Site-Specific Cleavage of a Yeast Chromosome by Oligonucleotide-Directed Triple-Helix Formation

Abstract

Oligonucleotides equipped with EDTA•Fe can bind specifically to duplex DNA by triple-helix formation and produce double-strand cleavage at binding sites greater than 12 base pair in size. To demonstrate that oligonucleotide-directed triple-helix formation is a viable chemical approach for the site-specific cleavage of large genomic DNA, an oligonucleotide with EDTA•Fe at the 5' and 3' ends was targeted to a 20-base pair sequence in the 340-kilobase pair cromosome III of Saccharomyces cerevisiae. Double-trand cleavage products of the correct size and location were observed, indicating that the oligonucleotide bound and cleaved the target site among almost 14 megabase pairs of DNA. Because oligonucleotide-directed triple-helix formation has the potential to be a general solution for DNA recognition, this result has implications for physical mapping of chromosomes.