We have reexamined the binding properties of the antitumor drug daunom
ycin using double-helical oligonucleotides 16 base pairs long that wer
e designed to contain preferred binding sites for the drug. The prefer
red sites are contained in a six base pair core which is flanked on th
e 5' and 3' ends by tracts of adenines. The flanking sequences, which
augment helix stability and reduce and effects, were chosen because da
unomycin is known to bind poorly to poly(dA).poly(dT). Four major sequ
ences were examined in the six base pair core: CGTACG, TAGCTG, TCATCC,
and (TA)3 and compared with calf thymus DNA. A randomly generated 16
bp sequence containing no A tracts and a sequence containing only trac
ts of As and Ts were also used. Fluorometric, absorption, calorimetric
, and stopped-flow techniques were used to examine the binding. The af
finity of the drug for oligomers containing known binding sites was co
mparable to or enhanced relative to that for calf thymus bulk DNA. Ass
ociation constants ranged from 1.0 x 10(8) to 3.0 X 10(7) M-1. The str
ongest core binding site found was CGTACG, but its affinity is only 2-
fold larger than that of other core sequences. Appreciable binding to
the flanking A tracts was observed. An oligonucleotide which incorpora
tes the CGTACG sequence in a short hairpin helix binds an order of mag
nitude more weakly. Complex lifetimes measured by stopped flow general
ly increase with equilibrium stability; the kinetics confirm the exist
ence of a set of weaker sites. The exothermic binding enthalpy for dau
nomycin with the CGTACG core sequence is more than twice as large as f
or the TATATA sequence. Binding to dA20.dT20 is endothermic, and a les
s exothermic component can be detected in the calorimetric binding cur
ve of the oligomers containing flanking A tracts.