The detection of oligonucleotide N3'-->P5' phosphoramidate/RNA duplexes with capillary gel electrophoresis.
Oligonucleotide N3'-->P5' phosphoramidates (3'-phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single-stranded RNA fragments utilizing 3'-phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3'-Phosphoramidate 9-mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3'-phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3'-phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utilized to verify duplex formation.
DeDionisio
L
L
Lynx Therapeutics Incorporated, Hayward, CA 94545, USA.
Raible
A M
AM
Gryaznov
S M
SM
D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015394:Molecular Structure; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*
D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D012313:RNA