ACTINOMYCIN-D BINDS TO METASTABLE HAIRPINS IN SINGLE-STRANDED-DNA

We have examined the role of DNA composition in the binding of actinom ycin D to single-stranded DNA. By using the fluorescent analogue 7-ami noactinomycin D, we were able to monitor binding of the drug to ssDNA with single base changes distant from the 5'-TAGT-3' site previously d etermined to be a high-affinity site for actinomycin D binding (Wadkin s et al. (1996) J. Mel. Biol. 262, 53-68). Our binding studies indicat ed that secondary structures in the ssDNA were likely to be responsibl e for binding the drug. A series of six low-melting DNA hairpins conta ining all or part of the 5'-TAGT-3' binding site were synthesized. The highest T-m observed for the melting of these hairpins was 34.2 +/- 0 .3 degrees C, and it depended on the length of the stem region. These metastable hairpins were stabilized by 7-aminoactinomycin D, with the drug shifting the T-m for the drug-hairpin complex to similar to 45 de grees C. The hairpins showed very high affinity (K-d similar to 0.1 mu M) for 7-aminoactinomycin D, with some dependence on stem length. Dig estion of the hairpins in the presence and absence of drug using mung bean nuclease, which specifically interacts with the loop region of ha irpin DNA, revealed that the stable hairpins (i) contain a number of n on-Watson-Crick base pairs, and (ii) undergo a conformational change i n the loop region upon binding 7-aminoactinomycin D. Our results sugge st that stabilization of unusual hairpins by actinomycin D may be an i mportant aspect of the potent transcription inhibition activity of thi s drug.