Probing the structure of long single-stranded DNA fragments with neocarzinostatin chromophore. Extension of the base-catalyzed bulge-specific reaction.

The base-catalyzed (bc) thiol-independent cleavage reaction of neocarzinostatin chromophore (NCS chrom) has been characterized with long single-stranded (ss) DNA in order to use this reaction as a selective probe for the tertiary structure of naturally occurring ss nucleic acids. The ss circular phi chi 174 phage and M13mp18 phage DNAs (approximately 5000 and 7500 bases, respectively) were shown to be bc NCS chrom reaction substrates, exhibiting the expected pH dependence. The ss DNA fragments (150-450 bases) were cleaved at six major sites; the lesions occurred at T-rich non-double-stranded sequences, as predicted from comparison with the minimal energy secondary structures. These sites exhibited the expected pH and drug: DNA ratio dependence shown to be required for this reaction. Optimization of the shortest sequence, which gave the highest cleavage yield, identified the minimal sequence requirements for the site (19-mer of the sequence 3'TACTGAGTCTCCTTTTGTA5', attacked residue in bold). Folding pattern analysis predicted that the oligonucleotide contained a two-base bulge at the cleavage site; this result was consistent with the observation that removing features which destabilize the bulged structure increased the cleavage yield. Furthermore, the derived 19-mer was shown to generate maximal amounts of the final drug product of the bc DNA cleavage reaction. Reaction of an RNA 339-mer containing the same sequence as one of the long ss DNA fragments showed it not to be a substrate for the bc reaction, while similar results were obtained for the RNA analog of shorter oligodeoxyribonucleotides identified in this and earlier studies. Through a combination of thermodynamic and kinetic assays, the observed difference in reactivity was shown to be the result of the low binding of the cleaving species to RNA.