Impact of 3′-Exonuclease Stereoselectivity on the Kinetics of Phosphorothioate Oligonucleotide Metabolism

Abstract

For the enzymatic digestion of a 25-mer phosphorothioate (PS) oligonucleotide, the reaction kinetics was previously determined to be the sum of two parallel processes: a fast and a very slow phase of digestion suggesting a two-exponential model. A characteristic metabolite profile was observed both in vitro and in vivo. This behavior is shown to be the result of the stereoselective cleavage of chiral R-configuration and S-configuration PS internucleotide linkages by 3'-exonucleases. The stereoselective nature of 3'-exonuclease action was analyzed by reverse-phase HPLC. The separation of eight diastereomers of the tetramer TTCT (5'-3') was used to follow the stereoselective course of exonuclease hydrolysis of PS internucleotide linkages. Degradation of the 25-mer parent compound having a 3' S-terminal internucleotide linkage was calculated to be more than 300 times slower than an analog with a 3'-terminal R-configuration. These results support an approach for protecting antisense oligonucleotides based on the chirality of only the 3'-end internucleotide linkage.

A Direct Comparison of Anti-microRNA Oligonucleotide Potency

“…The AMOs that are end-protected with PS linkages usually showed a small amount of truncated product consistent with 1-2 bases of exonuclease "nibbling"; this is expected as synthesis of PS-modified oligonucleotides leads to generation of two stereoisomers at each linkage, and the Sp isomer is significantly more nuclease resistant than the Rp isomer to exonucleolytic attack (45,46).…”

The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5′-nucleotidase

“…Although they are much more resistant to nucleolytic degradation than the PO oligos, reports indicating their stereodependent hydrolysis have been published recently. 2,3 Moreover, results published by Vaerman et al 4 indicate that the cytotoxicity of PO and PS oligos might have been, in part, caused by mononucleotides deoxyribonucleoside-5Ј-phosphates (dNMPs) or their phosphorothioate analogs, deoxyribonucleoside-5Ј-monophosphorothioates (dNMPSs) released during enzymatic degradation of the oligonucleotides. The toxicity of the dNMPs to leukemia cell lines depends on their concentration and/or the type of nucleobase.…”

A Dynamical Systems Model to Simulate the Perturbation Kinetics of Gene Expression by Antisense Oligonucleotides

“…The phosphorothioate modi"cation has been predominantly exploited for its nuclease stability and its ability to recruit RNase H. Despite this, non-stereospeci"c synthesis of phosphorothioates with 3 terminal chiral R con"guration exhibits marked sensitivity to 3 exonucleases (Gilar et al, 1998) and a signi"cant contribution to antisense reversibility. Changing the ASO t from 6 (unstable) to 72 hr (very stable) exhibited the principle e!ect of lengthening the duration of RNA recovery [ Fig.…”