Actinomycin D:
Binding of Actinomycin D to Single-Stranded DNA of Sequence
Motifs d(TGTCT(n)G) and d(TGT(n)GTCT).
Biophys J
2003 Jan;84(1):432-9
Chen FM, Sha F, Chin KH, Chou SH.
Department of Chemistry, Tennessee State University, Nashville, Tennessee
37209 USA and Institute of Biochemistry, National Chung Hsing University,
Taichung 40227, Taiwan.
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.
