Phosphodiesterase susceptibility of modified oligonucleotides studied in an integrated capillary electrophoresis system.

ABSTRACT

A capillary electrophoresis system that integrated an enzymatic reaction and capillary polymer sieving electrophoresis was used to check the enzymatic stability of oligonucleotides. Phosphodiesterase I was employed to assess the susceptibility to 3'-exonucleolytic breakdown of some unmodified and modified oligonucleotides. Before degradation, the purity of the synthetic oligodeoxynucleotides was checked by capillary electrophoresis with a replaceable hydroxyethyl cellulose polymer solution. Enzymatic breakdown was achieved inside the capillary by merging substrate and enzyme zones based on their difference in electrophoretic mobility. After reaction, oligonucleotide fragments were swept to the detector, where they were individually detected and the remaining substrate was quantified. The results from the in-capillary degradation were compared to an off-line incubation and separation.