STABILITY MEASUREMENTS OF ANTISENSE OLIGONUCLEOTIDES BY CAPILLARY GEL-ELECTROPHORESIS

The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For so me of the samples, matrix assisted laser desorption and ionization mas s spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after differen t incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The ki netic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Als o, a simple desalting method to improve the injection efficiency and s ensitivity of the method are described. Examples of measurements of ch emically modified antisense 19-mers are presented.