PROBING THE STRUCTURE OF LONG SINGLE-STRANDED-DNA FRAGMENTS WITH NEOCARZINOSTATIN CHROMOPHORE - EXTENSION OF THE BASE-CATALYZED BULGE-SPECIFIC REACTION

The base-catalyzed (be) thiol-independent cleavage reaction of neocarz inostatin chromophore (NCS chrom) has been characterized with long sin gle-stranded (ss) DNA in order to use this reaction as a selective pro be for the tertiary structure of naturally occurring as nucleic acids. The ss circular phi chi 174 phage and M13mp18 phage DNAs (similar to 5000 and 7500 bases, respectively) were shown to be be NCS chrom react ion substrates, exhibiting the expected pH dependence. The as DNA frag ments (150-450 bases) were cleaved at six major sites; the lesions occ urred at T-rich non-double-stranded sequences, as predicted from compa rison with the minimal energy secondary structures. These sites exhibi ted the expected pH and drug: DNA ratio dependence shown to be require d for this reaction. Optimization of the shortest sequence, which gave the highest cleavage yield, identified the minimal sequence requireme nts for the site (19-mer of the sequence 3'TACTGAGTCTCCTTTTGTA5', atta cked residue in bold). Folding pattern analysis predicted that the oli gonucleotide contained a two-base bulge at the cleavage site; this res ult was consistent with the observation that removing features which d estabilize the bulged structure increased the cleavage yield. Furtherm ore, the derived 19-mer was shown to generate maximal amounts of the f inal drug product of the be DNA cleavage reaction. Reaction of an RNA 339-mer containing the same sequence as one of the long ss DNA fragmen ts showed it not to be a substrate for the be reaction, while similar results were obtained for the RNA analog of shorter oligodeoxyribonucl eotides identified in this and earlier studies. Through a combination of thermodynamic and kinetic assays, the observed difference in reacti vity was shown to be the result of the low binding of the cleaving spe cies to RNA.