Thermodynamic Origins of Sequence-Recognition in Ligand-DNA Interactions

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Graves, David E.MCB-0092177The long-term objective of this research is to gain biophysical insight into the key aspects of sequence selective interactions of small molecules with nucleic acids. The model system that has been studied thus far involves the sequence-specific DNA binding agent, actinomycin D. This compounds shows a marked preference for DNA binding at the d(GpC) step through intercalation of the phenoxazone chromophore between the adjacent G and C base pairs. Concomitant with this intercalative binding is the interactions of the two cyclic pentapeptide sidechains with the floor of the DNA minor groove. This laboratory has recently demonstrated the bases that flank this intercalation site play a major role in directing the thermodynamic mechanism for complex formation. If the 5'-flanking sequence is T (-TGCA-), the change in binding enthalpy has been measured to be -2.5 kcal/mol. In contrast, when the 5'-flanking base is C (-CGCA-), the change in binding enthalpy is found to be -5.8 kcal/mol, nearly 3 kcal/mol more favorable than the other flanking sequences. 5 Historically, the binding of actinomycin D to DNA has been reported to be enthalpy driven as signified by large positive (favorable) binding enthalpies and binding enthalpies of near zero or -1 kcal/mol. These recent results from this laboratory indicate that the thermodynamic binding properties of actinomycin D to oligonucleotides with specifically designed single binding sites are quite varied, depending on the sequence of bases flanking the actinomycin D binding site. Based on the variances observed in the enthalpy and entropy components associated with complex formation, linkages between thermodynamic binding mechanism(s) and structural features of the actinomycin D-DNA complexeswil be explored. Specifically the structures of the actinomycin D complexes formed with the -TGCA- duplex and the -CGCA- duplex will be examined by high-resolution NMR methods to discern differences in the structural features of the complex that may explain the enhanced enthalpy contribution toward actinomycin D binding to the CGCA- duplex.The use of non-self complimentary deoxyoligonucleotides was serendipitous toward the finding ofsequence selective interactions of actinomycin D to single-strand DNA. The influence of DNA base sequence on the energetics and structural properties of actinomycin D-single strand DNA interactions is being examined. These observations could be of considerable benefit in gaining insight into how proteins bind single-strand DNA in a sequence-selective manner and expanded to biological roles of single-strand DNA interactions. The in vivo interactions of actinomycin D to double stranded DNA are characterized by high affinity and slow dissociation and have been suggested to carry out their biological function of specifically inhibiting transcription through blocking the progression of the RNA polymerase along the DNA template. With the recent findings of a high affinity of actinomycin D for single-stranded DNA, additional caveats may be added to this proposed mechanism, including the possible binding of the drug to single-strand DNA within the open complex, and/or to inhibiting reannealing of the single-strand DNA.