Activation of camptothecin derivatives by conjugation to triple helix-forming oligonucleotides.

Topoisomerase I (topo I) is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy. Camptothecins (CPTs) reversibly trap topo I in covalent complex with DNA but exhibit limited sequence preference. The utilization of conjugates such as triplex-forming oligonucleotides (TFOs) to target a medicinal agent (like CPT) to a specific genetic sequence and orientation within the DNA has been accomplished successfully.

Triple-helix directed cleavage of double-stranded DNA by benzoquinoquinoxaline-1,10-phenanthroline conjugates.

Oligonucleotide-directed triple-helix formation provides a rational means to interfere with genomic DNA targets and to direct modifications at specific sites. We have developed a new class of compounds that, at low concentrations, efficiently targets and damages double-stranded DNA specifically at the site where a triple-helical structure is formed. In these new compounds, a triple-helix-specific intercalator-benzoquinoquinoxaline (BQQ)-was coupled to one of two isomeric 1,10-phenanthrolinecarboxaldehyde derivatives.

Optimization of triple-helix-directed DNA cleavage by benzoquinoquinoxaline-ethylenediaminetetraacetic acid conjugates.

The formation of triple-helical structures of DNA is based on sequence-specific recognition of oligopyrimidine.oligopurine stretches of double-helical DNA. Triple-helical structures can be stabilized by DNA-binding ligands.

Spatial organization of topoisomerase I-mediated DNA cleavage induced by camptothecin-oligonucleotide conjugates.

Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3' or the 5' side of the triplex, and the location of the cleavage site. A previously identified cleavage site was inserted at different positions within two triplex site-containing 59 bp duplexes.

A directional nucleation-zipping mechanism for triple helix formation.

A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis.

Chemical modification of the third strand: differential effects on purine and pyrimidine triple helix formation.

DNA triple helices offer exciting perspectives toward oligonucleotide-directed control of gene expression. Oligonucleotide analogues are routinely used with modifications in either the backbone or the bases to form more stable triple-helical structures or to prevent their degradation in cells. In this article, different chemical modifications are tested in a model system, which sets up a competition between the purine and pyrimidine motifs.

Design and optimization of camptothecin conjugates of triple helix-forming oligonucleotides for sequence-specific DNA cleavage by topoisomerase I.

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.