Site-specific cleavage of single-stranded and double-stranded DNA sequences by oligodeoxyribonucleotides covalently...

An oligodeoxythymidylate, oligo [d(T8)], was covalently linked to an acridine derivative via its 3' end and to EDTA via its 5' end. The octathymidylate was targeted to a single-stranded DNA fragment 27 nucleotides in length containing an octadeoxyadenylate sequence. In the presence of Fe(II) and a reducing agent (dithiothreitol) cleavage reactions were induced in the nucleotide sequence. The extent of the reaction was dependent on oligo concentration, salt concentration and temperature. Dissociation of the complexes at high temperature or low salt concentration abolished the site-specific cleavage reactions. Treatment of the reacted DNA with piperidine or piperidine-formiate strongly enhanced the yield of cleavage reactions demonstrating that damages were induced on nucleic acid bases by the EDTA-Fe complex covalently linked to the octathymidylate. At high salt concentration (1 M NaCl) or in the presence of spermine and ethylene-glycol a triple helix was formed involving the 27-mer DNA fragment and two oligo[d(T8)]. One of the oligo[d(T8)] was bound parallel and the other antiparallel to the oligo[d(A8)] complementary sequence. Cleavage reactions were induced on both sides of this oligo[d(A8)] target sequence. When a 27-mer duplex was used as a target the oligo[d(T8)] was bound in a parallel orientation with respect to the oligo[d(A8)]-containing strand in the major groove of the double helix. Cleavage reactions were induced on the oligo[d(A8)]-containing strand by the EDTA-Fe chelate attached to the 5' end of the oligo[d(T8)].