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 some of the samples, matrix assisted laser desorption and ionization mass 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 different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented.
Bruin
G J
GJ
Corporate Analytical Research, Ciba, Basel, Switzerland.
Börnsen
K O
KO
Hüsken
D
D
Gassmann
E
E
Widmer
H M
HM
Paulus
A
A
D001483:Base Sequence; D004586:Electrophoresis / Q000379:methods*; D004720:Endonucleases / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D011071:Poly T / Q000737:chemistry*; D012910:Snake Venoms / Q000201:enzymology
D016376:Oligonucleotides, Antisense; D012910:Snake Venoms; D011071:Poly T; D004720:Endonucleases