Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides

Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothi oates (PS-oligos) synthesized via the oxathiaphospholane method has been us ed for assignment of;their diastereomeric purity, For this purpose, two wel l-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phosphodiesterase (svPDE) have been used. However, because of some di sadvantageous properties of svPDE, a search for other [R-P]-specific endonu cleases was undertaken, Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphor othioate bonds of the [R-P] configuration, whereas internucleotide [S-P]-ph osphorothioates are resistant to its action. Cleavage experiments carried o ut with the use of unmodified and phosphorothioate oligonucleotides of diff erent sequences demonstrate that the Serratia nuclease is more selective in recognition land hydrolysis of oligodeoxyribonucleotides than previously r eported. The substrate specificity exhibited by the enzyme is influenced no t only by the nucleotide sequence at the cleavage site but also by the leng th and base sequence of flanking sequences. The Serratia nuclease can be us eful for analysis of diastereomeric purity of stereodefined phosphorothioat e oligonucleotides, but because of its sequence preferences, the use of thi s enzyme in conjunction with svPDE is more reliable.