Drug-nucleic acid complexes have been studied extensively using lH-and 31P-nmr.l,2 While '3C-nmr has recently been used to investigate the conformational mobility of nucleic acid fragments,3-6 relatively few 13C-nmr studies of drug-nucleic acid complexes have been reported.3.7 In this communication, we present preliminary results of our l3C-nrnr analysis of the DNA complexes formed by two anticancer drugs, chromomycin A3 and actinomycin D (Fig. 1). Our results include the first observation of both ligand and DNA 13C resonances, thereby providing insight into the mechanism of binding and side-chain flexibility of the drugs.
Both chromomycin and actinomycin bind tightly to double-stranded DNA in the minor groove, with slow kinetics, and show a strong preference for sites containing G-C base pairs.8 The oligosaccharide and oligopeptide side chains of chromomycin and actinomycin, respectively, play an important role in the DNA binding process of these c o m p o ~n d s . g -~~ The drugs differ in that a great deal of experimental evidence supports intercalation of actinomycin between DNA base pairs, while data on chromomycin are inconclusive in this regard. Also, the DNA binding of chromomycin requires the presence of a divalent cation, such as Mg++.a
Calf thymus DNA (Sigma) was digested with deoxyribonuclease I1 (Sigma) for 6 h. After extraction with chloroform/amyl alcohol and precipitation with 70% ethanol solution, the resuspended DNA fragments were dialyzed and concentrated by ultrafiltration. The average length of these fragments was determined to be 70 f 30 base pairs by polyacrylamide gel electrophoresis. Nmr spectra were obtained with an excess of calf thymus DNA (42% G-C content) to ensure that all the drug was in the bound form. The use of short DNA fragments enhanced nmr resolution and minimized peak overlap. Thus, chemical shift changes and selective line-broadening were observable for both drug and DNA resonances.
While the DNA fragments may contain single-stranded ends, the specificity of' both drugs for double-stranded DNA plus the presence of an excess of DNA (19:L nuc1eotide:drug) ensure that the drug is bound to double-stranded regions of the fragments. The extremely slow kinetics for binding of both drugs to DNA'OJI guarantees that the drug-DNA systems are in slow exchange on the nmr time scale. Under conditions of excess DNA, then, we expect no signal from the free drug. Also, the observed linewidths reflect properties of the bound drug complex and are not affected by exchange rates between free and bound states.