Inhibition of Restriction Enzyme Ksp 632-I Via Triple Helix Formation by Phosphorothioate Oligonucleotides

Abstract

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 prepared double strand oligonucleotides corresponding to the major groove of SV40 DNA at 17 base pair homopurine-homopyrimidine sequences, and studied their interaction with homopyrimidine oligodeoxyribonucleotides including replacement of the PS group in the second nucleotide position from the 5′-terminus (SO-ODNs) and of the PS group at both the 3′- and 5′-ends (S₂O-ODNs). The resulting perfect DNA triplexes were detected by the 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).