title: Tritium labeling of antisense oligonucleotides by exchange with tritiated water.
abstract: We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides.
authors.affiliation: ISIS Pharmaceuticals, Carlsbad, CA 92008.
authors.forename: M J
authors.initials: MJ
authors.lastname: Graham
authors.forename: S M
authors.initials: SM
authors.lastname: Freier
authors.forename: R M
authors.initials: RM
authors.lastname: Crooke
authors.forename: D J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: R N
authors.initials: RN
authors.lastname: Maslova
authors.forename: E A
authors.initials: EA
authors.lastname: Lesnik
mesh_terms: D000818:Animals; D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008623:Mercaptoethanol; D008722:Methods; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D014316:Tritium; D014407:Tumor Cells, Cultured; D014867:Water / Q000737:chemistry*
chemical_list: D016376:Oligonucleotides, Antisense; D014867:Water; D014316:Tritium; D008623:Mercaptoethanol