To achieve a sequence-specific DNA cleavage by topoisomerase I, derivatives of the antitumor drug camptothecin have been covalently linked to triple helix-forming oligonucleotides that bind in a sequence-specific manner to the major groove of double-helical DNA. Triplex formation at the target sequence positions the drug selectively at the triplex site, thereby stimulating topoisomerase I-mediated DNA cleavage at this site. In a continuous effort to optimize this strategy, a broad set of conjugates consisting of (i) 16 -20-base-long oligonucleotides, (ii) alkyl linkers of variable length, and (iii) camptothecin derivatives substituted on the A or B quinoline ring were designed and synthesized. Analysis of the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced strong and highly specific cleavage at a site adjacent to the triplex. Furthermore, the drug-stabilized DNA-topoisomerase I cleavage complexes were shown to be more resistant to salt-induced reversal than the complexes induced by camptothecin alone. Such rationally designed camptothecin conjugates could provide useful antitumor drugs directed selectively against genes bearing the targeted triplex binding site. In addition, they represent a powerful tool to probe the molecular interactions in the DNA-topoisomerase I complex.The reaction between double-stranded DNA and topoisomerase I produces a covalent 3Ј-phosphorotyrosyl adduct, usually referred to as the cleavage complex (1, 2). Under physiological conditions, the covalent intermediate is barely detectable, because a fast religation step occurs after relaxation of the DNA constraints. A number of drugs, such as the antitumor alkaloid camptothecin (CPT), 1 can convert topoisomerase I into a cell poison by blocking the religation step, thereby enhancing the formation of persistent DNA breaks responsible for cell death (1, 3, 4). However, topoisomerase I poisons display a weak sequence specificity. Mainly one or two nucleotides on the 3Ј and 5Ј-side of the cleavage site (essentially thymine-guanine steps in the case of CPT) represent the only recognition elements. As a result, drugs like CPT induce massive nonspecific DNA damage in cells and can affect any gene within the genome. The identification, over the last decade, of genes that play a key role in the progression and maintenance of a specific disease, such as oncogenes and tumor suppressor, calls for the development of drugs able to regulate the expression and functions of these genes. For this reason, the design of molecules that bind to specific sequences in DNA is urgently needed. To direct the topoisomerase I enzymatic reaction to particular sites, topoisomerase I poisons, including camptothecin and rebeccamycin derivatives, have been covalently attached to sequence-specific DNA ligands, such as triplex-forming oligonucleotides (TFO) (5-7) and...
