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

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