Quantitative analysis of modified antisense oligonucleotides in biological fluids using cationic nanoparticles for solid-phase extraction.
Based on a novel method for solid-phase extraction using cationic polystyrene nanoparticles, the suitability of the extraction procedure for quantitation of terminally and backbone-modified antisense oligonucleotides was investigated. Extractions were carried out from both human plasma and urine. Quantitative analysis of the extracted samples was performed with capillary gel electrophoresis. In accordance with previous results obtained with phosphorothioate oligonucleotides in human plasma, high linearity and accuracy of the assay was demonstrated for an oligodeoxyribonucleotide-palmityl conjugate as well as for a modified oligoribonucleotide. Optimized extraction conditions allow the isolation of oligonucleotides in high yields and purity even for concentrations in the low nanomolar range, down to 5 nM. Comparing the results obtained from human plasma and urine, no significant differences in the absolute recovery rates which reach values up to 95% were observed. However, when the loading capacity of the nanoparticles was exceeded, selective recovery was observed for the coisolation of phosphodiester and phosphorothioate oligonucleotides. This effect can be explained by differences in the attractive forces between PO- and PS-oligonucleotides and the particle surface and appears to be valuable for a modification-dependent enrichment of oligonucleotides out of complex mixtures.
Gerster
M
M
Research Center for Nucleic Acid and Peptide Chemistry, University of Tübingen, Auf der Morgenstelle 18, Tübingen, D-72076, Germany.
Schewitz
J
J
Fritz
H
H
Maier
M
M
Bayer
E
E
D002138:Calibration; D002412:Cations / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D008863:Microspheres; D016376:Oligonucleotides, Antisense / Q000097:blood* / Q000652:urine*; D009843:Oligoribonucleotides / Q000737:chemistry; D011137:Polystyrenes / Q000378:metabolism
D002412:Cations; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; D011137:Polystyrenes