Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.

Abstract

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine-homopyrimidine sequences by forming local triple helices. Phenanthroline was covalently attached to the 5' end of an 1i-mer homopyrimidine oligonucleotide of sequence d(TT-TCCTCCTCT). Simian virus 40 DNA, which contains a single target site for this oligonucleotide, was used as a substrate for the phenanthroline-oligonucleotide conjugate. In the presence of copper ions and a reducing agent, a single specific doublestrand cleavage site was observed at 20'C by agarose gel electrophoresis. The efficiency of double-strand cleavage was >70% at 20'C and pH 7.4. Secondary cleavage sites were observed when binding of the oligonucleotide to mismatched sequences was allowed to take place at low temperature. The exact location of the cleavage sites was determined by polyacrylamide gel electrophoresis of denatured fragments by using both simian virus 40 DNA and a synthetic DNA fragment containing the target sequence. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove even though the phenanthroline linker was located in the major groove. Linkers of different lengths were used to tether phenanthroline to the oligonucleotide and their relative efficacies of DNA cleavage were compared. Based on these comparative studies and on model building, it is proposed that the phenanthroline ring carried by the oligonucleotide intercalates from the major groove and that copper chelation locks the complex in place from within the minor groove where the cleavage reaction occurs.

Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide.

“…Several possibilities have been described previously: azidoproflavine (4) and azidophenacyl (12) were used to photo-cross-link the oligonucleotide to one strand of the double helix; EDTA-Fe(II) chelate (5) or phenanthrolineCu(II) (13) chelate was shown to induce cleavage of both DNA strands in the presence of molecular oxygen and a reducing agent; an ellipticine-oligonucleotide conjugate was able to cleave double-helical DNA at the target sequence when irradiated with near-UV light (14). These reactions have rather low yields except for cleavage by the phenanthroline-Cu(II) chelate, which was shown to induce about 70% double-strand cleavage (13). However, this cleavage reaction requires addition of Cu(II) ions and a reducing agent, and it is not clear yet whether such a targeted reaction could be achieved inside living cells.…”

Cleaving DNA with DNA

“…This, coupled with the observation of substrate recognition by triplex formation, suggests that different DNA enzymes might be engineered to cleave duplex DNA substrates without the need for thermal denaturation. Such deoxyribozyme activity would be similar to that performed by a number of triplex-forming oligonucleotides that have been engineered to bind and cleave duplex DNA by using a chemically tethered metal complex such as Fe-EDTA (23)(24)(25)(26) or Cu-phenanthroline (27).…”

Repair of triple helix directed psoralen adducts in human cells

“…Thus, crosslinking of the two strands of the DNA by psoralen (6)(7)(8) and chlorambucil (9), and DNA cleavage by ellipticine (10), phenanthroline-Cu chelate (11), and EDTA-Fe(ll) (12) have been reported. Despite the great knowledge of triple helix formation in vitro little is known about the repair and mutagenic effects of these covalent modifications in vivo. In order to obtain a better understanding of the potential of triple helix forming oligonucleotides as site specific mutagens, we have examined the repair and mutagenesis of triple helix targeted psoralen monoadducts and crosslinks in human cells.…”