Stepwise phosphorolysis with polynucleotide phosphorylase: A novel method for sequence analysis of oligoribonucleotides

Abstract

Snake venom phosphodiesterase, bovine spleen phosphodiesterase and polynucleotide phosphorylase are currently used for sequence analysis of oligoribonucleotides. These exonucleases remove mononucleotide residues in a sequential order from either the 3’or 5’-end of an oligonucleotide. The nucleotide sequence of a given oligonucleotide is then derived by analyzing the partially degraded oligonucleotides which appear in the course of the reaction (cf. [l-3] ). These procedures, while very useful for analyzing labeled oligonucleotides, are inadequate when only trace amounts of non-labeled material are available. In the present report we describe a novel and sensitive method for sequence analysis of non-labeled oligoribonucleotides. The method is based on the property of polynucleotide phosphorylase to phosphorolyze short oligonucleotides from the 3’-end in a stepwise fashion by a nonprocessive mechanism [4-61. The final products of the reaction consist of a mixture of nucleoside diphosphates (NDP’s) and a limit oligonucleotide that is not further degraded by the enzyme. Depending on its base composition, this oligonucleotide is a dior trinucleotide. By following the order in which the released NDP’s appear during the phosphorolysis of a given oligonucleotide, it is possible to determine the nucleotide sequence from the 3’-end up to 2-3 residues from the 5’-terminus. The high sensitivity of this method is achieved by including 32Pi in the phosphorolysis medium. Upon digestion, the label is incorporated in the P-phosphate moieties of the generated NDP’s which can be easily