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) < or = 200 nM (oligomer)]. A sixth oligomer, d(GTAACCATATG), had moderately lower affinity (Kd = 370 nM), 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 (Kd = 125 nM). 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.
Yoo
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Department of Chemistry and Institute of Molecular Biophysics The Florida State University, Tallahassee, Florida, USA.
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D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013057:Spectrum Analysis; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D003609:Dactinomycin; C025942:7-aminoactinomycin D