Tritium labeling of antisense oligonucleotides by exchange with tritiated water.
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.
Graham
M J
MJ
ISIS Pharmaceuticals, Carlsbad, CA 92008.
Freier
S M
SM
Crooke
R M
RM
Ecker
D J
DJ
Maslova
R N
RN
Lesnik
E A
EA
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*
D016376:Oligonucleotides, Antisense; D014867:Water; D014316:Tritium; D008623:Mercaptoethanol