title: 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.
authors.affiliation: Hybridon, Inc., Cambridge, MA 02139, USA.
authors.forename: M
authors.initials: M
authors.lastname: Gilar
authors.forename: A
authors.initials: A
authors.lastname: Belenky
authors.forename: Y
authors.initials: Y
authors.lastname: Budman
authors.forename: D L
authors.initials: DL
authors.lastname: Smisek
authors.forename: A S
authors.initials: AS
authors.lastname: Cohen
mesh_terms: D005092:Exonucleases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D013873:Thionucleotides; D005092:Exonucleases; C022650:spleen exonuclease