Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis.
Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50cm neutrally coated capillary at 60 degrees C together with a 7M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions >or=1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.
Noll
Bernhard O
BO
Coley Pharmaceutical GmbH, Elisabeth-Selbert-Strasse 9, D-40764 Langenfeld, Germany. bnoll@coleypharma.com
Debelak
Harald
H
Uhlmann
Eugen
E
D000818:Animals; D001483:Base Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D020862:GC Rich Sequence / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000737:chemistry*; D051379:Mice; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D015203:Reproducibility of Results
D009838:Oligodeoxyribonucleotides