Is the strong actinomycin D binding of d(5'CGTCGACG3') the consequence of end-stacking?

It has been reported that ACTD binds strongly and cooperatively to a non-GC containing self-complementary octamer d(CGTCGACG) with a 2:1 drug to duplex ratio (Synder et al., 1989). If one views the classic intercalative preference of ACTD for the 5'GpC3' sequence to be the drug favoring the 3'-side of dG, the possibility exists that the drug molecules may in fact stack on the G.C base pairs at both ends of this oligomeric duplex. To investigate this possibility, d(CGTCGACG) and several related oligomers resulting from replacing the terminal base(s) or appending with dT and/or dA are used in a comparative study employing equilibrium titration, thermal denaturation, kinetic, and various spectral measurements. Absorbance titrations at 20 degrees C confirm the strong and highly cooperative nature of ACTD binding to this octamer. The stoichiometric association constants for the binding of the first and second drugs were found to be 1 x 10(5) and 3.2 x 10(7) M-1, respectively. The base replacements of dG and dC at the respective ends resulted in a much weaker ACTD binding affinity, the loss of binding cooperativity, and much faster association and dissociation kinetics. These are consistent with the inability of the drug to stack on the 3'-side of dG due to base replacements. Appending the end(s) with dA and/or dT resulted in some diminution of binding affinity and cooperativity, appearance of slower association kinetic components, and unusually strong 7-amino-ACTD fluorescence enhancement for oligomers with dA or dT attached to dG at the 3'-terminal. To further support our postulate, studies were also made with d(CGACGTCG), which is related to the parent octamer by inverting the A.T pairs. It was found that, despite the altered internal sequence, this oligomer exhibits cooperative ACTD binding and kinetic characteristics very similar to those of the parent octamer, consistent with its ability to end-stack on the 3'-side of dG.