Enzymatic degradation of various antisense oligonucleotides: monitoring and fragment identification by MECC and ES-MS.

Efficacy and sequence specific behaviour of antisense oligonucleotides in biological systems are attenuated by enzymatic degradation, which is predominantly dependent on the oligonucleotide modification. Quantitative data relating to the kinetics and pattern of enzymatic digestion are thus valuable for the interpretation of biological tests with novel antisense oligonucleotides. To study the stability of modified oligonucleotides against nuclease attack, in vitro experiments of enzymatic degradation have been carried out using micellar electrokinetic capillary chromatography (MECC) as a quantitative control and electrospray mass spectrometry (ES-MS) for fragment identification. In contrast to gel electrophoresis, which is commonly applied, monitoring of enzymatic digestion by MECC can be carried out directly from the incubated sample without the need for labeled substrate. Furthermore, exact quantitative analysis becomes possible. Phosphodiester oligonucleotides terminally conjugated with hexaethylene glycol have been prepared to investigate the stability and degradation process of 3'- and 5'-protected oligomers with natural backbones in serum-containing medium. The results demonstrate that 3'-protection is much more effective than 5'-protection for nuclease stability, both in fetal calf serum and in human blood serum. To examine the influence of backbone modification on nuclease stability, the digestion of dodecanucleotides containing different numbers of phosphorothioate groups has been investigated by MECC and ES-MS. Degradation rates vary by a factor of approximately 50. Most fragments have been identified and the degradation patterns allow conclusions about the variations of nucleolytic activity with changing substrates.