Sequence-specific cleavage of double helical DNA by triple helix formation

H. Moser, P. Dervan
1987 Science  
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more » ... imidine oligodeoxyribonucleotides with EDTAFe attached at a single position bind the corresponding homopyrimidine-homopurine tracts within large double-stranded DNA by triple helix formation and cleave at that site. Oligonucleotides with EDTA-Fe at the 5' end cause a sequence specific double strand break. The location and asymmetry of the cleavage pattern reveal that the homopyrimidine-EDTA probes bind in the major groove parallel to the homopurine strand of Watson-Crick double helical DNA. The sequence-specific recognition of double helical DNA by homopyrimidine probes is sensitive to single base mismatches. Homopyrimidine probes equipped with DNA cleaving moieties could be useful tools for mapping chromosomes. T t HE SEQUENCE-SPECIFIC CLEAVAGE OF DOUBLE HELICAL DNA by restriction endonucleases is essential for many techniques in molecular biology, including gene isolation, DNA sequence determination, and recombinant DNA manipulations (1, 2). With the advent of pulsed-field gel electrophoresis, the separation of large pieces of DNA is now possible (3, 4) . However, the binding site sizes of naturally occurring restriction enzymes are in the range of 4 to 8 base pairs, and hence their sequence specificities may be inadequate for mapping genomes over very large distances. The design of sequence-specific DNA cleaving molecules that go beyond the specificities of the natural enzymes depends on a detailed understanding of the chemical principles underlying two finctions: recognition and cleavage of DNA (5). Synthetic sequence-specific binding moieties for double helical DNA that have been studied are coupled analogs of natural products (5), transition metal complexes (6), and peptide fragments derived from DNA binding proteins (7, 8) . The DNA cleaving function used in our laboratories is EDTA-Fe(II), which cleaves the DNA backbone by oxidation of the deoxyribose with a short-lived diffusible hydroxyl radical (5, 9). The fact that hydroxyl radical is a relatively nonspecific cleaving species is useful when studying recognition because the cleavage specificity is due to the binding moiety alone, not some combination of cleavage specificity superimposed on binding specificity. The most sequencespecific molecules characterized so far, with regard to the natural product analog approach, is bis(EDTA-distamycin)fumaramide, which binds in the minor groove and cleaves at sites containing 9 bp of contiguous A,T DNA (10). A synthetic peptide containing 52 The authors are at the Arnold and Mabel Beckman
doi:10.1126/science.3118463 pmid:3118463 fatcat:5qkiclvx5vbhnd2ttpq64ktzhu