The Role of the Loop in Binding of an Actinomycin D Analog to Hairpins Formed by Single-Stranded DNA

Abstract

Our recent work has indicated that the potent antibiotic and antitumor agent actinomycin D has the ability to selectively bind and stabilize single-stranded DNA that is capable of adopting a hairpin conformation. This mechanism of DNA binding has been implicated in the drug's ability to inhibit transcription by HIV reverse transcriptase from single-stranded DNA templates. In this report, we studied the importance of the hairpin loop on the ability of the 7-amino analog of actinomycin D to selectively bind DNA hairpins. Binding dissociation constant (Kd) values were determined to be 0.22 ± 0.11 μM for the hairpin formed from the single-stranded DNA 5′-AAAAAAATAGTTTTAAATATTTTTTT-3′ (dubbed HP1). The hairpin stem without the loop resulted in binding with Kd = 2.6 ± 0.9 μM. The drug showed low affinity for the HP1 strand fully duplexed to its complementary sequence (estimated to be at least Kd > 21 μM). Evaluation of 7-aminoactinomycin D binding to a library of thermodynamically characterized DNA hairpins revealed an affinity for the hairpin-forming sequence 5′-GGATACCCCCGTATCC-3′ (dubbed ACC4) of Kd = 6.8 ± 2.2 μM. Replacement of the terminal guanines of this sequence to generate 5′-ATATACCCCCGTATAT-3′ resulted in a 10-fold increase in affinity for this hairpin compared to ACC4, to Kd = 0.74 ± 0.06 μM. A molecular model of the ACC4-actinomycin D complex reveals that significant interactions between the hairpin loop and the pentapeptide rings of the drug must occur during drug binding. Taken together, our data indicate that the composition of the stem–loop interface is critical for the selectivity of actinomycin D and its 7-amino analog for DNA hairpins and suggests that novel drugs may be designed based on selection for the desired hairpin composition.