Interaction of miracil D with double-stranded poly(adenylic acid)'poly(uridylic acid)

Miracil D [1-(2-diethylaminoethylamino)-4-methyl-10-thiaxanthenone] is a drug which is similar in structure to the acridines. It is an inhibitor of poly(nucleic acid) synthesis and is believed to exert its effect through noncovalent interactions with nucleic acids. High-resolution proton nuclear magnetic resonance (nmr) was used to study the interaction of Miracil D with double-stranded poly(adenylic acid)-poly-(uridylic acid) (poly(A) · poly(U)) of about 20-25 nucleotides in length, prepared by mixing equimolar amounts of singlestrand polymers. Formation of the double helix effected significant upfield shifts (0.7 ppm) for the base protons and produced a low-field nmr signal at 13.60 ppm in H2G indicative of hydrogen bonding between the base pairs. Upon addition of Miracil D to double-stranded poly(A) · poly(U), the resonance of the 4-methyl group on the thiaxanthenone ring was significantly broadened and shifted about 0.3 ppm upfield. By comparison, the resonances for the two terminal methyl groups on the diethylaminoethylamino chain were not shifted or broadened. As the concentration of Miracil D was increased, the poly(A) poly(U) base resonances were found to further broaden and shift to higher field. Double-stranded poly(A) · poly(U) appears to bind about 2-3 times more Miracil D than does an equivalent amount of single-stranded polymers. These results are consistent with a model in which the planar thiaxanthenone ring is intercalated with the stacked base pairs of poly(A) · poly(U), while the cationic chain portion of Miracil D extends to the outside of the helix, where it is probably bound to the anionic phosphate groups. Temperature studies indicate that Miracil D stabilizes the doublestranded poly(A) poly(U) structure with respect to heat denaturation. The interaction of Acridine Orange and triethylamine with poly(A) · poly(U) was also studied. These two compounds are structurally analogous to the ring and chain portions of Miracil D. Results of the nmr studies indicate that triethylamine is most likely bound (as triethylammonium ion) to the outside of the poly(A)·poly(U) helix, while Acridine Orange appears to be intercalated within the stacked base pairs. The results in general demonstrate the usefulness of polynucleotides of limited chain lengths for nmr studies, in contrast to more highly polymerized polynucleotides or nucleic acids.