three of the four deoxytetranucleotides; however, pdC-dC-dG-dG exhibited poor agreement between the actual and nearest-neighbor calculated spectra, which suggests that pdC-dC-dG-dG may exist in an unusual conformation. A nearest-neighbor method was also used to calculate the extinction coefficients for each of the deoxytetranucleotides: these values differ substantially from values recently published by Patel and Canuel [(1977), Proc. Natl. Acad. Sci. U.S.A. 74, 2624--2628] in which the method used for the determination of the extinction coefficients was not stated. The magnitude of the extinction coefficients is important in determining the stoichiometry of complex formation as well as the relative sequence preferences for ethidium binding. The visible absorption titrations, the fluorescence titrations, and the circular dichroism titrations with ethidium bromide clearly show that two ethidiums will intercalate into a (pdC-dG-dC-dG).(pdC-dG-dC-dG) double helix presumably at the two (dC-dG).(DC-dG) sequences. Results from the pdG-dC-dG-dC titrations with ethidium bromide are not as definitive. Raising the temperature from approximately 2 to 26 degrees C diminishes the strength of complex formation for the EthBr plus pdC-dG-dC-dG system and makes it more difficult to unequivocally determine the stoichiometry of the complex formation. These data thus confirm and extend our earlier observation that ethidium bromide preferentially binds to pyrimidine-purine sequences as opposed to purine-pyrimidine sequences. Experiments monitoring the binding of ethidium bromide to pdC-dC-dG-dG and pdG-dG-dC-dC indicate that ethidium will bind strongly to the (dG-dG).(dC-dC) sequence. We conclude that the relative ordering of the sequence preferences for the binding of ethidium to the three sequences available in the tetranucleotides studied is (dC-dG).(dC-dG) congruent to (dG-dG).(dC-dC) greater than (dG-dC).(dG-dC).