Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT).
Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3'-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTCTTTTG), d(TGTTTTGTC), and d(TGTTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3'-terminus of d(TGTCTTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTCTTTTGTC) and d(TGTCTTTTGTCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3'-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.
Chen
Fu-Ming
FM
Department of Chemistry, Tennessee State University, Nashville 37209, USA. fchen@tnstate.edu
Sha
Feng
F
Chin
Ko-Hsin
KH
Chou
Shan-Ho
SH
D000903:Antibiotics, Antineoplastic / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D003596:Cytosine / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013941:Thymine / Q000737:chemistry
D000903:Antibiotics, Antineoplastic; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D003609:Dactinomycin; D006147:Guanine; C025942:7-aminoactinomycin D; D003596:Cytosine; D013941:Thymine