Inhibition of restriction endonuclease cleavage site via triple helix formation by homopyrimidine phosphorothioate oligonucleotides.

The ability of pyrimidine rich oligonucleotide phosphorothioate to form stable triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme Ksp 632-I was examined. First, we synthesized double strand oligonucleotides corresponding to the SV40 sites and studied their interaction with homopyrimidine oligodeoxyribonucleotides including replacement of the other chain either PS group (SO-ODNs) in second nucleotide position (from 5'-terminus) and end capped with the PS group at both 3'- and 5'-ends (S2O-ODNs). The resulting perfect DNA triplexes were detected by gel-mobility shift. The phosphorothioate oligonucleotide analogues (SO-ODNs) and (S2O-ODNs) were shown to inhibit enzymatic cleavage under conditions that allow for triple helix formation. Inhibition is sequence-specific and occurs in the micromolar concentration range. Of particular interest is the Sp-phosphorothioate analogue (Sp-SO-ODNs) which inhibited endonuclease more than the other phosphorothioate oligonucleotide analogues (Rp-SO-ODNs or S2O-ODNs).