EFFECT OF RIBONUCLEOTIDE SUBSTITUTION ON NUCLEIC-ACID BULGE RECOGNITION BY NEOCARZINOSTATIN

Bulged RNA structures are not as good substrates for cleavage by the e nediyne antibiotic neocarzinostatin chromophore in the general base-ca talyzed reaction as are DNA bulges. In an effort to determine why this is so, we have systematically substituted ribonucleotide residues in a DNA bulged structure (CCGATGCG.CGCAG (T) under bar TCGG) (cleaved re sidue is underlined) known to be an excellent substrate. It was found that ribonucleotide substitution at the bulge target site, as well as at other regions involving duplex formation had a small effect on the cleavage reaction, unless either of the two strands was entirely of th e ribo form. By contrast, changing the A.T base pair on the 5' side of the target nucleotide ((T) under bar residue) to ribo A.U resulted in an 87% decrease in cleavage; in fact, conversion of the A alone to th e ribo form caused a 68% loss in cleavage. This result can be understo od from the recent solution structure of the complex formed between an analogue of the drug radical species and a bulged DNA (Stassinopoulos , A.; Ji, J.; Gao, X.; Goldberg, I. H. Science 1996, 272, 1943), since the 2' hydroxyl group of the ribo A would be expected to clash steric ally with the 7 ''-O-methyl moiety of the drug. Additional studies on substrate bulge-dependent drug product formation and protection agains t spontaneous drug degradation support the cleavage experiments, and i mply that bulge-specific drug binding is required for efficient cleava ge. (C) 1997 Elsevier Science Ltd.