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Analysis of an oligonucleotide N3′→ P5′ phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis
Author(s) -
DeDionisio Lawrence A.,
Raible Anette M.,
Nelson Jeffrey S.
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150191623
Subject(s) - phosphoramidate , capillary electrophoresis , oligonucleotide , chemistry , nucleotide , chromatography , polyacrylamide gel electrophoresis , combinatorial chemistry , dna , biochemistry , gene , enzyme
N3′→ P5′ phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, “second generation” chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18‐mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3′→ P5′ phosphoramidate (amidate) linkages on the 5′‐end and five amidate linkages ont he 3′‐end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3′→ P5′ phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase ‐ high performance liquid chromatography (RP‐HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.