Actinomycin D Binding to Unstructured, Single‐stranded DNA

Actinomycin D is an anticancer antibiotic best know for inhibiting transcription by binding double‐stranded DNA. Tight, sequence selective binding of actinomycin to single‐stranded DNA is also known, however, and is implicated in biological activities including inhibition of (−) strand transfer by HIV reverse transcriptase. Oligonucleotide d(GTTAACCATAG) is one of the rare single‐stranded DNAs that lack GC steps yet have high affinity for actinomycin. Oligonucleotide sequence and length requirements for drug binding were investigated by monitoring association of the fluorescent surrogate, 7‐aminoactinomycin D, to d(GTTAACCATAG) and 31 related oligomers. The TAG‐3′ terminal sequence was essential for high‐affinity binding, but was not sufficient. Five oligomers with TAG sequences on or near the 3′‐end had high affinity [ K d  ≤ 200 n M (oligomer)]. A sixth oligomer, d(GTAACCATATG), had moderately lower affinity ( K d  = 370 n M ), and other homologous oligomers had much lower affinity. The minimum length sequence for tight binding of 7‐aminoactinomycin D was identified as only eight nucleotides, corresponding to d(AACCATAG). This octanucleotide is unstructured in the absence of actinomycin, and has the highest drug affinity of all oligomers examined ( K d  = 125 n M ). These studies show that high‐affinity binding of 7‐aminoactinomycin, and actinomycin D by extension, to single‐stranded DNA does not require pre‐existing secondary structure or any apparent propensity for secondary structure. It is proposed that actinomycin D binds to certain single‐stranded DNA sequences by an induced‐fit mechanism favored by participation of at least eight nucleotides, or the equivalent of four base pairs. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: AMD actinomycin D7AAMD 7‐aminoactinomycin DdsDNA double‐stranded DNAssDNA single‐stranded DNA.