Actinomycin D binding to unstructured, single-stranded DNA

Actinomycin D is an anticancer antibiotic best know for inhibiting transcri ption by binding double-stranded DNA, Tight, sequence selective binding of actinomycin to single-stranded DNA is also known, however, and is implicate d in biological activities including inhibition of(-) strand transfer by HI V reverse transcriptase, Oligonucleotide d(GTTAACCATAG) is one of the rare single-stranded DNAs that lack GC steps yet have high affinity for actinomy cin, Oligonucleotide sequence and length requirements for drug binding were investigated by monitoring association of the fluorescent surrogate, 7-ami noactinomycin D, to d(GTTAACCATAG) and 31 related oligomers, The TAG-3' ter minal sequence was essential for high affinity binding, but was not suffici ent. Five oligomers with TAG sequences on or near the 3'-end had high affin ity [K-d less than or equal to 200 nM (oligomer)], A sixth oligomer, d(GTAA CCATATG), had moderately lower affinity (K-d= 370 nM), and other homologous oligomers had much lower affinity. The minimum length sequence for tight b inding of 7-aminoactinomycin D was identified as only eight nucleotides, co rresponding to d(AACCATAG). This octanucleotide is unstructured in the abse nce of actinomycin, and has the highest drug affinity of all oligomers exam ined (K-d = 125 nM), These studies show that high-affinity binding of 7-ami noactinomycin, 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 si ngle-stranded DNA sequences by an induced-fit mechanism favored by particip ation of at least eight nucleotides, or the equivalent of four base pairs. Copyright (C) 2001 John Wiley & Sons, Ltd.