Enzymatic Assignment of Diastereomeric Purity of Stereodefined Phosphorothioate Oligonucleotides

Abstract

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.

Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability

“…Chemically synthesised phosphorothioate linkages are a mixture of two enantiomers and it is likely that the multiphase kinetics observed were due in part to the different susceptibility of the two isomers (Rp and Sp) to the nucleases present in the serum [37,38]. Mrz12/12 K has 2'-amino modification at U 4 , is very stable in serum, and is only 7-fold less active than the unmodified parent.…”

Data for isolation and properties analysis of diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide

“…Oligonucleotides were incubated with SVDPE at 37 °C. Reaction micture contaned 3 μg of SVDPE, 20 mM Tris-HCl, pH 9.0, 5 mM MgCl 2 and 0.1 mM oligonucleotide [4]. Aliquots of 20 μl withdrawn at 0.5, 1, 24, 48 and 150h, heated to 65 °C for 30 min, then analyzed by RP-HPLC.…”

“…Abbreviations [4].#Cluster - Cluster number starting from 0 (0 is most populated).Frames - number of frames in cluster.Frac - Size of cluster as fraction of total trajectory.AvgDist - Average distance between points in the cluster.Stdev - Standard deviation of points in the cluster.AvgCDist - Average distance of this cluster to every other cluster.…”

Significance of Stereochemistry of 3′-Terminal Phosphorothioate-modified Primer in DNA Polymerase-mediated Chain Extension

“…The R P -PS-oligonucleotides are preferably cleaved by several nucleolytic enzymes including snake venom phosphodiesterase (svPDE) [14], endonuclease from Serratia marcescens [15], and transposases [16], while nuclease P1 (nP1) preferentially hydrolyzes the S P -PSdiastereomers [17]. It was also reported from our laboratory that R P PS-oligos are better substrates for the 3 0 -exonuclease present in human plasma [18].…”