Design of a triple-helix-specific cleaving reagent

Background: Double-helical DNA can be recognized sequence specifically by o ligonucleotides that bind in the major groove, forming a local triple helix . Triplex-forming oligonucleotides are new tools in molecular and cellular biology and their development as gene-targeting drugs is under intensive st udy. Intramolecular triple-helical structures (H-DNA) are expected to play an important role in the control of gene expression. There are currently no good probes available for investigating triple-helical structures. We prev iously reported that a pentacyclic benzoquinoquinoxaline derivative (BQQ) c an strongly stabilize triple helices. Results: We have designed and synthesized the first triple-helix-specific D NA cleaving reagent by covalently attaching BQQ to ethylenediaminetetraacet ic acid (EDTA). The intercalative binding of BQQ should position EDTA in th e minor groove of the triple helix. In the presence of Fe2+ and a reducing agent, the BQQ-EDTA conjugate can selectively cleave an 80 base pair (bp) D NA fragment at the site where an oligonucleotide binds to form a local trip le helix. The selectivity of the BQQ-EDTA conjugate for a tripler structure was sufficiently high to induce oligonucleotide-directed DNA cleavage at a single site on a 2718 bp plasmid DNA. Conclusions: This new class of structure-directed DNA cleaving reagents cou ld be useful for cleaving DNA at specific sequences in the presence of a si te-specific, triple-helix-forming oligonucleotide and also for investigatin g triple-helical structures, such as H-DNA, which could play an important r ole in the control of gene expression in vivo.