Site-specific intercalation at the triplex-duplex junction induces a conformational change which is detectable by hypersensitivity to diethylpyrocarbonate

Abstract

Using site-specific intercalation directed by intermolecular triplex formation, the conformation of an intercalation site in DNA was examined by footprinting with the purine-specific (A »G) reagent diethylpyrocarbonate. Site specific intercalation was achieved by covalently linking an intercalator to the 5′ end of a homopyrimidine oilgodeoxynucleotide, which bound to a homopurinehomopyrimidine stretch in a recombinant plasmid via intermolecular triplex formation. This directs intercalation to a single site in 3kb of DNA at the 5′ triplex-duplex junction. Footprinting with diethylpyrocarbonate and dimethylsuphate revealed strong protection from modification of adenine residues within the triple-helix in concordance with their Hoogsteen pairing with the third strand, and a strong hypersensitivity to diethylpyrocarbonate at the first adenine of the duplex. This result indicates that intercalation at this site induces a conformational change at the 5′ triplex-duplex junction. Furthermore, the same diethlypyrocarbonate hypersensitivity was observed with an unmodified triple-strand forming oligonucleotide and a range of intercalating molecules present in solution. Thus the 5′ triplex-duplex junction is a strong binding site for some intercalating molecules and the junction undergoes a conformational change which is sensitive to diethylpyrocarbonate upon insertion of the planar aromatic chromophore. This conformational change can be used to direct a single-strand cut in duplex DNA to a defined site. © 1991 Oxford University Press.