An acridine derivative was covalently linked to the 5' end of a homopyrimidine oligonucleotide. Specific binding to a homopurine homopyrimidine sequence of duplex DNA was demonstrated by spectroscopic studies (absorption and fluorescence) and by "footprinting" experiments with a copper phenanthroline chelate used as an artificial nuclease. A hypochromism and a red shift of the acridine absorption were observed. Triple-helix formation was also accompanied by a hypochromism in the ultraviolet range. The fluorescence of the acridine ring was quenched by a stacking interaction with a GC base pair adjacent to the homopurine-homopyrimidine target sequence. The intercalating agent strongly stabilized the complex formed by the oligopyrimidine with its target duplex sequence. Cytosine methylation further increased the stability of the complexes. Footprinting studies revealed that the oligopyrimidine binds in a parallel orientation with respect to the homopurine-containing strand of the duplex. The intercalated acridine extended by 2 base pairs the region of the duplex protected by the oligopyrimidine against degradation by the nuclease activity of the copper phenanthroline chelate. Random intercalation of the acridine ring was lost due to the repulsive effect of the negatively charged oligonucleotide tail. Intercalation occurred only at those double-stranded sequences where the homopyrimidine oligonucleotide recognized the major groove of duplex DNA.
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.
Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. The oligonucleotide is bound parallel to the homopurine strand of the duplex. This binding can be revealed by a footprinting technique using copper-phenanthroline as a cleaving reagent. Oligonucleotide binding in the major groove prevents cleavage by copper-phenanthroline. The cleavage patterns on opposite strands of the duplex at the boundaries of the triple helix are asymmetric. They are shifted to the 3'-side, indicating that the copper-phenanthroline chelate binds in the minor groove of the duplex structure. Binding of the chelate at the junction between the triple and the double helix is not perturbed on the 5'-side of the bound homopyrimidine oligonucleotide. In contrast, a strong enhancement of cleavage is observed on the purine-containing strand at the triplex-duplex junction on the 3'-side of the homopyrimidine oligonucleotide.
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