Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides.
Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-oligos) synthesized via the oxathiaphospholane method has been used for assignment of their diastereomeric purity. For this purpose, two well-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phosphodiesterase (svPDE) have been used. However, because of some disadvantageous properties of svPDE, a search for other [Rp]-specific endonucleases was undertaken. Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphorothioate bonds of the [Rp] configuration, whereas internucleotide [Sp]-phosphorothioates are resistant to its action. Cleavage experiments carried out with the use of unmodified and phosphorothioate oligonucleotides of different sequences demonstrate that the Serratia nuclease is more selective in recognition and hydrolysis of oligodeoxyribonucleotides than previously reported. The substrate specificity exhibited by the enzyme is influenced not only by the nucleotide sequence at the cleavage site but also by the length and base sequence of flanking sequences. The Serratia nuclease can be useful for analysis of diastereomeric purity of stereodefined phosphorothioate oligonucleotides, but because of its sequence preferences, the use of this enzyme in conjunction with svPDE is more reliable.
Koziołkiewicz
M
M
Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry, Lódź.
Owczarek
A
A
Gendaszewska
E
E
D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; C021835:Serratia marcescens nuclease; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom