Sequence-targeted cleavage of single- and double-stranded DNA by oligothymidylates covalently linked to 1,10-phenanthroline

Abstract

The nuclease activity of 1,10-phenanthroline copper ion was targeted to a specific sequence by attachment of the ligand to the 5' or 3' end of octathymidylates. An acridine derivative was also attached to the other end of the oligothymidylate-phenanthroline conjugate. The duplex formed by the oligothymidylate with its complementary sequence was stabilized by intercalation of the acridine derivative. The reaction induced by 3-mercaptopropionic acid led to a very localized cleavage of a 27-nucleotide-long DNA fragment containing a (dA)8 sequence. At high NaCl concentration or in the presence of spermine, cleavage of the single-stranded 27-mer fragment occurred on both sides of the target sequence. This was ascribed to the formation of a triple helix involving two 1,10-phenanthroline-octathymidylate strands that adopt an antiparallel orientation with respect to each other. When a 27-mer duplex was used as a substrate, cleavage sites were observed on both strands. The location of the cleavage sites led us to conclude that the octathymidylate was bound to the (dA)8.(dT)8 sequence in a parallel orientation with respect to the (dA)8-containing strand. This result reflects the ability of thymine to form two hydrogen bonds with an adenine already engaged in a Watson-Crick base pair. This study shows that it is possible to design DNA-binding oligodeoxynucleotides that could selectively recognize and cleave polypurine-polypyrimidine sequences in double-stranded DNA.