SEQUENCE-SPECIFIC DOUBLE-STRAND ALKYLATION AND CLEAVAGE OF DNA MEDIATED BY TRIPLE-HELIX FORMATION

Attachment of the nondiffusible electrophile N-bromoacetyl to the 5-position of a thymine at the 5'-end of a pyrimidine oligodeoxyribonucleotide affords sequence specific alkylation of a guanine two base pairs to the 5'-side of a local triple-helix complex in >96% yield. N-Bromoacetyloligodeoxyribonucleotides bind adjacent inverted purine tracts on double-helical DNA by triple-helix formation and alkylate single guanine positions on opposite strands at 37-degrees-C (pH 7.4). After depurination, double-strand cleavage at a single site within plasmid DNA (4 kp in size) occurs in greater than 85% yield. The resulting DNA fragments from site-specific alkylation and cleavage can be ligated with DNA fragments generated by restriction endonuclease digestion. This nonenzymatic approach which couples sequence-specific recognition with sequence-dependent cleavage affords double-strand site-specific cleavage in megabase size DNA. A yeast chromosome, 340 000 base pairs in size, was cleaved at a single site in 85-90% yield.