The role of the loop in binding of an actinomycin D analog to hairpins formed by single-stranded DNA

Our recent work has indicated that the potent antibiotic and antitumor agen t actinomycin D has the ability to selectively bind and stabilize single-st randed DNA that is capable of adopting a hairpin conformation. This mechani sm of DNA binding has been implicated in the drug's ability to inhibit tran scription by HIV reverse transcriptase from single-stranded DNA templates. In this report, we studied the importance of the hairpin loop on the abilit y of the 7-amino analog of actinomycin D to selectively bind DNA hairpins. Binding dissociation constant (K-d) values were determined to be 0.22 +/- 0 .11 muM for the hairpin formed from the single-stranded DNA 5'-AAAAAAATAGTT TTAAATATTTTTTT-3' (dubbed HP1). The hairpin stem without the loop resulted in binding with K-d = 2.6 +/- 0.9 muM. The drug showed low affinity for the HP1 strand fully duplexed to its complementary sequence (estimated to be a t least K-d > 21 muM). Evaluation of 7-aminoactinomycin D binding to a libr ary of thermodynamically characterized DNA hairpins revealed an affinity fo r the hairpin-forming sequence 5'-GGATACCCCCGTATCC-3' (dubbed ACC4) of K-d = 6.8 +/- 2.2 muM. 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 K-d = 0.74 +/- 0.06 muM. A molecular model of the ACC4-actinomycin D complex reveals that significant interacti ons between the hairpin loop and the pentapeptide rings of the drug must oc cur during drug binding. Taken together, our data indicate that the composi tion of the stem-loop interface is critical for the selectivity of actinomy cin 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. (C ) 2000 Academic Press.