CLEAVAGE OF PHOSPHOROTHIOATE-CONTAINING OLIGONUCLEOTIDES AND DNA BY RESTRICTION ENDONUCLEASES

Abstract

The oligonucleotide d(GGsAATTCC) containing the recognition sequence of the Eco R1 restriction endonuclease with a phosphorothioate group at the site of cleavage was synthesized by two approaches both based on the polymer support phosphoroamidite method. Only the Rp-diastereomer was cleaved by Eco R1, at a rate approximately 20 times slower than the all-phosphate-containing octamer. To study the interaction of restriction endonucleases with phosphorothioate internucleotidic linkages double stranded Mi3mp2 DNA was prepared in which the (+)strand contained only phosphate groups but the (-)strand phosphate groups as well as base specifically introduced phosphorothioate groups of the Rp-configuration. This hybrid DNA was cleaved by several restriction enzymes. The results obtained allow the classification of these enzymes into three groups: those which produce nicked DNA as an isolatable intermediate, those where the nicked DNA is the final product and a third where only linearised DNA can be detected. Particularly the second class of enzymes should be of interest for the manipulation of DNA.